key: cord-277611-3iynrfzq authors: buetti, niccolò; patrier, juliette; le hingrat, quentin; loiodice, ambre; bouadma, lila; visseaux, benoit; timsit, jean-françois title: risk factors for sars-cov-2 detection in blood of critically ill patients date: 2020-09-02 journal: clin infect dis doi: 10.1093/cid/ciaa1315 sha: doc_id: 277611 cord_uid: 3iynrfzq nan m a n u s c r i p t 2 dear editor, data on detection of sars-cov-2 viral rna within covid-19 patients' blood are scarce [1] [2] [3] . the recently published article by veyer et al. described plasma sars-cov-2 viral load in 58 noncritically and critically ill patients [4] . in their multivariate analysis the authors showed that sars-cov-2 rnaaemia was strongly associated with the clinical class, with higher level rnaaemia among critically ill patients. however, to date, risk factors for detectable sars-cov-2 rnaaemia in critically ill patients remain unknown. therefore, we conducted a similar study using prospectively collected data at the bichat university hospital, france, in order to identify risk factors for sars-cov-2 detection in blood in critically ill intubated patients. all included patients had a sars-cov-2 positive nasopharyngeal swab before intensive care (icu) admission. during the icu stay, all patients underwent a regularly monitoring of sars-cov-2 rnaaemia. all blood specimens were sent to the virology laboratory and used for rna extraction, using magnapure large volume total na kit (roche), and amplification by real time polymerase chain reaction (rt-pcr) techniques using realstar sars-cov-2 rt-pcr ruo assay (altona). of note, such rt-pcr assay presents a low limit of detection at 625 copies/ml [5] , probably slightly higher than droplet pcr assay used by veber et al. in order to identify risk factors for sars-cov-2 detection in blood, we used univariable and multivariable mixed-effect logistic models for clustered data (proc glimmix of sas) and we adjusted for the time between symptoms' onset and date of sampling. this model takes into account the clustering effect of multiple sampling per patient. from march to april 2020, in 42 patients 81 blood samples for sars-cov-2 detection were collected; 30 samples (37%) were positive. thirty-four (81%) patients were male and the median age was 58 (iqr: 46; 67); 22 (52%) had a cardiovascular comorbidity and eight (19%) were immunosuppressed. twenty-two (52%) and 18 (43%) patients received corticosteroids and lopinavir/ritonavir. the median time to negativity (i.e., time between onset of symptoms and viral clearance process from blood) was 17 days (iqr: 12; 21). using univariable mixed-effect models after adjusting for the time interval between onset of symptoms and date of sampling, we showed that immunosuppression (or 12.16, 95% ci 1.74-84.93, p=0.013) and chronic renal failure (or 5.98, a c c e p t e d m a n u s c r i p t 3 95% ci 1.14-31.35, p=0.035) increased the risk for sars-cov-2 detection in blood (table) . interestingly, sars-cov-2 detection in blood was not associated with 6-week mortality. in the multivariable analysis, immunosuppression significantly increased the risk for sars-cov-2 detection in blood (or 8.95, 95% ci 1.17; 68.38, p=0.035, table) . veyer et al. showed that sars-cov-2 rnaaemia was strongly correlated with disease severity [4] . with our data and using classical rt-pcr, we observed that especially immunosuppressed critically ill patients tended to be viremic with sars-cov-2. in contrast to veyer et al., we did not observe any association with mortality. further larger multicentric cohorts are urgently needed to investigate risk factors for rnaemia using classical and ultrasensitive rt-pcr methods in severe and non-severe covid-19 patients. m a n u s c r i p t m a n u s c r i p t 8 legend. *adjustment for the time interval between onset of symptoms and date of sampling. or: odds ratio. ci: confidence interval. sofa: sequential organ failure assessment score. icu: intensive care unit. a sensitivity analysis forcing the variables "sofa" and "chronic respiratory failure" in the multivariable analysis showed similar results (or for immunosuppression 8.35, 95% ci 0.89-78.29, p=0.063). clinical features of patients infected with 2019 novel coronavirus in wuhan, china detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients detectable 2019-ncov viral rna in blood is a strong indicator for the further clinical severity. emerging microbes & infections 2020 highly sensitive quantification of plasma sars-cov-2 rna shelds light on its potential clinical value. clinical infectious diseases : an official publication of the infectious diseases society of america evaluation of the realstar(r) sars-cov-2 rt-pcr kit ruo performances and limit of detection a c c e p t e d m a n u s c r i p t 6 a c c e p t e d m a n u s c r i p t key: cord-264660-tfktgy57 authors: creech, c buddy title: it’s true even in a pandemic: children are not merely little adults date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa680 sha: doc_id: 264660 cord_uid: tfktgy57 nan m a n u s c r i p t 2 though separated from family and friends due to physical distancing, my wife's grandfather celebrated his 104 th birthday in april 2020. born in 1916, in the middle of the first world war, he is now experiencing his 2 nd global pandemic and the attendant reality of human mortality. in his toddler years, he belonged to a high-risk population for pandemic influenzachildren less than 5 years old, adults >65, and uniquely, young adults 20-40 years of age were disproportionately affected. now, a century later, he finds himself again in a high-risk populationadults >65 years of age and living in a long-term care facility. strangely, and to his delight, his great-great grandchildren (of whom there are many) do not seem to share this increased risk for disease. his endurance and repose during the intervening years point to a principle that each new global threat to human health brings with it something old and something new. pandemic influenza and coronavirus share transmission characteristics that allow for rapid and dense spread through communities. they share a predilection for severe disease, particularly in high risk hosts, while they also benefit from asymptomatic hosts and minimally symptomatic individuals who are capable of transmission prior to obvious signs of disease. an unusual and new feature of sars-cov-2 is the apparent reduced infectivity and relatively low frequency of severe disease in children, an unusual characteristic of a highly contagious respiratory virus. understanding these similarities and differences will be critically important as we move forward through this pandemic. in this issue of cid, mehta et al (cid paper) provide a systematic review of pediatric covid-19, evaluating the available literature to date to glean characteristics of disease and transmission. though limited by the availability of case reports and case series, many from epicenters in china and italy, the authors were able to identify a number of studies that were of sufficient quality and granularity to warrant inclusion in the review. consistent with local and national experiences, children appear to be less affected by covid-19 than adults. the authors report that children represent only 5% or less of diagnosed covid cases and the data available at the time of review suggest that children are less likely to develop either severe pneumonia or the laboratory alterations commonly associated with severe disease, such as lymphopenia and elevated inflammatory markers. a c c e p t e d m a n u s c r i p t 3 the authors also report that intrauterine transmission appears to be extremely uncommon and that newborns born to infected mothers are likely to experience either asymptomatic disease or mild disease. taken together, it would appear that children experience a very different response to sars-cov-2 infection than adults and raises the hypothesis that dysregulated host responses may be the primary driver of disease severity. in fact, targeting the host response to infection has become the focus of multiple clinical trials, including baricitinib, tocilizumab, ravulizumab, eculizumab, and a host of other drugs aimed at blunting an exuberant immune response. using a similar approach, zimmermann et al recently evaluated available cases of disease in children and neonates [1] . in this review, approximately 35% were asymptomatic and coinfections (namely mycoplasma and influenza) have been identified in many children, complicating how providers attribute symptoms to sars-cov-2. pregnancy outcomes were similarly encouraging, though fetal distress was reported in up to 30% of pregnancies and preterm delivery complicated approximately 35% or reported cases. the authors raise a number of hypotheses that may explain the differences in phenotypes from adults, including differences in immune response, fewer co-morbidities, microbial interactions in the nasal mucosa, differences in ace2 receptor density in the nasal epithelium [2] , and others. the report, and emerging data from the uk and us, particularly new york city, are particularly intriguing given previous hypotheses regarding the potential role of coronaviruses in the pathogenesis of kawasaki disease [4] . in addition, larger case series of pediatric cases are becoming available [5] , giving even greater clarity on epidemiology, transmission characteristics, and disease manifestations. teachers, administrators, and support staff in local schools and childcare facilities belong to high risk groups as a result of age or comorbidities. we have a duty to protect these individuals as much as possible. moreover, we must recognize that some proportion of children that are infected will experience either moderate disease, severe disease, or immunologically mediated complications, such as mis-c. defining the true burden of disease across pediatrics will be essential as we consider best practices for the start of the 2020-21 academic year. my wife's grandfather would likely summarize the issue by saying there is 'nothing new under the sun.' unfortunately, the vast majority of us have not been around the sun as many times as he has. we are still learning what we know, what we don't know, and what we think we know that just isn't so. as it relates to children, early reports are encouraging, but the emergence of immunologically mediated complications must give us pause. in this pandemic, at least thus far, children are not merely mirrors of the adult experience but bring their own challenges in diagnosis and treatment. a c c e p t e d m a n u s c r i p t children, pregnancy and neonates nasal gene expression of angiotensin-converting enzyme 2 in children and adults an outbreak of severe kd-like disease at the italian epicentre association between a novel human coronavirus and kawasaki disease epidemiology of covid-19 among children in china key: cord-254458-bbcef8xt authors: ali, farhana; sweeney, daniel a title: throat wash testing and covid-19 disease: should we put our money where our mouth is? date: 2020-04-30 journal: clin infect dis doi: 10.1093/cid/ciaa511 sha: doc_id: 254458 cord_uid: bbcef8xt nan m a n u s c r i p t accurate and efficient identification of infected persons is necessary if the covid-19 pandemic is to be optimally managed and contained. unfortunately, patient reported symptoms and physician examinations, especially early in the clinical course, have not been consistently helpful in diagnosing covid-19 disease. moreover, asymptomatic or pre-symptomatic transmission of covid-19 disease has been described [1] . the tragedy that is the us aircraft carrier theodore roosevelt, in which 60 percent of the 600 sailors who tested positive for sars-cov-2 infection were asymptomatic, further highlights the need for accurate laboratory diagnostics for covid-19 disease [2] . the us centers for disease control and prevention (cdc) currently recommends that initial diagnostic testing for covid-19 disease be performed using a nasopharyngeal (np) swab [3] . sars-cov-2 has been detected in a variety of clinical samples including sputum and bronchoalveolar lavage fluid. and when compared directly to oropharyngeal (op) samples, nasopharyngeal testing for sars-cov-2 was not consistently shown to be superior [4] [5] [6] . there are other obvious drawbacks to using a nasopharyngeal swab-based test compared to a throat wash-based test both to the individual patient and the healthcare system. to begin with, the swab sample collection method is an unpleasant experience for the patient. moreover, personal protective equipment (ppe) is required for clinicians collecting np swabs, resulting in increased utilization of ppe and supply shortages. similarly, there have also been shortages of nasopharyngeal swabs, leading to delays in covid-19 testing [7] . the limitations of this study are worth noting. this is a single center investigation with a relatively small number of participants (n=11) enrolled. the technique by which samples were collected ("asking the patient to oscillate over posterior pharyngeal wall with 20 ml of sterile saline") may have differed among study participants. and whether the test results are due to pharyngeal sampling or oral rinsing at the time of expectoration cannot be determined. in terms of the actual laboratory testing, the details of the assay kits used, including which primers were employed, were not disclosed. finally, the authors also assume that rt-pcr testing did not yield any false positive results. although rare, false positive results with rt-pcr testing can occur secondary to sample contamination. these shortcomings aside, guo et al. make the case that testing of throat wash samples may be more sensitive than testing of np swabs for covid-19 disease. np testing compared to throat wash testing failed to detect sars-cov-2 nucleic acid in 86% a c c e p t e d m a n u s c r i p t (6/7) of paired samples. interestingly, there is biologic plausibility that a sample collected via the oral cavity (such as a throat wash) may yield higher results than a np swab test based on a study by xu et al. which demonstrated high ace2 receptor expression on the epithelial cells of oral mucosa and the base of the tongue which is a part of the oropharynx [8] . whether two consecutive rt-pcr tests using np swab specimens (currently recommended by the cdc as part of a test-based strategy for discontinuing isolation precautions) results in a sensitivity comparable to one throat wash is not known [9] . admittedly, the sensitivity of throat wash testing almost seems "too good to be true;" nonetheless, the ramifications of failing to identify covid-19 positive patients would have dire consequences amid a pandemic. regardless of testing method employed, this study is further remarkable for the fact that sars-cov-2 nucleic acid was detected in persons late in their clinical course and had presumably recovered. of the paired samples, 29% (7/24) were notably positive in individual 48 days after the onset of symptoms. these findings are consistent with another study showing that the median duration of viral shedding in a cohort of 41 patients with severe covid-19 disease was 31 days from onset of illness [10] . other investigators have also described the clinical course of 191 patients with covid-19 disease and reported the median duration of viral shedding was 20 days in survivors and shedding continued to the time of death in non-survivors [11] . while the presence of detectable viral rna does not necessarily mean that replicating virus is present or that a patient is infectious, it does raise the question of whether current isolation guidelines are adequate to contain the spread of disease. for example, the duration for transmissionbased precautions can be as brief as seven days based on current cdc recommendations a c c e p t e d m a n u s c r i p t [9] . ultimately, the period of sars-cov-2 infectivity can only be determined with serial viral culture testing (unlikely to be performed except at centers where laboratory containment standards are in place) or possibly estimated using longitudinal serology testing. improved laboratory detection of sars-cov-2 infection is critical if the medical community is going to effectively contain and curtail the covid-19 pandemic. the use of an np swab sample has its drawbacks. putting their money where their mouth is, guo and colleagues suggest that rt-pcr testing of a throat wash sample--which represents a non-invasive, painless, self-administered (without the need for ppe), swab-free method-may be a more sensitive test for covid-19 disease. finally, the authors' detection of virus late in the patients' clinical course emphasizes the uncertainty faced by policy makers as they try to determine the optimal length of isolation for an individual recovering from covid-19 disease. neither author has any potential conflicts of interest. presumed asymptomatic carrier transmission of covid-19 coronavirus clue? most cases aboard u.s. aircraft are symptom free virological assessment of hospitalized patients with covid-2019 saliva is more sensitive for sars-cov-2 detection in covid-19 patients than nasopharyngeal swabs evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections testing swabs run in short supply as makers try to speed up production high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa discharging hospitalized patients the duration of viral shedding of discharged patients with severe covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study a c c e p t e d m a n u s c r i p t key: cord-279932-bilr71ay authors: plotkin, stanley a title: the value of human challenges in severe acute respiratory syndrome coronavirus 2 vaccine development date: 2020-07-16 journal: clin infect dis doi: 10.1093/cid/ciaa1013 sha: doc_id: 279932 cord_uid: bilr71ay nan 1 pediatrics, university of pennsylvania, vaxconsult, doylestown, pennsylvania, usa, and 2 sanofi pasteur, doylestown, pennsylvania, usa the epidemic of coronavirus disease 2019 (covid-19) rolls on, at least in the rulerecalcitrant population of the united states, but also in countries that are too poor and disorganized to provide conditions for self-isolation. aside from the increasing toll of death and disability, the economic and social effects of the epidemic are disastrous and grow day by day, exceeding anything seen since the 1918-19 influenza outbreak or the depression of the 1930s. we have only 2 hopes for salvation: the exhaustion of susceptible individuals by natural infections or the development of a preventive vaccine. the former hope would require acceptance of many deaths and overflowing hospitals, while the latter hope depends on an effective vaccine being licensed for widespread use. normally, licensure of a vaccine requires many years of development, including a large safety and efficacy trial in which vaccinees are compared to placebo recipients with respect to reactions and to the incidence of the disease for which the vaccine was developed. in this pandemic, that would mean a requirement for sufficient covid-19 disease and possibly deaths in the placebo group, with less disease in the vaccine group. meanwhile, those not in a trial would still be subject to the ravages of the disease. is there another way to confirm that a vaccine against the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is protective, and thus to accelerate its use? a number of people, including nguyen et al [1] in this issue and others [2] [3] [4] [5] [6] [7] elsewhere, have proposed the use of human challenge trials as a way of confirming the protective ability of candidate vaccines, in order to allow emergency use in high-risk groups and to facilitate the way to eventual licensure and use in the general population. however, there are obvious objections to such a strategy, summarized by the adage that all physicians learn: "above all, do no harm. " it is repugnant to think that something we do may cause disease and perhaps death, even if the goal in the long term is to save lives. the idea behind human challenge trials is to recruit young, healthy volunteers who have the lowest chance of serious disease, who would be given vaccine candidates and then be challenged with sars-cov-2 in order to determine whether the vaccines protect. however, there would also have to be prior challenges of unvaccinated volunteers to determine the optimal infectious dose, and also challenges of some volunteers who have had prior covid-19 infections, to confirm that immune responses can give subsequent protection. obviously, the ethical issues around human challenge trials are many. however, numerous ethicists have weighed in on this issue. although some have disagreed [8, 9] , the majority have accepted human challenge trials done in informed volunteers as ethical under the current circumstances. some ethicists have pointed out that organ donation by volunteers is an accepted practice, despite a low but definite risk of death or disability, which is equivalent to the risk of covid-19 in the young age group of volunteers [10] . the organization to which the authors of the paper by nguyen et al [1] belong, one day sooner, represents literally thousands of volunteers for sars-cov-2 challenges. aside from the ethical issues, the principal objection to human challenge trials with sars-cov-2 is the absence of a reliable rescue medication for the treatment of serious disease. to mitigate disease severity, one could begin treatment with remdesivir, convalescent serum, and steroids as soon as a lower respiratory tract infection is confirmed, but one must bear in mind that as of this writing, no therapy is confirmed to be completely effective. nevertheless, by confining studies to young (18-25 years old) and healthy volunteers, current data suggest a very low risk of severe disease. in evaluating the idea of human challenges with sars-cov-2, one must balance the risks of those challenges against the risks associated with the usual process of vaccine development and evaluation. table 1 attempts to contrast the advantages and disadvantages of each approach. clearly, there are unknowns that should obviate dogmatism, but it appears to this observer that human challenge experiments are worth preparing for and potentially putting in motion, particularly considering the large number of current vaccine candidates and the difficulties of conducting phase 3 trials for all of them. clearly, human challenges in a pandemic disease with associated fatality, such as covid-19, must be seriously evaluated before launching them. the manufacture of a challenge virus will take some weeks, as will organizing a site in which the challenged volunteers can be isolated and receive medical care. however, the objection of some that those steps will take so long that the value of human challenge studies will be negated is a self-fulfilling prophecy: the longer we argue, the less the value of the proposed idea. if vaccines are rapidly tested and made available through the usual phases 1-3, human challenge trials might be obviated and could always be cancelled. however, a committee of the world health organization and a committee of the national institutes of health have both given provisional approval to human challenges [11, 12] . the longer we hesitate, the less the value of human challenge trials. note potential conflicts of interest. s. a. p. is a consultant to numerous companies and biotechs, none of which have indicated interest in human challenge trials. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. evaluating use cases for human challenge trials in accelerating sars-cov-2 vaccine development human challenge studies to accelerate coronavirus vaccine licensure extraordinary diseases require extraordinary solutions why challenge trials of sars-cov-2 vaccines could be ethical despite risk of severe adverse events ethics of controlled human infection to address covid-19 covid-19 human challenge studies: ethical issues a strategic approach to covid-19 vaccine r&d severe acute respiratory syndrome coronavirus 2 human challenge trials: too risky, too soon an ethical path to a covid vaccine estimates of the severity of coronavirus disease 2019: a model-based analysis accelerating development of sars-cov-2 vaccines -the role for controlled human infection models who advisory group tasked to consider the feasibility, potential value, and limitations of establishing a closely monitored challenge model of experimental covid-19 infection and illness in healthy young adult volunteers key: cord-252761-ro5tj0tx authors: marriott, deborah; beresford, rohan; mirdad, feras; stark, damien; glanville, allan; chapman, scott; harkness, jock; dore, gregory j; andresen, david; matthews, gail v title: concomitant marked decline in prevalence of sars-cov-2 and other respiratory viruses among symptomatic patients following public health interventions in australia: data from st vincent’s hospital and associated screening clinics, sydney, nsw. date: 2020-08-25 journal: clin infect dis doi: 10.1093/cid/ciaa1256 sha: doc_id: 252761 cord_uid: ro5tj0tx our australian hospital tested almost 22,000 symptomatic people over 11 weeks for sars-cov-2 in a multiplex pcr assay. following travel bans and physical distancing, sars-cov-2 and other respiratory viruses diagnoses fell dramatically. increasing rhinovirus diagnoses as social control measures were relaxed may indirectly indicate an elevated risk of covid-19 resurgence a c c e p t e d m a n u s c r i p t diagnoses increased rapidly to a peak of 460 daily cases on 28 th march, before declining to fewer than less than ten per day by mid-april. stage 3 requirements were relaxed by the end of april and by mid-may restaurants and businesses had largely re-opened. by 14 th may a total of 6,989 covid-19 cases had been diagnosed, with almost half in new south wales (nsw). 6 a screening service for sars-cov-2, using a multiplex pcr assay was established on 9 th march at st. vincent"s hospital, a university-affiliated hospital in inner-sydney, nsw. two community satellite testing services were opened in april. we report the prevalence of sars-cov-2 and other respiratory pathogens including co-infection, and evaluate the a c c e p t e d m a n u s c r i p t 4 temporal pattern of respiratory infections alongside the introduction, and subsequent relaxation, of physical distancing measures. nasopharyngeal (np) swabs were collected by trained nurses using appropriate infection control measures at the dedicated covid-19 clinic at st vincent"s hospital and satellite clinics at bondi beach and east sydney. testing was performed in the hospital emergency department (ed) when clinically indicated. a small proportion of specimens were referred from other sites such as rural laboratories, correctional facilities and general practitioners. testing was carried out in accordance with nsw health policy during the study period. initially, testing was offered to individuals with respiratory symptoms who had returned from overseas, had severe respiratory illness, had been in contact with a known covid-19 case, or had healthcare employment. these criteria were subsequently expanded to include anyone with fever or respiratory symptoms in local "hot spots" (30 th march 2020), defined over the eleven-week period from 12 th march to 27 th may 2020, 21,808 people were tested. the proportion of people in whom any respiratory pathogen was detected declined markedly during the study period, from 32.5% in week one to 3.1% in week eight before rising again to 12.9% in week 11 (figure) (p<0.001 for each subsequent week, compared to baseline, in a two-sample test of proportions). in most cases a greater than 10-fold reduction was observed: rhinovirus, 19.9% to 1.7% (before increasing again to 11.8% by late-may); parainfluenza, a c c e p t e d m a n u s c r i p t 6 3.0% to 0.1%; and non-sars-cov-2 coronaviruses, 2.3% to less than 0.1%. of note, nonviral respiratory pathogens such as bordetella pertussis, mycoplasma pneumoniae and pneumocystis jiroveci did not demonstrate marked reduction. (supplementary table) . at a major hospital serving the initial geographical epicentre of australian sars-cov-2 diagnoses, 175 cases were identified over an 11-week period. despite the broadening of testing criteria, an increase in total testing numbers, and a move into cooler months, sarscov-2 cases to similarly rise supports the absence of circulating sars cov-2 in the nsw community during that period, but the increased rhinovirus transmission does raise concern that sars cov-2 may also spread readily if reintroduced. singaporean report 10 showed a 76% reduction over historical rates. to our knowledge, ours is the first report from the southern hemisphere demonstrating a reduction during a move into the cooler months, and also the first to document simultaneous trends in other infectious respiratory pathogens. among individuals with sars-cov-2, 5% had co-infection with other respiratory pathogens, with rhinovirus most common. limited data have been reported on co-infection between sars-cov-2 and other respiratory viruses. in a seattle surveillance study, 11 4 of 25 people (16%) with sars-cov-2 had rhinovirus coinfections. interestingly, in that study (as in ours) no coinfections between sars-cov-2 and non-pandemic coronaviruses were found. this raises the hypothesis that co-exposure to different human coronaviruses may lead to only one establishing infection, or that shared viral epitopes lead to some degree of cross-immunity within the coronavirus group. a californian report, 12 however, has shown occasional coinfections between sars-cov-2 and non-pandemic coronaviruses. there are some key limitations of our study. firstly, during the eleven-week period the sars-cov-2 testing criteria changed, with broadened testing initially in local geographical "hotspots" then our entire catchment area. however, guidance was consistent that only people with fever or other respiratory tract symptoms should be tested. secondly, detailed data on a c c e p t e d m a n u s c r i p t 8 the timing of testing in relation to symptom onset were not available. in general, however, most people were tested within a few days of symptom onset, when sars-cov-2 should have remained detectable. thirdly, we are unable to determine which sars-cov-2 prevention measures had the greatest impact on respiratory virus prevalence, as they were introduced in rapid succession. it is also unclear how much impact a shift in sars-cov-2 cases from largely returned overseas travellers (predominantly from north america and europe) to locally acquired cases had on temporal trends in respiratory viral pathogen prevalence. higher prevalence of all respiratory viral pathogens would have been expected among returned travellers from the northern hemisphere late in the northern winter, and these may have reduced naturally over time even without local covid-19 prevention measures. our study period is too short however to assess any seasonal trends. in conclusion, the introduction of multiple public health measures to minimise sars-cov-2 transmission in australia from mid to late-march 2020 had a major impact on the prevalence of all respiratory viral infections highlighting the effectiveness of this approach. changes in the prevalence of circulating respiratory viruses may provide a useful reflection of the success of ongoing measures including physical distancing restrictions. m a n u s c r i p t 11 9. kuo s-c et al (2020 covid-19) from publicly reported confirmed cases: estimation and application summary of a report of 72 314 cases from the chinese center for disease control and prevention viral dynamics in mild and severe cases of covid-19. yang liu et al. lancet infectious diseases analytical sensitivity and efficiency comparisons of sars-cov-2 qrt-pcr primer-probe sets interpreting diagnostic tests for sars-cov-2, nandini sethuraman et al suppressing the epidemic in new south wales. jeremy m. mcanulty and kate ward viruses associated with acute respiratory infection in a community-based cohort of healthy new zealand children a c c e p t e d m a n u s c r i p t key: cord-258304-86gqxajw authors: bahl, prateek; de silva, charitha; bhattacharjee, shovon; stone, haley; doolan, con; chughtai, abrar ahmad; macintyre, c raina title: droplets and aerosols generated by singing and the risk of covid-19 for choirs date: 2020-09-18 journal: clin infect dis doi: 10.1093/cid/ciaa1241 sha: doc_id: 258304 cord_uid: 86gqxajw choral singing has become a major risk during covid-19 pandemic due to high infection rates. our visualisation and velocimetry results reveal that majority of droplets expelled during singing follow the ambient airflow pattern. these results points toward the possibility of covid-19 spread by small airborne droplets during singing. singing in group settings has become an apparent risk for outbreaks of covid-19 [1] . while social distancing is effective in normal social interactions, singing can produce a substantially larger number of respiratory droplets and aerosols than speaking, as it is louder and sustained for longer durations [2] . this may require further measures to be put into place to mitigate risks. in contained smaller spaces, the transmission risk may be higher, as respiratory aerosols may saturate the whole indoor environment [3] . despite this, at present there is not a collective approach or response to the potential risks of group singing. certain countries, including germany and the netherlands, have banned all group singing activities [4] . choir-related outbreaks of covid-19 in berlin, amsterdam, and washington state had high attack rates of 75.6%, 78.5% and 86.9% respectively [1, 4, 5] . since restrictions have eased globally, a rise in outbreaks related to singing has been reported [6] . a large proportion of these documented outbreaks associated with singing (approx. 69%), were reported from the united states resulting in over 544 cases [1] . covid-19 is assumed to be transmitted through respiratory and contact routes; and transmission by respiratory droplets is believed to occur only in close contact (within 1-2 m) with someone who is infectious [7] . however, there is uncertainty about the dynamics of respiratory emissions during singing. additionally, studies have shown that droplet and airborne transmission may not be mutually exclusive modes of transmission and exist as a continuum [8] . hence, unravelling the spread of respiratory particles during singing, especially in closed environments, could inform infection control policy and practice. to quantify and understand the spread of infection during singing we performed a detailed flow visualization of aerosols and droplets expelled during singing using an image-based flow diagnostic system. the visualisation technique employs a led based light source (gs vitec multiled pt) with a spherical lens to control the divergence of the light beam, along with a high-speed camera (nac memrecam hx-7s) to capture the light scattered by the droplets expelled during singing. to capture the video, the head of the subject was adjusted in front of black backdrop/background and the light was positioned in a forward scatter arrangement to maximize the scattering from expelled droplets ( figure 1 ). once the position is adjusted, the subject was asked to sing a major scale using the solfège system and the high-speed video was captured with an exposure of 600 µs per frame at a resolution of 2 megapixels. in addition to singing, the subject also counted from 1 to 10 and coughed voluntarily. the horizontal field of view captured in the high-speed video, from the mouth of the subject, was 26 cm approximately. frames obtained from the video were first calibrated and a 2-axis stabilisation was applied to subject's head (further details on this procedure can be found in [9] ). thereafter, all the frames were processed with a hybrid particle tracking velocimetry technique [10] . this technique first performs a particle image velocimetry (piv) procedure to estimate flow velocity and these estimates are subsequently refined using a particle tracking algorithm applied to each droplet. specifically, for the piv step we used a variable interrogation window with an initial size of 96 × 96 pixels and a final size of 48 × 48 pixels with an overlap of 75%. for particle tracking we used a correlation window of 64 pixels and particle size range of 2 to 100 pixels in order to cover the entire size range of visible droplets. the flow visualisation together with detailed particle tracking results are included in supplementary video. the results of detailed particle tracking (in supplementary video) reveals that the maximum velocity of droplets expelled, specifically for certain syllables such as 'do', 'fa' and 'ti', is approx. 6 m/s, which is similar to the velocities reported for speaking [11] . upon further examining the motion of droplets at a distance of 15 cm from the mouth, we observed that almost 90% of the droplets are moving at velocities less than 1 m/s (figure 2a) . the droplets moving with velocities greater than 1 m/s are moving in the direction between 120° -240° (θ = 0° towards subject (figure 1) ) and move away from the mouth. moreover, approx. 75% of droplets observed are moving at velocities less than 0.5 m/s and the motion is equally distributed in all the directions, which implies that they do not settle rapidly and may follow the ambient airflow pattern. these results points toward high aerosol generation, as the behaviour of these droplets is like airborne particles [12] . nevertheless, to accurately quantify the size, future work using particle counters, is essential to better understand the dynamics of these droplets. figure 2b shows the distribution of droplet velocities obtained at 15 cm from the mouth for syllable 'fa' and the direction in which these droplets are moving. approximately 50% of the droplets are moving at velocities less than 0.5 m/s and more than 75% are moving away from mouth (θ = 120° to 240°), which is also evident in the supplementary video. figure 2c shows the velocity distribution of droplets that are visible while the subject was singing syllable 'sol' & 'la' and the direction in which these droplets are moving. it can be observed that all droplets are moving at velocities less than 0.5 m/s and are equally distributed in all directions. the direction in which these droplets are moving is important because it implies that for a normal choir configuration with multiple rows and heights, these droplets can pose a risk to those in the adjacent rows as well as to those in the distant rows. figure 2d and 2e shows the distribution for counting and coughing, respectively. in the case of coughing approximately 50% of the detected droplets were moving at velocities greater than 6 m/s whereas in case of speaking only 15% were moving at velocities greater than 6 m/s. we note, the loudness measured during singing was within the range of 66 -72 decibels. further, it is also worth noting that some degree of variability is expected in the number of droplets expelled between different individuals, and due to other parameters, such as loudness, notes, consonants, and duration of each note sung. nevertheless, the droplets observed do not appear to be settling down rapidly and without adequate ventilation, these droplets can potentially saturate the indoor environment which can likely explain the very high attack rates of covid-19 seen in choirs in the us and europe (almost 87% in skagit county, washington) [1] . we note the present study only provides visual evidence of the droplets and aerosols expelled during singing and compare the associated velocities and directions with speaking and coughing. however, these droplets have the capacity to potentially transmit viruses such as sars-cov-2. we only used a basic major scale for our experiments and during singing various other factors comes into play, such as pitch, rhythm, diction, etc. and it would be valuable to investigate all these aspects for future studies to have a better understanding of droplet and aerosol generation while singing. nonetheless, the data presented combined with high infection rate among the choir members (60 -90%) [1] points towards the possibility of airborne spread of covid-19 during singing events, hence, should be considered when designing safety guidelines for public singing events. these findings could inform safety guidelines for restarting choirs during and after the covid-19 pandemic and other similar respiratory infection outbreaks. for example, rehearsals could be done with fewer people, greater physical distancing between singers, or face coverings and masks to reduce droplet and aerosol expulsion [13] . in addition to that either well ventilated large spaces or outdoor performances should be utilised to minimize the risk of infection. high sars-cov-2 attack rate following exposure at a choir practice -skagit county aerosol emission and superemission during human speech increase with voice loudness indoor aerosols: from personal exposure to risk assessment german choirs silenced as singing branded virus risk concertgebouw chorus is devastated after pre-covid bach passion church-related coronavirus outbreaks reported as trump pushes for reopening modes of transmission of virus causing covid-19 : implications for ipc precaution recommendations airborne or droplet precautions for health workers treating coronavirus disease an experimental framework to capture the flow dynamics of droplets expelled by a sneeze a hybrid digital particle tracking velocimetry technique characterization of expiration air jets and droplet size distributions immediately at the mouth opening aerosol technology: properties, behavior, and measurement of airborne particles face coverings and mask to minimise droplet dispersion and aerosolisation: a video case study none of the authors has any potential conflicts to disclose. key: cord-280915-yk872yaz authors: flaherman, valerie j; afshar, yalda; boscardin, john; keller, roberta l; mardy, anne; prahl, mary k; phillips, carolyn; asiodu, ifeyinwa v; berghella, w vincenzo; chambers, brittany d; crear-perry, joia; jamieson, denise j; jacoby, vanessa l; gaw, stephanie l title: infant outcomes following maternal infection with sars-cov-2: first report from the priority study date: 2020-09-18 journal: clin infect dis doi: 10.1093/cid/ciaa1411 sha: doc_id: 280915 cord_uid: yk872yaz infant outcomes after maternal sars-cov-2 infection are not well-described. in a prospective u.s. registry of 263 infants born to mothers testing positive or negative for sars-cov-2, sars-cov-2 status was not associated with birth weight, difficulty breathing, apnea or upper or lower respiratory infection through 8 weeks of age. maternal viral infection in pregnancy and the peripartum and postpartum periods can adversely affect infant outcomes. while studies have reported that maternal sars-cov-2 infection increases the risk of preterm birth 1 and can be vertically transmitted, [2] [3] [4] [5] overall risks for infants born to mothers with sars-cov-2 are not yet well-described. currently, national and international guidelines for management of infants born to mothers with sars-cov-2 [6] [7] [8] are based on limited data without outcomes reported past the neonatal period. a more complete understanding of infant outcomes after maternal sars-cov-2 infection would inform guidelines and policies to manage this important and growing segment of the population. to address this urgent need, we report here early findings from infants born to mothers enrolled in the pregnancy coronavirus outcomes registry (priority), an ongoing nationwide study of pregnant or recently pregnant women who have confirmed or suspected sars-cov-2. priority is a prospective cohort study enrolling u.s. individuals 13 years old with suspected or confirmed sars-cov-2 during pregnancy or in the first 6 weeks after pregnancy. this manuscript reports infant outcomes for live births occurring to 179 mothers who had a positive test for sars-cov-2 and 84 mothers who had a negative test for sars-cov-2 and excludes live births of 10 mothers suspected of sars-cov-2 who were not tested. maternal outcomes from priority will be reported separately. mothers were recruited nationally through outreach by professional organizations, traditional media, social media, and word of mouth to healthcare providers. once recruited, informed consent was obtained by the study team from the mother, for herself and her infant; births occurred at over 100 hospitals across the u.s. priority was approved by the university of california san francisco institutional review board (irb #20-30410). table 1 for infant questionnaire items and the dates that their collection was initiated. priority enrollment and follow up is ongoing; for this manuscript, we report data available by june 22, 2020. we calculated the incidence and associated 95% confidence intervals for adverse outcomes using exact binomial techniques. we used chi-square analysis and fisher's exact test to compare the proportion of outcomes between infants whose mothers tested positive for the virus and those whose mothers tested negative. our cohort of 263 infants included 179 and 84, respectively, born to mothers testing positive or negative for sars cov-2. among those testing positive, 146 (81.6%) were symptomatic, while among those testing negative, 53 (63.1%) were symptomatic (p=0.001). a c c e p t e d m a n u s c r i p t 5 see the table for other clinical and demographic characteristics by maternal sars cov-2 status. in this cohort of 263 infants, 44 infants (17%) were admitted to the nicu; fast breathing or difficulty breathing was reported for 14 (11%) of 127 infants surveyed after expansion of the birth questionnaire, and apnea was reported for 2 (1.6%). these characteristics did not differ between mothers testing positive for sars-cov-2 compared to those who tested negative. among infants born to mothers who first tested positive 0-14 days prior to delivery, 20 (26.0%) of 77 were admitted to the nicu compared to 10 (12.2%) of 82 born to mothers who first tested positive more than 14 days prior to delivery (p=0.04). infants born to mothers who first tested positive 0-14 days prior to delivery were also born earlier as compared to infants born to mothers who first tested positive more than 14 days prior to delivery (mean 37.5 versus 39 week gestation, p=0.0009). additionally in this cohort, 16 mothers first tested positive for sars-cov-2 after delivery; the positive test for this subgroup occurred a median of 6 days after delivery with an interquartile range of 1-12 days after delivery. infants born to mothers who first tested positive 0-14 days prior to delivery were less likely to room in with mothers than were those born to mothers who first tested positive more than 14 days prior to delivery or after delivery (see supplemental table 2 ). two infants born to mothers who tested positive for sars-cov-2 in the third trimester were reported to have birth defects, each with multiple congenital anomalies reported. one of these had cardiac, vertebral, renal and pulmonary anomalies while the other had facial, genital, renal, brain and cardiac anomalies. one mother who tested negative for sars-cov-2 also reported an infant with gastrointestinal, renal and cardiac anomalies. among 263 initial infants enrolled in the priority study, adverse outcomes, including preterm birth, nicu admission, and respiratory disease did not differ between those born to mothers testing positive for sars-cov-2 and those born to mothers testing negative. no pneumonia or lower respiratory tract infection was reported in this cohort through 6-8 weeks of age. among infants born to mothers who tested positive for sars-cov-2, the estimated incidence of a positive infant sars-cov-2 test was low at 1.1% (0.1%, 4.0%), and infants had minimal symptoms. overall, these results are reassuring and suggest that infants born to mothers infected with sars-cov-2 generally do well in the first 6-8 weeks after birth. our study has several limitations. first, we are unable to estimate the incidence of infant sars-cov-2 infection because infant testing was incomplete and might be biased by both false-positive and false-negative results. further research is needed to report infant incidence of sars-cov-2 after maternal infection. second, since priority's control group includes both symptomatic and asymptomatic women testing negative for sars-cov-2, it may not be representative of all u.s. pregnancies. however, these inclusion criteria allowed sampling of control mothers who were more similar to the exposed group in all respects a c c e p t e d m a n u s c r i p t 7 except for sars-cov-2 test results, which may enhance causal inference for the effect of sars-cov-2 on infant outcomes. third, priority's current racial and ethnic distribution underrepresents maternal latina ethnicity and black race compared to a concurrent cdc assessment of u.s. pregnant women infected with sars-cov-2 that reported race/ethnicity as 46% hispanic, 22% black and 23% white. 9 barriers to registry participation are expected given the historical harm related to research participation and systemic racism experienced by black, indigenous, people of color communities and the current burden of sars-cov-2 in these communities and may impact the generalizability of our findings. 10 in may 2020, priority launched a reproductive health equity and birth justice core to increase enrollment of underrepresented groups and engage with partners in highly impacted communities. fourth, the timing of maternal testing in this cohort was determined at the clinical sites and may not have coincided with the onset of illness. therefore, while we found that nicu admission and earlier gestational age were more common for infants born to mothers testing positive for sars-cov-2 0-14 days before delivery than for those testing positive at other times, these associations may reflect hospital practices for management of mothers testing positive for sars-cov-2 rather than infant physiology. overall, priority's initial findings regarding infant health are reassuring. further investigation with longer follow up periods and larger sample sizes will be needed to make a definitive determination of the risk of vertical transmission, neonatal illness, and the incidence of congenital anomaly and are planned for the priority cohort. covid-19 in pregnancy was associated with maternal morbidity and preterm birth covid-19 in children, pregnancy and neonates: a review of epidemiologic and clinical features association between mode of delivery among pregnant women with covid-19 and maternal and neonatal outcomes in spain delivery room preparedness and early neonatal outcomes during covid19 pandemic neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china. jama pediatr world health organization. clinical management of covid-19 faqs: management of infants born to mothers with suspected or confirmed covid-19 evaluation and management considerations for neonates at risk for covid-19 characteristics of women of reproductive age with laboratory-confirmed sars-cov-2 infection by pregnancy status -united states a systematic review of barriers and facilitators to minority research participation among african americans, latinos, asian americans, and pacific islanders acknowledgements: we thank all of the study participants. a c c e p t e d m a n u s c r i p t 9 key: cord-275454-an8xvow3 authors: clark, andrew e; lee, francesca m title: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) screening with specimen pools: time to swim, or too deep for comfort? date: 2020-09-28 journal: clin infect dis doi: 10.1093/cid/ciaa1145 sha: doc_id: 275454 cord_uid: an8xvow3 nan the outbreak and global spread of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) continues to challenge the way physicians, laboratories, and public health officials diagnose and track cases. testing was initially relegated to reference laboratories and academic medical centers with the expertise to rapidly design and validate laboratorydeveloped diagnostic assays to detect a novel virus. these laboratories quickly became overburdened, resulting in turnaround times beyond what is clinically actionable. the availability of commercial sars-cov-2 diagnostics decentralized coronavirus disease 2019 (covid-19) testing algorithms, resulting in increased testing capacity on a national level. however, despite the herculean efforts of physicians, public health officials, and clinical laboratory personnel, significant challenges and case backlogs remain. as we learn more about covid-19 transmission, countries worldwide have tried to curtail disease spread via containment efforts, including quarantines, closure of municipal spaces, and compulsory social distancing. due to limited availability, testing initially focused on the identification of symptomatic individuals. as parts of the world report decreased caseloads, the focus has shifted to include screening of asymptomatic individuals in community settings in order to prevent future outbreaks. similar to routine diagnostics, screening tests need to be both analytically and clinically sensitive and easy to perform. screening tests also carry an inherent requirement to be reasonably inexpensive and able to identify large numbers of affected yet asymptomatic patients. the optimal specimen type, diagnostic technology, sampling format, and screening strategy for sars-cov-2 remain unknown; this uncertainty is compounded by unstable testing supply chains. we read with interest the study appearing in this issue by li et al, who utilized a pooled sample strategy and a point-of-care (poc) reverse transcriptase-polymerase chain reaction (rt-pcr) assay for screening asymptomatic airline passengers arriving from areas of high sars-cov-2 prevalence. the study demonstrates sample pooling as a mechanism for both cost reduction and resource conservation, using a commercial molecular platform with a rapid turnaround time and strong analytical sensitivity. while we view the results of this work as an overwhelmingly positive step forward, it is important to consider the context in which this strategy was deployed and the impact of the location and population on the results. sample pooling is a method of increasing the throughput of diagnostic assays. in this approach, small volumes of samples from multiple patients are combined into a single test, resulting in substantial reagent savings. if the pooled sample returns a negative result, all patients with specimens comprising that pool are considered not to be infected. a positive result requires that the pool be deconvoluted, or split, into its representative parts, and each component sample is tested individually to identify the infected patient(s). sample pooling was evaluated for tracking sars-cov-2 in a community setting early in the us outbreak [1] , and has been used historically as a public health tool for the detection of other viral agents, including human immunodeficiency virus. at the time of this writing, 2 reference laboratories in the united states (quest diagnostics and labcorp) have received emergency use authorizations from the us food and drug administration to use pooled specimens for sars-cov-2 detection [2] . in this work, pooling was performed in a 10:1 ratio, meaning 10 patient specimens were combined and tested using a single sars-cov-2 assay. the number of sars-cov-2 specimens per pool varies in the limited number of investigations reported to date [1, [3] [4] [5] . while the potential financial and reagent savings are obvious, careful and rigorous investigation is necessary to assure the pooling of specimens does not impact the analytical sensitivity of the assay [3, 4] . this is particularly important in a screening test used for asymptomatic individuals, who may not have high levels of detectable viral material present in their clinical specimens. indeed, in this work, rt-pcr cycle threshold (ct) values of nasopharyngeal swabs from the 2 positive, asymptomatic patients were 40. 6 and 41.6 when tested alone, and 43.8 and 43.9 when tested in the context of a 10:1 pool. these values are at the extreme end of the analytical sensitivity of this assay, highlighting the need for careful experimental design. li et al pooled specimens from patients traveling from high-risk areas in mainland china to hainan island as part of a sars-cov-2 disembarkation screening process at sanya airport. a particularly remarkable aspect of this work is that all testing (including positive pool deconvolution) was performed on site at the airport. most investigations of sars-cov-2 sample pooling utilize laboratory-developed assays that must be performed in approved biosafety level 2 settings, require specialized instrumentation, require trained technical staff, and are not portable. by taking advantage of the ease of operation of the cepheid platform, sample analyses were able to be performed in real time, allowing for rapid intervention. this approach may be feasible for an island featuring limited points of entry and a more isolated population. it is, however, challenging to extrapolate this study to an effective screening protocol for use in schools, workplaces, and sporting events in geographical areas with more porous borders. additionally, for sample pooling to be an effective strategy, the test population must have a low prevalence rate. the total number of covid-19 cases in hainan at the time of writing was 170. this number is significantly lower than the daily number of cases reported by some us cities, highlighting the utility of this approach in a low-prevalence setting. locales with higher sars-cov-2 prevalences will require more frequent deconvolution of positive patient pools, thus chipping away at or negating the benefits of a pooling approach. there are several practical points to resolve before a test-based screening policy can be deployed. beyond the obvious financial concerns, a major issue is the testing turnaround time. to date, most of the available "rapid" poc platforms repeatedly demonstrate lower levels of analytical sensitivity. in the united states, this means the molecular testing would need to be performed in a clinical laboratory improvement amendments (clia)certified laboratory, which could again lead to the overburdening of clinical laboratories; a notable exception is the cepheid platform for sars-cov-2, which is clia-waived and has good analytical sensitivity, hence its use in this study. moreover, the simple reality of specimen collection, transportation, testing, and reporting further prolongs turnaround times in settings without access to an on-site laboratory (ie, schools, business, etc.). this might imply that patients need to be tested the day before results are to be reviewed; in other words, get swabbed on sunday to go to work on monday. this leads to a second and possibly more important question: what does a negative or positive result mean? at our institution, we are aware of patients who underwent preprocedure sars-cov2 screening utilizing the same assay deployed in this work, only to be diagnosed with active, symptomatic covid-19 within 5 days of testing. as knowledge about covid-19 increases, it appears that the predictive value of a negative test in an asymptomatic person depends on the timing of sample collection in the context of the patient's disease course [6] . therefore, if an institution utilizes a weekly screening protocol, and screened individuals are still in their incubation period, it is possible they could test negative on monday, become infectious by wednesday, but not show symptoms until friday. if people misinterpret negative test results as justification to relax their nonpharmacologic interventions (mask wearing), especially at traditionally social times like lunch and coffee breaks, the magnitude of disease spread could be significant. conversely, in the absence of robust data concerning viral shedding, infectivity, and correlation to rt-pcr ct values, testing of asymptomatic patients using an assay with high analytical sensitivity begs the question as to whether such patients are truly contagious. of the 2 positive patients detected in this work, both exhibited immunoglobin g seropositivity and had nasopharyngeal samples with elevated ct values; 1 had an exposure history spanning up to 6 months before testing. it is not possible to know whether these patients are truly infectious without additional studies. thus, until we learn more about the biology and transmission kinetics of sars-cov-2, it is difficult to ascertain the clinical significance of such results. it might be reasonable to deploy a test-based screening strategy in a setting with controlled points of entry, a rapid turnaround time, effective means of contacting those who are tested, a low disease prevalence, and high compliance with masking. as these prerequisites are unlikely to be met in the contiguous united states, we either need to reconsider the utility of test-based screening or find faster, cheaper, and more sensitive poc tests. until this happens, pooled testing is an option to reduce costs and speed results. potential conflicts of interest. the authors: no reported conflicts of interest. both authors have sample pooling as a strategy to detect community transmission of sars-cov-2 united states food and drug administration pooling of samples for testing for sars-cov-2 in asymptomatic people assessment of specimen pooling to conserve sars cov-2 testing resources coronavirus (covid-19) update: fda issues first emergency authorization for sample pooling in diagnostic testing • cid 2020:xx (xx xxxx) • editorial commentary variation in false-negative rate of reverse transcriptase polymerase chain reactionbased sars-cov-2 tests by time since exposure editorial commentary • cid 2020:xx (xx xxxx) • 3 submitted the icmje form for disclosure of potential conflicts of interest. key: cord-007049-02p8ug67 authors: mcgeer, allison title: let him who desires peace prepare for war: united states hospitals and severe acute respiratory syndrome preparedness date: 2004-07-15 journal: clin infect dis doi: 10.1086/421784 sha: doc_id: 7049 cord_uid: 02p8ug67 nan on 12 march 2003, the world health organization (who) first posted a worldwide alert concerning an outbreak of severe acute respiratory syndrome in vietnam, hong kong, and guandong province, china [1] . in june 2003, the centers for disease control and prevention (cdc) surveyed members of the infectious disease society of america emerging infections network (ein) about sars preparedness in their hospitals. it is a measure of the rapid globalization of both the outbreak and the outbreak response that, !3 months after the outbreak was recognized, 30% of responding members of the ein reported that their hospital had cared for a patient meeting the case definition of sars and that 90% had plans in place to address sars [2] . the major characteristics of the plans were remarkably uniform, given the very short time for their development and the rapidly changing data. it is a credit to who, cdc, and the outbreak management teams in each country that as much information flowed as rapidly as it did. the variability in the plans illustrates both the uncertainties inherent in the data available at the time of the survey and the significant challenges in sars preparedness. the most important and most difficult component of sars preparedness programs is the identification of infected patients. sars is a febrile respiratory illness that is often clinically indistinguishable from other causes of fever and pulmonary infiltrates [3] [4] [5] . identification of cases depends on prompt recognition of epidemiological risk and clustered infections. of the 456 ein members responding to the survey in this issue of clinical infectious diseases [2] , 381 (83%) reported that patients with respiratory symptoms in their emergency department (ed) would be screened for a travel history. routine screening in the ed is a substantial investment for most hospitals and one that some may judge to be of dubious benefit, given that only 8 laboratory-confirmed cases of sars were diagnosed in the united states [6] . on the other hand, the hospitals of 17% of respondents that have not implemented screening are dependent on their admitting physicians to consider sars in the differential diagnosis and to order appropriate precautions. this latter system has been shown to repeatedly fail to identify tuberculosis, another cause of acute respiratory disease [7, 8] . sars is much more likely than tuberculosis to be transmitted and to result in disease. in the event of another outbreak of sars, systematic screening of ed patients, at least those who are to be admitted to the hospital, should be part of every plan. some aspects of the plans from june 2003 will likely now have changed. for instance, the relatively low percentage of plans (70%) that incorporated follow-up of exposed patients and visitors reflected the focus on health care worker infections early in the outbreak. documentation indicating that exposed visitors and patients who became ill were the major source of transmission should result in the incorporation of prompt patient and visitor follow-up into all plans [9, 10] . similarly, at the height of the outbreak, uncertainties about transmission led many institutions to impose quarantine on returning travelers (and led some travelers to self-impose quarantine). however, transmission of sars was almost invariably linked to households and hospitals and did not occur before the onset of symptoms [10] [11] [12] . thus, quarantine of travelers is not a necessary measure. recognizing the power of denial, however, some hospitals may continue to require daily checks of returning workers until the full incubation period has passed. as noted by srinivasan et al. [2] , the survey also highlights more-general issues in infection control in hospitals. it may still be possible to manage sars safely in the significant minority of hospitals (17%) and eds (29%) that lack airborne isolation rooms. however, it is not possible to manage chickenpox, measles, or tuberculosis without appropriate airborne isolation precautions. because 30% of the responding hospitals admitted a traveler from an area of sars endemicity despite travel restrictions, it seems unlikely that they can avoid caring for all diseases spread by the airborne route. the issue of whether protection from sars requires airborne precautions will continue to generate controversy. a careful assessment of exposures in sars outbreaks, particularly those due to superspreading events and transmission despite compliance with isolation precautions, is needed to determine whether airborne spread occurs [10, [13] [14] [15] . in addition, continued work on the science of health care worker respiratory protection is clearly needed. national standards vary widely in the developed world. in the united states, the standards of the occupational safety and health association recommend annual fit testing for n95 respirators [16] . the results of this survey suggest that compliance with this recommendation is the exception rather than the rule. in canada, the canadian standards association, in the absence of data, has made no recommendation for protection against infectious agents [17] . in some countries of the european union, fit testing is required before use but is not required annually; in others, fit testing of individuals is not currently recommended [18] . the issue of how best to assure protection for each ward nurse in the middle of a weekend night is real. so is the fear that the logistical problems associated with always having a supply of fit-tested masks for all health care workers will push in-vestigators to downplay the risk of airborne spread. at least 2 issues of importance in hospital preparedness for sars were not touched on in the survey reported by srinivasan et al. [2] . disaster preparedness plans allow most facilities to contact all staff rapidly at the beginning of an emergency. outbreaks of disease, however, require plans for regular (sometimes twice daily) information updates for hospital staff, patients, visitors, and related medical service professionals. preparedness for sars also requires clear delineation of the roles and responsibilities of hospitals and government agencies in many areas of outbreak management. for instance, it is essential before outbreaks to determine who will set hospital policy (e.g., restrictions on hospital admissions, requirements for managing infected patients, and decisions as to which hospitals will admit patients with sars), who will establish work restriction policies for exposed health care workers, and who is responsible for follow-up of exposed patients, staff, and visitors. a number of ein members surveyed expressed concerns about health care worker compliance with precautions. at least 2 analyses of risks associated with health care worker infection despite the use of precautions now identify that 12 h of infection-control training and confidence that precautions would be protective are associated with substantial reductions in the risk of infection (toronto sars hospital investigation, unpublished data; lau et al. [19] ). management personnel at all hospitals should now be asking themselves how confident they are that clinical staff clearly understand infectioncontrol precautions and how they can be sure that, if sars should reemerge, all health care workers have the training necessary to comply with precautions. one challenge for hospitals is to maintain the impetus to integrate the rapidly expanding new knowledge about sars into the best prevention programs. a second is to reassess the management of ex-posure to droplet-spread pathogens in hospitals. the cdc is currently recommending that all hospitals consider offering masks to all coughing patients and using droplet precautions (placing patients for whom such precautions are required in a private room, if possible; masking health care workers within 1 m of such patients or upon room entry; and limiting the movement of such patients outside of their room), in addition to standard precautions, for all patients with symptoms of respiratory infections [20] . although, as srinivasan et al. [2] suggest, these isolation precautions may have benefits that extend to situations beyond sars outbreaks, it is also true that isolation may have risks, as recently demonstrated by redelmeier et al. [21] . as life returns to a "new normal" after sars, we urgently need a better understanding of how to minimize the risk of transmission of viral respiratory diseases without creating adverse events associated with additional infection-control precautions. world health organization. cases of severe respiratory illness may spread to hospital staff. released 12 hospital preparedness for severe acute respiratory syndrome in the united states: views from a national survey of infectious diseases consultants discriminative ability of laboratory parameters in severe acute respiratory syndrome (sars) analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis council of state and territorial epidemiologists. sars investigative team, cdc. revised u.s. surveillance case definition for severe acute respiratory syndrome (sars) and update on sars cases-united states and worldwide environmental infection control of tuberculosis nosocomial transmission of multidrug-resistant mycobacterium tuberculosis sars transmission and hospital containment investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto, canada. hospital outbreak investigation team epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of 144 patients with sars in the greater toronto area possible sars coronavirus transmission during cardiopulmonary resuscitation superspreading sars events cluster of sars in medical students exposed to a single patient institute for occupational safety and health. tb respiratory protection program in health care facilities: administrator's guide. september 1999 selection, use and care of respirators. document severe acute respiratory syndrome (sars) sars transmission among hospital workers in hong kong public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) safety of patients isolated for infection control key: cord-269973-sntnmqqd authors: to, kelvin kai-wang; chan, wan-mui; ip, jonathan daniel; chu, allen wing-ho; tam, anthony raymond; liu, raymond; wu, alan ka-lun; lung, kwok-cheung; tsang, owen tak-yin; lau, daphne pui-ling; to, wing-kin; kwan, mike yat-wah; yau, yat-sun; ng, anthony chin-ki; yip, cyril chik-yan; chan, kwok-hung; tse, herman; hung, ivan fan-ngai; yuen, kwok-yung title: unique sars-cov-2 clusters causing a large covid-19 outbreak in hong kong date: 2020-08-05 journal: clin infect dis doi: 10.1093/cid/ciaa1119 sha: doc_id: 269973 cord_uid: sntnmqqd after two months of relative quiescence, a large covid-19 outbreak occurred in hong kong in july 2020 after gradual relaxation of social distancing policy. two unique sars-cov-2 phylogenetic clusters have been identified among locally-acquired cases, with most genomes belonging to cluster hk1 which is phylogenetically related to sars-cov-2 reported overseas. the coronavirus disease 2019 (covid-19) pandemic has overwhelmed the healthcare system in many parts of the world. to combat the pandemic, social distancing measures have been implemented to reduce community transmission. however, resurgence of covid-19 cases has been seen in many parts of the world after the relaxation of these social distancing measures. hong kong was one of the first places in the world to report covid-19 cases [1] . however, the number of covid-19 cases remained relatively low due to the early implementation of stringent public health measures, including border control, voluntary community-wide wearing of face masks, hand hygiene and social distancing, prompt isolation of suspected cases, and testing and quarantine of close contacts and travelers from epidemic areas [2, 3] . the first wave, consisting of imported cases from china and limited local transmission, occurred in january and early march, 2020. the second wave, mainly related to imported cases outside asia and associated local transmission, occurred in mid-march to may, 2020. after stepping up public control measures, only sporadic local cases were reported in may and june. hence, most social distancing restrictions were lifted on june 18, 2020. however, a third wave started to occur since early july 2020. locally-acquired cases re-appeared since july 5, 2020. a total of 617 locally-acquired laboratory-confirmed cases have been reported between july 5 and 21 [4] . in this study, we use whole genome sequencing to investigate the genomic epidemiology of this large summer outbreak. archived nasopharyngeal swab or posterior oropharyngeal saliva from covid-19 patients were retrieved. this study was approved by the the institutional review board of the nanopore sequencing was performed as described previously with modifications [1, 5] . rna was extracted from clinical specimens with qiagen viral rna mini kit, and was amplified with sequence-independent single-primer amplification (sispa) or tiled primers. nanopore sequencing was performed with the oxford nanopore minion platform. bioinformatic analysis was performed as described previously with modifications [1] . sanger sequencing was performed on specimens with low coverage at the cluster-defining mutations. details on library preparation, bioinformatics analysis, and sanger sequencing are included in the supplementary methods section. genomes from hong kong and selected genomes from overseas were included for the phylogenetic tree analysis. nucleotide position was numbered according to the reference genome wuhan-hu-1 (genbank accession number mn908947.3). please refer to supplementary methods section for details. in this study, a total of 116 high-quality whole genomes of sars-cov-2 were table s1 ). ten genomes from the first wave were reported previously [1, 5, 6] . for the third wave, we included imported cases up to 2 weeks prior to the first locally-acquired case. spike protein d614g mutation was not found in any genomes during the first wave, which mainly involved travelers from mainland china or other parts of asia, or the linked local cases. however, d614g mutation was present in 73.8% (31/42) of the genomes in the second wave, which mainly involved travelers returning from europe or north america ( figure 1a ). in the third wave, 32 specimens collected from locally-acquired cases between july 7 and 14, 2020, and 18 specimens collected from imported cases collected between june 22 and july 14, 2020, were included for analysis. the majority of the locally-acquired cases (29/32) table s2 ). phylogenetically, cluster hk1 is most closely related to 4 imported cases from the philippines but at least 2 of the hk1-defining mutations were not found in these cases. another local cluster during the third wave, consisting of 3 members in a household, also belongs to gisaid clade gr, nextstrain clade 20b, and pangolin lineage b.1.1. this cluster is characterized by 3 unique mutations, including a19702g (nsp15 n28d), t22020c (spike protein m153t) and c28269t (located between orf8 and np gene) (referred to as cluster hk2) (supplementary table s2 ). cluster hk2 is most closely related to imported a c c e p t e d m a n u s c r i p t cases from kazakhstan, but those from kazakhstan only had a19702g (nsp15 n28d) and t22020c (spike m153t) but not c28269t mutation. in july, 2020, hong kong has experienced the third wave of covid-19, which represented the largest local covid-19 outbreak since the beginning of the pandemic. in this study, we have identified two unique clusters causing this covid-19 summer outbreak in hong kong. the majority of genomes from locally-acquired cases (91%) during this third wave belong to a cluster hk1, a unique cluster within the gr clade, which is characterized by 4 non-synonymous mutations (nsp3 a85v, nsp15 a231v, spike protein s12f, np a12g) and 1 synonymous mutation (np c29144t). genomes from a local household cluster with 3 members form another unique cluster (cluster hk2), which is characterized by 2 nonsynonymous mutations (nsp15 n28d, spike protein m153t) and c28269t. both clusters, especially cluster hk1, were phylogenetically more closely related to imported cases than the strains collected in hong kong during the previous waves before june 2020, suggesting that this summer covid-19 outbreak is unlikely to be related to silent carriers from previous waves. instead, our results suggest that the current outbreak may be related to imported cases. cluster hk1 is most closely related to genomes of patients traveling from the philippines, while cluster hk2 is most closely related to those from kazakhstan. however, there are important differences between our locally-acquired clusters and these imported cases. all the genomes from cases imported from the philippines did not contain nsp3 a85v or np a12g that define cluster hk1, while those from kazakhstan lack the mutation c28269t that define cluster hk2. hence, our results suggest that there is a missing link between these locally-acquired cases in hong kong and those cases from the philippines or both cluster hk1 and hk2 possess d614g, which is now the predominant clade worldwide. d614g, located on the surface of the spike protein promoter, has become a predominant mutation worldwide [8] . d614g mutation is associated with a higher viral load in patients, and is associated with better viral replication in a pseudovirus assay [8] . several factors may explain this explosive summer outbreak in hong kong. first, the sudden increase in social gatherings especially at eateries after the stepping down of public health control measures facilitated person-to-person transmission. second, this outbreak occurred in the summer, when both the temperature and humidity are high. whether these climate conditions affect virus transmissibility require further investigations. third, the unique mutations in these clusters, especially in cluster hk1, may have increased the survival or transmissibility of the virus. the mutations specific to cluster hk1 occur in different proteins, including two nonstructural proteins (nsp3 and nsp15) and two structural proteins (spike protein and nucleoprotein). nsp3 is a putative pl-pro domain. nsp15 is a xendou: poly(u)-specific endoribonuclease, and has been shown to be a potent interferon antagonist [9] . the surface spike protein is responsible for receptor binding, and the nucleoprotein mainly participates in viral genome transcription, replication and virion assembly. spike protein s12f is located in the signal sequence, while the nucleoprotein a12g is located in the n1a domain, a linker domain [10] . cluster hk2 contains two non-synonymous mutations, nsp15 n28d and spike protein m153t. m153t is at the n-terminal domain. this residue is located within a region that is newly found in the sars-cov-2 strains when compared with sars-cov [11] .whether these mutations affect the function of these proteins remains to be determined. a c c e p t e d m a n u s c r i p t there are several limitations in this study. first, the current third wave is still ongoing, and there may be other clusters that have not been identified. second, since whole genome sequencing required samples with relatively high viral load, those with lower viral load could not yield good quality sequence for phylogenetic analysis. two unique sars-cov-2 clusters have been identified during this large summer outbreak in hong kong shortly after the easing of social distancing policies. further studies are required to determine how environmental, host or viral factors contribute to this outbreak. source control at the borders and airport are important to prevent imported cases. since transmission from asymptomatic or mildly symptomatic patients are common [12, 13] , community-wide wearing of face mask and social distancing measures, especially at eateries, are required for prevent local spread. a c c e p t e d m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t figure 1b temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study the epidemiology of covid-19 cases and the successful containment strategy in hong kong the role of community-wide wearing of face mask for control of coronavirus disease 2019 (covid-19) epidemic due to sars-cov-2 centre for health protection. latest situation of cases of covid-19 (as of 15 july gene as the target of sars-cov-2 real-time rt-pcr using nanopore whole-genome sequencing comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study clade and lineage nomenclature aids in genomic epidemiology studies of active hcov-19 viruses sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus sars-cov-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists architecture and self-assembly of the sars-cov-2 nucleocapsid protein evolutionary relationships and sequence-structure determinants in human sars coronavirus-2 spike proteins for host receptor recognition asymptomatic transmission during the covid-19 pandemic and implications for public health strategies sars-cov-2 shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series we gratefully acknowledge the originating and submitting laboratories who a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-288432-n2y9cunc authors: liu, kun; ai, siqi; song, shuxuan; zhu, guanghu; tian, fei; li, huan; gao, yuan; wu, yinglin; zhang, shiyu; shao, zhongjun; liu, qiyong; lin, hualiang title: population movement, city closure in wuhan and geographical expansion of the 2019-ncov pneumonia infection in china in january 2020 date: 2020-04-17 journal: clin infect dis doi: 10.1093/cid/ciaa422 sha: doc_id: 288432 cord_uid: n2y9cunc background: the unprecedented outbreak of 2019-ncov pneumonia infection in wuhan city caused global concern, the outflowing population from wuhan was believed to be a main reason for the rapid and large-scale spread of the disease, so the government implemented a city closure measure to prevent its transmission considering the large amount of travelling before the chinese new year. methods: based on the daily reported new cases and the population movement data between january 1 and 31, we examined the effects of population outflow from wuhan on the geographical expansion of the infection in other provinces and cities of china, as well as the impacts of the city closure in wuhan in different scenarios of closing dates. results: we observed a significantly positive association between population movement and the number of the 2019-ncov cases. the spatial distribution of cases per unit outflow population indicated that some areas with large outflow population might have been underestimated for the infection, such as henan and hunan provinces. further analysis revealed that if the city closure policy was implemented two days earlier, 1420 (95% ci: 1059, 1833) cases could have been prevented, and if two days later, 1462 (95% ci: 1090, 1886) more cases would be possible. conclusions: our findings suggest that population movement might be one important trigger for the transmission of 2019-ncov infection in china, and the policy of city closure is effective to control the epidemic. in december 2019, an unprecedented pneumonia outbreak caused by a novel coronavirus, namely 2019-ncov, emerged in wuhan, the capital city of hubei province in china [1] . similar with severe acute respiratory syndrome (sars), the outbreak was highly suspected to be linked to the wild animals in the seafood market, although the definitive source was not clear yet [2] . as of january 31, 2020, the infection has been transmitted to all the provinces in china and a few other countries. epidemiology evidence showed that most of the cases outside wuhan had a history of living or travelling to wuhan, and human-to-human transmission route was possible [3] , which might be the reason for a rapid increasing rate of infection across the country and globally [4] . considering the person-to-person transmission and the large travel volume during the traditional chinese new year (the largest annual population movement in the world), it is expected that the population movement would lead to further expansion of the infection, so the government imposed a lockdown on wuhan city at 10:00 am on january 23, as well as some other cities later on [5] . however, an estimated 5 million individuals had already left wuhan for the holiday or travelling, some of which rushed out after the lockdown announcement [6] . in addition, the novel coronavirus is infectious during the incubation period and when the symptoms are not obvious, which is likely to make the huge floating population potential sources of infection [7] . therefore, it is reasonable to hypothesize that the population transported a c c e p t e d m a n u s c r i p t 6 from wuhan may have a significant impact on the potential outbreaks in other parts of china. recent studies on the novel coronavirus pneumonia focused more on its etiology [8, 9] , transmission route [10, 11] , and epidemiological characteristics [12, 13] , there is still a lack of investigating the relationship between the migrating population and the outbreak, which is of great importance for making intervention policies. thus, we conducted this study with the following objectives: 1) to evaluate the impacts of the population movement on the spatial transmission of the 2019-ncov cases at the provincial and city levels in china; 2) to estimate the potential outbreak risk at areas with the population outflowed from wuhan; 3) to evaluate the effectiveness of the city closure measures on the epidemic control. the data on the daily number of 2019-ncov pneumonia cases from january 1 to 31 were derived from the real-time update of the china health commission (http://www.nhc.gov.cn/), 2019-ncov epidemic report on the websites of phoenix and dingxiangyuan. the diagnosis and definition of the case have been described elsewhere [2, 3] . in brief, a confirmed case was defined as a pneumonia case that was laboratory confirmed 2019-ncov infection with related respiratory symptoms and a travel history to wuhan or direct contact with patients from wuhan. a c c e p t e d m a n u s c r i p t 7 as the city closure took place at 10:00 am on january 23, 2020, and the incubation period of the infection was considered to be about 3-7 days [14] , we obtained the daily index of population outflow from wuhan and the proportion of the daily index from wuhan to other provinces and top 50 cities, from january 1 to 31 in 2020, the information was retrieved through the spring festival travel information of china released by baidu qianxi. the data came from baidu location based services (lbs) and baidu tianyan based on location and traffic information systems, which could provide real-time dynamic information on regional population outflow. data of baidu qianxi was freely available to the public (http://qianxi.baidu.com). the daily index of population outflow from wuhan to other provinces and top 50 cities was obtained by multiplying the daily index of population outflow within c is the cumulative 2019-ncov cases in each province from january 1 to 31: d is the total index of population inflow from wuhan to other provinces: we finally calculated the average number of cases per unit outflow population for each province in china: the net loss index of outflow population caused by advanced wuhan city closure for each province: 11 a c c e p t e d m a n u s c r i p t 10 similarly, we evaluated the impacts of one-day and two-day delayed city closure. we took the average index of the population outflow between january 21 and 23 as the daily index of population outflow before the city closure, and used the same calculation method to estimate the index of population outflow within wuhan increased by the delayed city closure on january 24 and january 25 (the delayed outflow index). we multiplied the delayed outflow index by the average proportion and one corresponding unit to estimate the increased number of cases caused by one-day and two-day delayed city closure of wuhan for each province in china. (table 1) . on the contrary, if the closure measures was delayed for one to two days, the number of cases would increase by 722 and 1462, respectively. our study provided timely evidence for the formulation of efficient strategies to prevent diseases from spreading out. on the one hand, the result could help assess the effectiveness of the prevention and control efforts. for example, the cases in zhejiang and guangdong are apparently more than estimated, which indicated a better health emergency response system (i.e. higher detection efficiency) or inadequate isolation, whereas the cases reported in henan were much lower than expected. two possible explanations should be considered: (1) strong prevention and control measures had been adopted in henan; (2) the epidemic in henan has been underestimated and enhanced screening efforts should be enforced. on the other hand, exploring the association was expected to help identify high-risk areas and guide health strategy a c c e p t e d m a n u s c r i p t 15 formulation [19, 20] . take henan as an example, great difference between estimated and reported data may imply a great increase of cases in the future, which required enhancement of the surveillance system and rational allocation of resources [17] . the medicine supply, personal protective equipment, hospital supplies, and the human resources necessary to respond to an outbreak should be always ensured [21] . in addition, this study could be used to guide the assessment of the risk of disease transmission and help raise public awareness. as a large number of infected people had transported to all of 31 provinces, epidemics across the country may be inevitable. to halt the spread of the epidemic, harsh measures including quarantine and isolation of exposed persons, cancellation of mass gatherings, school closures, and travel restriction were needed to reduce transmission in affected areas. furthermore, screening of people who have been to wuhan recently was of crucial importance, especially cities with close ties to wuhan. considering the impact of population movements on the outbreak, the wuhan government announced the suspension of public transportation on january 23, 2020, with a closure of airports, railway stations, and highways, to prevent further disease transmission [22] . despite inconsistent reports on the role of the lockdown in halting the disease transmission across china [11, 20, 21] , the unprecedented measure might play an important role in slowing the epidemic spread, especially when an effective vaccine was developed [23, 24] . in addition, to explore the impact of date selection, we estimated the changes of cases when the measure was implemented on different a c c e p t e d m a n u s c r i p t 16 date. the results varied significantly, 1420 cases could be prevented with the measure implemented two days earlier, and the number of cases will increase by 1462 with the lockdown implemented two days later, suggesting that the effect of the lockdown depending on the choice of date greatly, which could provide a reference for the future outbreaks. since the political and economic effects were not considered, further studies on secondary impacts of the measure, like socioeconomic impacts, were also warranted. though we estimated that some cases would possibly be prevented if the policy was implemented earlier, it was actually hard to make such a huge decision given the whole picture of the infection was not clear at that stage. the authors believe that the current policy was appropriate at this complex situation. there were a few limitations of our study. firstly, we used the index of population outflow to reflect the general real-time magnitude of population movements, so it was not an accurate representation of the actual population flow data. secondly, some possible influencing factors, such as socio-economic factors and demographic characteristics, were not included in the analysis because of data inaccessibility. thirdly, it is assumed that the infected travelers in the population were randomly distributed [25] and that there was no significant difference in the surveillance capability between cities [17] , which would result in some difference between the estimated value and the actual situation. in addition, daily data used in this study was reported infection data, rather than the actual number of incident cases. in summary, our study indicates that the population outflow from wuhan might m a n u s c r i p t we appreciated the support by national key r&d program of china (grant no: 2018yfa0606200). we thank our colleagues for their careful reading and editing of this manuscript. the funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the authors declare they have no 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estimation of epidemiological parameters and epidemic predictions the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan, china new coronavirus pneumonia diagnosis and treatment program pneumonia of unknown etiology in wuhan, china: potential for international spread via commercial air travel modeling impact of temperature and human movement on the persistence of dengue disease assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia population movement patterns among the democratic republic of the congo, rwanda, and uganda during an outbreak of ebola virus disease: results from community engagement in two districts -uganda forecast and control of epidemics in a globalized world nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study commission cnh. announcement from the headquarter for novel coronavirus pneumonia prevention and control delaying the international spread of pandemic influenza empirical evidence for the effect of airline travel on inter-regional influenza spread in the united states predicting the international spread of middle east respiratory syndrome (mers) a c c e p t e d m a n u s c r i p t 20 a c c e p t e d m a n u s c r i p t 22 key: cord-007325-g8ke9rfg authors: koskiniemi, marjaleena title: cns manifestations associated with mycoplasma pneumoniae infections: summary of cases at the university of helsinki and review date: 1993-08-17 journal: clin infect dis doi: 10.1093/clinids/17.supplement_1.s52 sha: doc_id: 7325 cord_uid: g8ke9rfg cns manifestations appear in one of 1,000 patients with mycoplasma pneumoniae-associated infections. encephalitis is the most frequent manifestation, but cases of meningitis, myelitis, and polyradiculitis, as well as many other symptoms (e.g., coma, ataxia, psychosis, and stroke), have been reported. the onset of these manifestations is usually acute, with lowered consciousness, convulsions, pareses, and other neurological signs. severe, even fatal, cases are known. the pathophysiology of cns manifestations is unknown. to our knowledge, m. pneumoniae has never been isolated from brain tissue, but instead it has been recovered from csf specimens in at least seven cases. besides direct invasion of m. pneumoniae into the brain, neurotoxic or autoimmune reaction within the brain tissue is suspected. at neuropathological examination, edema, demyelination, and microthrombi have been described. improved diagnostic methods may reveal the pathophysiology of cns manifestations associated with m. pneumoniae infection. antibody to m. pneumoniae was observed in 10 further patients in whom encephalitis apparently was associated with another microbe (coxsackie virus b in five and various microbes in five). the routine schema for examining the patients included blood and urine analysis, csf examination, electroencephalogram (eeg) recording, and electroneuromyography, if needed. neuroradiological examinations, including computed tomography (ct) of the brain since the late 1970s and magnetic resonance imaging (mri) of the brain since the early 1980s, were performed if needed. screening for antibody was done by the cf test with 15-17 different antigens. these antigens included adenovirus, coronavirus (until 1982), coxsackie virus b5, cytomegalovirus, hepatitis b virus (since 1970), herpes simplex virus, influenza a and b viruses, parainfluenza 1 and 3 viruses, poliovirus, respiratory syncytial virus, rotavirus (since 1976), varicella zoster virus, chlamydia group antigen, m. pneumoniae, and toxoplasma gondii. screening for arbovirus was dropped in the early 1980s because no cases were found. acute encephalitis. all pediatric patients (45) had encephalitis. of the adult patients, 10 (63%) had encephalitis (figure 2). most patients (78%) had meningeal symptoms and signs: headache, nausea or vomiting, and neck stiffness (table 1) . many patients had high temperatures, though some patients' temperatures were normal. one-half of the patients had convulsions on admission or during their stay in the hospital. more than two-thirds of the patients were unconscious or somnolent on admission. in addition, focal neurological signs appeared, e.g., ataxia, deviation of the eye, hemiparesis, or paresthesia. more than one-third (38%) of the patients had an associated pneumonia or other respiratory disease. in some patients, carditis or reye's syndrome was present. the symptoms were usually acute; in one-half of the cases, they lasted day (figure 3). a large proportion (30%) of the patients needed intensive care, usually for a long period (mean period, 12 days). the hospital stay ranged from 1 day to 3 months (mean stay, 2-3 weeks). other diseases. besides encephalitis, adult patients had meningitis, myelitis, and polyradiculitis, two cases of each (figure 2). cases of meningitis were mild, resembling the clinical course of a usual case of serous meningitis. one patient with myelitis became tetraplegic; plasma exchange was done, and after 2 months she was able to walk. the other patient with myelitis had transverse involvement at the t-8 level; this patient recovered in 1 month with minor sequelae. both patients with polyradiculitis became tetraplegic; one was treated with plasma exchange, and the other was not. both of these patients recovered in 6 weeks. findings of electroneuromyography were consistent with polyradiculitis, while the protein concentration in the csf was only slightly increased. in our series, 60% of cases had normal findings for csf (white blood cell count, 0-230 x 10 6/l; protein concentration, 180-1,640 mg/l) at the onset of the manifestation. most samples were taken on the first 3 days, and if results of csf analysis were normal, specimens were not obtained later. thus these findings are relevant to the beginning but not to the later course of the disease. enterovirus was cultured concomitantly from feces from two patients, and herpes simplex virus antigen was detected in throat specimens from three patients. results of eeg recording were usually severely (51%) or moderately (27%) disturbed during the first week of neurological illness, and the disturbance was often seen for weeks after the onset. findings of ct/mri were unremarkable in our cases, though these tests were performed for one-third of the patients. evidence of edema, but not of focal lesions, was seen on some patients' scans. five patients (8%) died. fourteen patients (23%) had severe sequelae: mental retardation, choreoathetosis, convulsions, an.lmovement disorders. the duration of neurological symptoms before hospital admission was not associated with the outcome. long-lasting unconsciousness was associated with poor outcome, though even after 8 days one patient recovered from unconsciousness. if an abnormality revealed by eeg recording was present for >4 weeks, sequelae were more common than on average [17] . m. pneumoniae-associated cns manifestations are variable (table 2) . encephalitis seems to be the main type in children [17, 18] ; in adults the clinical picture is more diverse [19] [20] [21] . encephalitis may be diffuse or focal [22] , often expansive [23, 24] . it may resemble encephalitis lethargica [25] . this manifestation may concentrate in the cerebellum or pons regions [26, 27] and may cause hydrocephalus [28] . sometimes encephalitis may be recurrent [29] . meningitis is usually a mild manifestation [11, 30] . myelitis may appear in different forms: diffuse, transverse [1 9 11, 31, 32], or poliolike [33] . in addition, there are descrip[38] [39] [40] . neuropathy in cranial or peripheral nerves and myositis have been described as well [41] . the primary diagnosis of m. pneumoniae-associated disease is clinical. the first serology available was the test for cold agglutinins in 1941 [42] . this test, however, is positive in only one-or two-thirds of cases. titers of cf antibodies may decline on admission to the hospital because often the patient has had the disease for some time. throat cultures are positive in 65% of cases [43] . antibody titers in the serum as well as those in the csf may be nonspecific [44] apparently because of differences between antigens; lipid-containing antigen may be cross-reactive with brain antigens, making the results uncertain [45] [46] [47] . a ct scan may reveal hypodensity in parietal areas [48] m. pneumoniae has been isolated from csf specimens in at least seven cases [51] [52] [53] [54] [55] , but isolation will never be the [11] . isolation of the organism from the csf suggests that m. pneumoniae invades the cns. however, to our knowledge, m. pneumoniae has never been isolated from brain tissue. thus the theory of direct invasion of m. pneumoniae into brain tissue is somewhat loose. the rna hybridization test (gen-probe, san diego) has been diagnostic for throat specimens, tracheal aspirates, and lung tissue [56] , but whether it is for csf or brain tissue is unknown. as a new approach, the polymerase chain reaction is a promising method [57] [58] [59] [60] , although no reports on determinations of csf have been published yet. both erythromycin and tetracycline are effective against m. pneumoniae in vitro and in vivo [61] . the results, however, are variable, and antibiotic therapy may be beneficial [50] or have no effect [5, 11, 22] . in cns diseases associated with m. pneumoniae, the problem of therapy is unresolved. corticosteroids, anti-inflammatory drugs, and antidiuretics have been used as well as antibiotics [22, 24, 62] . plasma exchange has been reported in at least one case, and it appeared to be beneficial [63] . in our series, plasma exchange was done for one patient with myelitis and one with polyradiculitis, and both patients recovered; however, the other two patients with myelitis and polyradiculitis who did not undergo plasma exchange recovered as well. the pathophysiology behind the cns symptomatology in m. pneumoniae-associated diseases remains hypothetical [64, 65] . m. pneumoniae itself may invade the cns (in the csf at least) because the organism has been isolated from csf [52] [53] [54] [55] . still, involvement in brain tissue remains uncertain. some neurotoxin may be released or there may be an autoimmune complex or vasculopathy (table 3) at neuropathological examination, the brain may be edematous throughout or on basal parts, and scattered areas of hemorrhages may be seen [49] . there may also be perivascu-lar infiltration, microthrombi, and areas of demyelination [24, 49] . it is evident that cns manifestations associated with m. pneumoniae infections are real, but exact diagnostic methods are rarely available. diagnostic difficulties include crossreactivity of the antigen [46, 47, 68] . newer technology may help us to make specific diagnoses. m. pneumoniae may be a concomitant or predisposing factor [69] . the pathophysiology of m. pneumoniae-associated cns manifestations is largely unknown. in spite of the diagnostic problems associated with titers of cf antibody, we argue for the existence of cns manifestations in association with m. pneumoniae infections. in our large series, m. pneumoniae accounted for 13% of known or suggested etiologies in childhood encephalitides [70] and 7% in adult encephalitides [71] . our present series of 61 patients with diagnostic or high titers of antibody to m. pneumoniae showed clinical diseases similar to those previously reported. the age spectrum of patients who have neurological diseases associated with m. pneumoniae infection seems to be a little higher than that of patients with respiratory diseases caused by m. pneumoniae [4, 12, 72 ]. the death rate and sequelae we observed resemble those observed by other investigators [3] . these results, many signs of persistent syndromes [25, 27] , persistent brain stem dysfunction [50] , and prolonged immunologic response [62] all emphasize the need for further study of m. pneumoniae-associated cns manifestations. central nervous system complications of primary atypical pneumonia mycoplasma pneumoniae infection in the united kingdom: 1967-1973 mycoplasmas as agents of human disease respiratory infections due to mycoplasma pneumoniae in infants and children central nervous system disease associated with mycoplasma pneumoniae infection: report of five cases and review of the literature central nervous system manifestations associated with serologically verified mycoplasma pneumoniae infection studies on mycoplasma pneumoniae infection in sweden aseptic meningitis and meningoencephalitis in cold agglutinin 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infection complicated by intravascular coagulation cerebral infarction associated with mycoplasma pneumoniae neurological complications of primary atypical pneumonia mycoplasma pneumoniae as a determinant of the guillain-barre syndrome mycoplasma antibody in guillain-barre syndrome and other neurological disorders cranial neuropathy, myeloradiculopathy, and myositis: complications of mycoplasma pneumoniae infection cold agglutinins accompanying mycoplasma pneumoniae infection mycoplasma pneumoniae infections in children serological diagnosis of mycoplasma pneumoniae infection by enzyme immunoassay antibodies to brain and other tissues in cases of mycoplasma pneumoniae infection immunoglobulin m antibody response against mycoplasma pneumoniae lipid antigen in patients with acute pancreatitis increase in titers of antibodies to mycoplasma pneumoniae in patients with purulent meningitis postinfectious meningoencephalitis complicating mycoplasma pneumoniae in a child postinfectious leukoencephalitis complicating mycoplasma pneumoniae infection neurological complications of mycoplasma pneumoniae infection nachweis von mycoplasma pneumoniae im liquor bei akuter polyneuritis neurologic disease associated with mycoplasma pneumoniae pneumonitis: demonstration of viable mycoplasma pneumoniae in cerebrospinal fluid and blood by radioisotopic and immunofluorescent tissue culture techniques isolation and characterization of mycoplasma pneumoniae from cerebrospinal fluid of a patient with pneumonia and meningoencephalitis direct invasion of the central nervous system by mycoplasma pneumoniae.• a report of two cases isolation of mycoplasma pneumoniae from pleural fluid and/or cerebrospinal fluid: report of four cases dna probes for detection and identification of mycoplasma pneumoniae and mycoplasma genitalium detection of mycoplasma pneumoniae and cns s57 mycoplasma pneumoniae by using the polymerase chain reaction detection of mycoplasma pneumoniae in simulated clinical samples by polymerase chain reaction evaluation of dna probe test for rapid diagnosis of mycoplasma pneumoniae infections comparison of gen-probe commercial kit and culture technique for the diagnosis of mycoplasma pneumoniae infection the effect of antibiotics on mycoplasma pneumoniae in vitro and in vivo mycoplasma pneumoniae disease: clinical spectrum, pathophysiology, epidemiology, and control neurological deficit associated with mycoplasma pneumoniae reversed by plasma exchange neurological syndromes and mycoplasmal infections mycoplasma-tissue cell interactions disseminated vasculomyelinopathy: an immune complex disease the possible role of pleuropneumonia-like organisms in neurological disease questionable specificity of lipid antigen in the mycoplasma pneumoniae complement fixation test in patients with extrapulmonary manifestations possible association of mycoplasma and viral respiratory infections with bacterial meningitis effect of measles, mumps, rubella vaccination on pattern of encephalitis in children acute encephalitis of viral origin age-related prevalence of complement-fixing antibody to mycoplasma pneumoniae during an 8-year period key: cord-001381-b0tlco4t authors: howie, stephen r. c.; morris, gerard a. j.; tokarz, rafal; ebruke, bernard e.; machuka, eunice m.; ideh, readon c.; chimah, osaretin; secka, ousman; townend, john; dione, michel; oluwalana, claire; njie, malick; jallow, mariatou; hill, philip c.; antonio, martin; greenwood, brian; briese, thomas; mulholland, kim; corrah, tumani; lipkin, w. ian; adegbola, richard a. title: etiology of severe childhood pneumonia in the gambia, west africa, determined by conventional and molecular microbiological analyses of lung and pleural aspirate samples date: 2014-09-01 journal: clin infect dis doi: 10.1093/cid/ciu384 sha: doc_id: 1381 cord_uid: b0tlco4t molecular analyses of lung aspirates from gambian children with severe pneumonia detected pathogens more frequently than did culture and showed a predominance of bacteria, principally streptococcus pneumoniae, >75% being of serotypes covered by current pneumococcal conjugate vaccines. multiple pathogens were detected frequently, notably haemophilus influenzae (mostly nontypeable) together with s. pneumoniae. pneumonia remains the leading cause of death in children worldwide. a better understanding of the range of pneumonia pathogens is needed to reduce child mortality further [1] , but this is hampered by limitations at the bedside and in the laboratory. pneumonia etiologic studies have depended largely on blood culture, which is insensitive [2] . direct percutaneous aspiration from the site of lung infection, rarely done despite its good safety record, yields the highest-quality clinical specimen, but even then bacterial culture identifies a pathogen in no more than half of cases [3] . molecular methods have advantages over conventional methods for the detection and characterization of pathogens in clinical samples, and hold promise for improving diagnostic sensitivity [4] . we describe the application of both approaches to the detection of pathogens in lung and pleural aspirates obtained from children with severe pneumonia in a west african setting. the objective of the study was to elucidate the etiology of severe pneumonia in this group more comprehensively than has been possible previously. the gambia is a west african country of 1.8 million people with a human immunodeficiency virus (hiv) infection prevalence of <2%. a study of the etiology of severe childhood pneumonia was undertaken in the coastal area of the gambia (supplementary appendix 1). at the time of the study (2007-2009), there was high coverage with conjugate haemophilus influenzae type b (hib) vaccine but no routine usage of pneumococcal conjugate vaccine (pcv), which is the case currently in many african countries. cases were children aged 2-59 months with severe pneumonia defined clinically by modified world health organization (who) criteria (cough or difficulty in breathing, plus any of the following: lower chest wall indrawing, nasal flaring, or an oxygen saturation of <90% on pulse oximetry). participants were recruited from the medical research council (mrc) hospital in fajara; the royal victoria teaching hospital in banjul; and the major health centers at fajikunda, serekunda, and brikama. children with a cough of ≥2 weeks, or severe anemia (hemoglobin level <6 g/dl) or confirmed wheeze were excluded. radiological pneumonia was defined using who criteria (ie, endpoint consolidation or pleural effusion). hiv testing was done if informed consent was given after standard counseling. a standard who guideline-based antibiotic regimen was used. written informed consent was obtained for participation in the study from parents or guardians. the study was approved by the gambian government/mrc joint ethics committee (scc/ec1062). details of lung aspiration in our setting over a period of 25 years, during which there have been no associated serious adverse events, have been described previously [5] . participants underwent lung aspiration if they had accessible consolidation adjacent to the chest wall, had no contraindications, and written informed consent had been obtained. pleural aspiration was undertaken in those with pleural effusions. culture, nonmolecular serotyping, hiv testing, singleplex polymerase chain reaction (pcr) for lyta and cpsa (for streptococcus pneumoniae) and glpq (for h. influenzae), 16s rrna pcr, multilocus sequence typing (mlst), molecular serotyping, and multiplex fast-track 33 pcr, all using standard methods (supplementary appendix 2) [2, [6] [7] [8] [9] [10] [11] [12] [13] , were performed at the mrc unit in the gambia, and multiplex masstag pcr [14] was performed at columbia university. multiplex pathogen targets are listed in supplementary appendix 3. laboratory analyses were done blinded and independent of each other. summary results of organisms identified to genus or species level using 1 or more detection methods were compiled. demographic and clinical data were double-entered into an sql database (microsoft corporation) and verified, and laboratory data were entered into an access (microsoft corporation) database, cross-checked, and verified. statistical analyses were done using stata software, version 11 (statacorp). fifty-five children, representing 74% of the radiological severe pneumonias and 26% of all clinical severe pneumonia cases identified in the study period, underwent lung aspiration (n = 47) or pleural fluid aspiration (n = 9); 1 participant underwent 2 aspirates at different time points (supplementary appendix 4). hiv testing was done in 33 of 55 (60%) participants, 2 of whom were positive for hiv type 1. the characteristics of those who underwent lung or pleural aspiration were similar to the radiological pneumonia group from which they were drawn (supplementary appendix 5). pathogens were identified to genus or species level in 53 of 56 (95%) samples by 1 or more laboratory methods (table 1 ). an organism was cultured from 21 of 56 (38%) specimens: s. pneumoniae in 14 (25%), h. influenzae (all non-type b on latex agglutination) in 3 (5%), and staphylococcus aureus in 3 (5%). ziehl-neelsen staining was done in 37 of 56 (66%) lung aspirate samples (all negative); 35 of 37 (95%) underwent culture for mycobacterium tuberculosis, and all were negative. streptococcus pneumoniae was detected by 1 or more molecular assays (supplementary appendix 6) in 48 of 53 (91%) samples and in 2 or more assays in 36 of 53 (68%) samples. molecular pneumococcal serotyping (40 serotypes; supplementary appendix 7) showed the following prevalences: serotype 1, 22%; serotype 4, 18%; serotype 14, 18%; serotype 5, 16%; serotype 6a/b/c, 4%; and serotype 9v/a, 2%. ten-and 13-valent pcvs both include serotypes that account for 76%-80% of serotypes identified in the samples in this study. haemophilus influenzae was detected in 12 of 53 (23%) samples. four of the 12 had sufficient deoxyribonucleic acid (dna) for full mlst typing, 3 being nontypeable h. influenzae (nthi), whereas 1 in a child with hiv infection had a serotype b infection. two of the 12 had sufficient loads for 5 or 6 of the standard 7 alleles of mlst to be defined, and these were suggestive of nthi, 1 being in a culture-positive case confirmed as being non-type b by latex agglutination assay. staphylococcus aureus was detected in 3 of 53 (6%) samples, all of which were pleural fluid specimens. one or more other bacteria were identified to genus or species level in 8 of 53 (15%) samples. viruses, led by respiratory syncytial virus, adenovirus, and bocavirus, were detected by 1 or more methods in 10 of 53 (19%) samples. half (28/53 [53%]) of samples had >1 organism detected to genus or species level, predominantly 2 or more bacterial species. codetection of s. pneumoniae and h. influenzae occurred in 11 of 53 (21%) samples, h. influenzae having the higher bacterial load in 4 of 11 samples. in the 3 specimens positive for s. aureus, low loads of s. pneumoniae were detected in 2 and of h. influenzae in the other. this study showed that s. pneumoniae was the predominant pathogen in this group of children with radiologic pneumonias, followed by h. influenzae, s. aureus, a range of gram-negative bacteria, and a few viruses. potentially causative pathogens were found in all but 1 sample (98%), in contrast to the use of culture alone, which yielded an organism in 38% of specimens. streptococcus pneumoniae and h. influenzae, predominantly nthi, were detected together in around 1 in 5 cases in this hib-vaccinated population. current pcvs include >75% of serotypes identified in these samples. gambian studies of pneumonia etiology in the pre-hib vaccine era showed a predominance of s. pneumoniae followed by hib, as have other low-income country studies, including a recent lung aspirate study from malawi using pcr [15, 16] . the role of nthi in pneumonia has been raised before [17] and has been controversial [15] . it is likely that this study's findings are relevant to similar patient groups in other developing-country settings, especially those where conjugate hib vaccine is routinely used and where pcvs have not yet been introduced. the multicountry pneumonia etiology research for child health (perch) study will provide important data in this respect [4] . the prominence of s. pneumoniae but low frequency of hib in our study appears to contrast with recent global burden of disease 2010 estimates [18] . the strength of this study is its detailed analysis of the best possible specimens for diagnosing the cause of pneumonia using sensitive methods from a well-defined patient group that is representative of radiologic pneumonias. multiple assays have provided several lines of supporting evidence, and 2 laboratories analyzed the raw clinical specimens, strengthening the findings. the predominance of s. pneumoniae along with its serotype distribution is supported by a high level of concordance between independent laboratories [19] . the study's weaknesses are that the best possible specimens are still subject to sampling error, that the study is relatively small, and that the identification of potential pathogens does not in itself confirm that these are the causative agents, a fundamental challenge for the field. nevertheless, if there are any clinical samples for which pathogen detection alone is sufficient to assign causation, then these are lung and pleural aspirates. the findings of this study emphasize the importance of bacteria, prominently s. pneumoniae, as a cause of severe pneumonia, and the potential for current pcvs to reduce the burden of this disease. this study also confirms that molecular methods are able to detect potential pathogens far more readily than culture and have a role in defining the etiology of pneumonia. the challenge of accurately assigning causation to the pathogens detected remains, particularly when using more generally available specimen types, and this will require the additional development of robust biomarkers of pathogen-specific disease. notes contributed to study oversight and interpretation of data. m. a., o. s., e. m. m., and m. d. contributed to laboratory design, conduct, or interpretation. j. t. contributed to data analysis and interpretation. w. i. l. and t. b. oversaw and contributed to analyses at columbia university and contributed to interpretation. k. m., b. g., p. c. h., and t. c. contributed to design and interpretation. m. j. and m. n. contributed to the design and conduct of the study. all authors contributed to critical review of the manuscript. disclaimer. the bill & melinda gates foundation had no role in drafting the manuscript or the decision to publish. financial support. this work was supported by the mrc and the bill & melinda gates foundation. potential conflicts of interest. r. a. a. is an employee of glaxosmith-kline vaccines in belgium and received previous grant awards for studies of bacterial diseases while working as an employee of the mrc unit, the gambia. all other authors report no potential conflicts. breathing new life into pneumonia diagnostics the etiology of pneumonia in malnourished and well-nourished gambian children the value and complications of percutaneous thoracic lung aspiration for etiologic diagnosis of community-acquired pneumonia the pneumonia etiology research for child health project: a 21st century childhood pneumonia etiology study use of percutaneous transthoracic lung aspiration for the etiologic diagnosis of pneumonia: a 25 year experience from the gambia serotyping streptococcus pneumoniae by multiplex pcr a detailed analysis of 16s ribosomal rna gene segments for the diagnosis of pathogenic bacteria a multilocus sequence typing scheme for streptococcus pneumoniae: identification of clones associated with serious invasive disease characterization of encapsulated and noncapsulated haemophilus influenzae and determination of phylogenetic relationships by multilocus sequence typing a novel campylobacter jejuni sequence type from a culture-negative patient in the gambia determination of bacterial load by real-time pcr using a broad-range (universal) probe and primers set measuring nasal bacterial load and its association with otitis media longitudinal molecular microbial analysis of influenzalike illness diagnostic system for rapid and sensitive differential detection of pathogens epidemiology and etiology of childhood pneumonia pcr improves diagnostic yield from lung aspiration in malawian children with radiologically confirmed pneumonia aetiology of pneumonia in children in goroka hospital global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study serotype analysis of streptococcus pneumoniae in lung and nasopharyngeal aspirates from children in the gambia by masstag pcr acknowledgments. the authors thank the participants and their parents/guardians; the clinical, field, laboratory, data, and administrative teams at the medical research council (mrc) unit in the gambia; the staff of the centre for infection and immunity, columbia university; simon donkor for his contribution to managing data; nuru adams and essa jarra, who assisted with the molecular work in the gambia; ramu sarge-njie and her team, who performed the hiv testing; christian bottomley and yin bun cheung, who assisted with statistics advice; and pamela collier njai, charles onyeama, danlami garba, uduak okomo, and augustin fombah, who contributed to the clinical aspects of the study.author contributions. s. r. c. h. proposed and led the study; designed epidemiological and clinical aspects; contributed to interpretation; completed the first draft of the article; and finalized the article. g. a. j. m. led gambian-based molecular analysis design and conduct; contributed to interpretation; and contributed to the first draft of the article. r. t. undertook laboratory analyses at columbia university and contributed to interpretation. b. e. e., r. c. i., o. c., and c. o. collected lung and pleural aspirates and clinical data from participants. r. a. a. contributed to the conception of the study; led conventional microbiological aspects; and all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-260630-vvpzp73r authors: mandell, lionel a. title: etiologies of acute respiratory tract infections date: 2005-08-15 journal: clin infect dis doi: 10.1086/432019 sha: doc_id: 260630 cord_uid: vvpzp73r nan in this issue of clinical infectious diseases, there are 2 articles that provide us with some insight into the various etiologic agents that can cause acute respiratory tract infection (arti) in general practice patients in the netherlands [1] and into the significance of the human metapneumovirus (hmpv) in patients with community-acquired pneumonia (cap) and exacerbations of chronic obstructive pulmonary disease (copd) in quebec, canada [2] . if we examine the broad spectrum of infectious and noninfectious illnesses experienced by people of all ages worldwide, it is clear that artis are, without question, the most common maladies encountered, regardless of age or sex [3] . the objectives of the dutch study were to estimate the incidence of influenza-like illnesses (ilis) and of other artis in patients visiting their general practitioners (to determine the etiologic agents) and to test the hypothesis that asymptomatic persons with subclinical infection may act as sources of transmission [1] . the objective of the canadian study was to examine the role of hmpv infection in adults with cap and adults with exacerbations of copd [2] . a case-control method was used in the dutch trial, and ili was defined as an "acute onset of illness" (duration of prodromal stage, у3-4 days) and the presence of at least 1 of the following symptoms: cough, rhinitis, sore throat, frontal headache, retrosternal pain, or myalgia. arti was defined as an acute respiratory illness other than ili with at least 1 of the following symptoms: cough, rhinitis, or sore throat. nose and throat swab specimens were obtained from case patients and control subjects, and viral cultures and pcr tests were performed for detection of adenovirus, coronavirus, enterovirus, hmpv, influenza virus, parainfluenza virus, rhinovirus, and respiratory syncytial virus (rsv), as well as for mycoplasma pneumoniae, chlamydophila pneumoniae, and chlamydophila psittaci. bacterial cultures of throat swab specimens were also performed to detect all bacterial pathogens that are known to potentially cause "community-acquired" respiratory infection. the findings were then semiquantitatively classified into 5 groups: no colonies, sporadic colonies, few colonies, several colonies, or many colonies. the spectre of respiratory illnesses and of epidemic respiratory illnesses in particular, such as can be seen with influenza, was heightened recently by the epidemic of severe acute respiratory syndrome (sars) during 2002-2003 and widespread publicity about concerns regarding a possible epidemic or even pandemic attrib-utable to avian influenza [4, 5] . the more we learn and understand about the epidemiology and etiology of artis, the better position we will be in to prevent and treat them. essentially, the article by van gageldonk-lafeber et al. [1] shows us a number of things. the overall incidences of consultations for ilis and other artis are 132 and 413 consultations per 10,000 personyears, respectively. the diagnoses made for case patients with artis other than ilis were primarily the common cold (36% of patients), acute pharyngitis (30%), and acute tonsillitis (20%). the main symptom for case patients with ili was fever (90% of patients), and for those with other artis, it was sore throat (76%). for the control subjects, the most common symptoms were joint-muscle complaints (21% of patients) and skin disorders (14%). approximately 20% of control subjects consulted their family physicians for other reasons, such as to pick up prescriptions, to undergo a routine physical examination, and to accompany relatives. not surprisingly, no pathogens were detected in 35% of case patients; in contrast, pathogens were found in 31% of control subjects without airway complaints. in the case patients, the pathogens were viruses (58% of patients), group a b-hemolytic streptococci (11%), and mixed pathogens (virus plus group a b-hemolytic streptococci; 3%). case patients were defined as persons with ili or an arti other than ili who had not used antimicrobials in the previous 2 weeks, and the findings reported above are certainly in keeping with the general supposition that artis are primarily caused by viruses. a breakdown of the most common pathogens in case patients and control subjects revealed that influenza a viruses were the most common pathogens in case patients with ili (42% of patients), and rhinovirus was the most common pathogen in case patients with arti (25%). in control subjects, rsv (17% of subjects) was the most common pathogen. the main conclusions of van gageldonk-lafeber et al. [1] are that most artis are viral, rhinovirus was the most common pathogen in both case patients and control subjects, and patients without symptoms suggestive of artis may harbor pathogens and could be a potential source of transmission of respiratory pathogens. the majority of respiratory tract infections in general and of viral infections in particular are caused by rhinoviruses and coronaviruses [3] . in the category of respiratory infections, influenza was reported more frequently than the common cold, despite the fact that colds occur more frequently [3] . this is further supported by data on viruses associated with the common cold in which influenza virus was reported to occur in 25%-30% of cases [6, 7] . in another community-based study, the tecumseh study [8] , the viruses that caused all respiratory infections were reported. here too, as in the dutch study, rhinoviruses were the most frequently found pathogens. the pathogens capable of causing viral respiratory disease vary in their ability to both initiate and transmit infection. if one examines the relative role or impact of different respiratory viruses in producing artis, the top 3 etiologic agents (expressed as estimated percentages of all respiratory illnesses) are as follows: rhinovirus, 34%; coronaviruses, 14%; and influenza viruses, 9% [3] . the viral pathogens also vary in their ability to transmit infection. coronavi-ruses are generally transmitted by large droplet spread, whereas influenza viruses are spread by airborne methods, including both aerosol and droplet spread [9] . the exact mode of transmission of rhinoviruses, however, is not clear, and it is still debated whether rhinovirus is transmitted primarily by direct contact (e.g., droplet nuclei) or by indirect contact. for example, it has been shown that these viruses can survive on surfaces and can be spread by inoculation of nose or eyes with one's fingers, and others have shown that spread by droplet transmission can take place as well [10] . one of the most recent outbreaks that drew worldwide attention to the potential of a pandemic was the outbreak of the sars. this began in the guandong province of china in november 2002 and subsequently spread to hong kong and, ultimately, to far reaches of the world. the etiologic pathogen was determined to be a new coronavirus (sars-cov) that presumably spread from animals that are sometimes eaten as part of exotic banquets by humans. as happens with other coronaviruses, the sars-cov probably first mutated and then spread to new and susceptible species (e.g., humans). it is likely that spread among humans happened primarily by close contact that probably included droplet spread, direct contact, or fomite transmission. there were also several instances that may have involved small particle airborne or fecal/oral transmission. usually, 1 case would only result in spread of the virus to a few other individuals, but there were several well-documented cases of "super transmitters," in which 1 person could infect у10 other persons [11, 12] . one of the interesting findings of the dutch study was the fact that the researchers found pathogens in 30% of control subjects. this was higher for the youngest age groups and might act as a potential source of infection of others. they drew attention to the fact that only 2 other studies have addressed this issue, one that involved children and adults and the other that examined only adults [13, 14] . the former showed that results of virologic assessments of asymptomatic subjects were positive for 12% of children and 4% of adults for rhinovirus and enterovirus, respectively [13] . the latter study showed that only 4% of asymptomatic subjects aged у60 years tested positive for rhinovirus or coronavirus [14] . these figures are significantly lower than those reported by the dutch study [1] . the frequencies of subclinical infection and of asymptomatic subjects, by age group, were as follows: children (age, 0-15 years), 68%; adults, 55%; and persons aged у65 years, 51%. a number of possible explanations are offered, but the important point still remains that asymptomatic persons, whether young or old, may act as unsuspected sources of infection. the article by hamelin et al. [2] is an attempt to assess the role that hmpv plays in adults with cap and exacerbations of copd. hmpv is a newly discovered viral respiratory pathogen for which, thus far, there are limited epidemiologic data to describe it. it was first isolated in the netherlands from nasopharyngeal secretions of 28 children collected over a 20-year period. the virus itself is a single-stranded negative-sense rna virus, and sequence studies of isolates have identified 2 main lineages. it belongs to the order mononegavirales, family paramyxoviridae, and genus metapneumovirus [15, 16] . as mentioned in the study by van gageldonk-lafeber et al. [1] , a significant percentage of patients with artis never have an etiologic pathogen identified. this is true not only for ilis and other artis but also in serious cases of cap that require admission to the hospital and in nosocomial and ventilator-associated pneumonia that is managed in medical or surgical wards and the intensive care units in tertiary care university hospitals, despite the availability of the best equipment and access to excellent diagnostic laboratories. the article by hamelin et al. [2] assesses 2 patient populations: patients with exacerbations of copd aged у40 years (with or without cap) who sought help at one of the emergency departments at study hospitals, and patients aged у18 years without copd who had cap that required admission to the hospital. three hospitals were involved for the periods of 17 january 2002 through 6 may 2002 and 6 january 2003 through 6 may 2003. statistical analysis involving regression modeling showed an association between artis and excess winter deaths, particularly for influenza and rsv, but it was also implied that other agents may have been involved as well [17] . the large number of negative results of culture, pcr, and serologic testing to identify a causative agent certainly bears this out as well. identification of the different pathogens that contribute to the various respiratory illnesses is very important not only from an epidemiological point of view but also from a treatment and prevention point of view, because such information may also help in the development of specific treatments aimed at these pathogens, as well as the development of preventive measures through the use of chemoprophylaxis or immunoprophylaxis (i.e., vaccines). hamelin et al. [2] studied nasopharyngeal aspirate specimens for the presence of influenza viruses a and b, rsv, and hmpv by pcr, and they studied paired blood samples (obtained at a 3-4-week interval) for antibody to hmpv (by elisa) and antibody to rsv and influenza (both by complement fixation), using serologic testing. all of the patients with cap and 81% of the patients with exacerbations of copd were admitted to the hospital. among the 145 patients from the study period, the incidence of hmpv infection was 4.1%, the incidence of influenza a was 6.2%, the incidence of influenza b was 0%, and the incidence of rsv infection was 9%, as determined by pcr and/or serologic testing. of the 6 patients in whom hmpv was identified, 4 had cap (2 of whom also had copd), and 2 had acute exacerbations of copd (but not cap). other features of note are that all 6 patients pre-sented to the emergency department with cough and sore throat, 5 had a mean elevated oral temperature of 38.3њc, 5 of 6 were dyspneic, 4 of 6 had nasal congestion and/or rhinorrhea, 3 of 6 had purulent sputum, 3 of 6 had wheezing, and 2 of 6 had myalgias. one of the largest studies of patients with hmpv infection is that by williams et al. [18] published in the new england journal of medicine. nasal wash specimens were obtained over a 25-year period from otherwise healthy children who presented to the pediatric division of infectious diseases at vanderbilt university medical center (nashville, tn) with acute respiratory tract illness. the samples were studied for the presence of hmpv, which was found in 49 of 248 specimens. it is difficult to compare the vanderbilt cohort and the quebec cohort, because the former consisted exclusively of otherwise healthy infants and children, whereas the latter included adults only, many of whom had comorbid illnesses. also, the sample sizes were significantly different: the quebec study had a total of only 145 subjects, 6 (4.2%) of whom were hmpv positive. in the vanderbilt study, the authors concluded that 12% of all lower respiratory tract infections were most likely caused by hmpv, whereas the figure is only 4.1% in the quebec study. the symptoms of diarrhea and vomiting were seen in 17% and 10% of the children in the vanderbilt study, respectively, whereas these symptoms were not even mentioned in the canadian study, and although fever occurred in 5 (83.3%) of the 6 subjects in the quebec study, it was only found in 52% of children in the vanderbilt study. hamelin et al. [2] claimed that hmpv is associated with a significant number of cases of cap and exacerbations of copd in adults during the early spring. i would take issue with this claim, unless we interpret the statement literally (i.e., if we consider the etiology to be specifically in the early spring [all 6 cases occurred in april]). otherwise, it is hard to imagine that 4% is a particularly significant figure. the study, however, is an important one, and it is definitely a step in the right direction. to date, we have few data on hmpv, and anything that sheds light on its role as a pathogen is welcome. what is very important is the fact that the quebec data show that, in the elderly population-particularly for persons with underlying lung disease-hmpv infection can lead to severe infection that requires hospitalization (mean duration, 10 days). hamelin and colleagues' finding that ∼85% of the adult population in their study had preexisting hmpv antibody is consistent with the premise that hmpv infection is almost universal by the age of 5 years. a case-control study of acute respiratory tract infection in general practice patients in the netherlands human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease epidemiology of viral respiratory infections coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome virologic studies of acute respiratory disease in young adults. iv. virus isolations during four years of surveillance rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes the tecumseh study of illness. xiv. occurrence of respiratory viruses, 1976-1981 rhinovirus transmission: one if by air, two if by hand aerosol transmission of rhinovirus colds severe acute respiratory syndrome-singapore transmission dynamics and control of severe acute respiratory syndrome use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection human metapneumovirus infections in young and elderly adults a newly discovered human pneumovirus isolated from young children with respiratory tract disease impact of influenza and respiratory syncytial virus on mortality in england and wales for january 1975 to december 1990 human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children potential conflicts of interest. l.a.m.: no conflicts. key: cord-279550-7u2hksxm authors: wang, kai; long, quan-xin; deng, hai-jun; hu, jie; gao, qing-zhu; zhang, gui-ji; he, chang-long; huang, lu-yi; hu, jie-li; chen, juan; tang, ni; huang, ai-long title: longitudinal dynamics of the neutralizing antibody response to sars-cov-2 infection date: 2020-08-03 journal: clin infect dis doi: 10.1093/cid/ciaa1143 sha: doc_id: 279550 cord_uid: 7u2hksxm background: coronavirus disease 2019 (covid-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. little is known about the longitudinal dynamics of sars-cov-2-specific neutralizing antibodies (nabs) in covid-19 patients. methods: blood samples (n=173) were collected from 30 covid-19 patients over a 3-month period after symptom onset and analyzed for sars-cov-2-specific nabs, using the lentiviral pseudotype assay, coincident with the levels of igg and proinflammatory cytokines. results: sars-cov-2-specific nab titers were low for the first 7–10 d after symptom onset and increased after 2–3 weeks. the median peak time for nabs was 33 d (iqr 24–59 d) after symptom onset. nab titers in 93·3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34·8% (iqr 19·6–42·4%). nab titers increased over time in parallel with the rise in igg antibody levels, correlating well at week 3 (r = 0·41, p & 0·05). the nab titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including scf, trail, and m-csf. conclusions: these data provide useful information regarding dynamic changes in nabs in covid-19 patients during the acute and convalescent phases. coronavirus disease 2019 (covid-2019) is a novel respiratory disease that is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). since the outbreak of sars-cov-2 last year, it has spread rapidly and caused a global pandemic. 1 as of july 28, 2020, over 16 million people worldwide have been reportedly infected and more than 650,800 individuals have died of covid-19. 2 currently, considerable progress is being made to understand sars-cov-2 pathogenesis, epidemiology, antiviral drug development, and vaccine design. however, no licensed specific antiviral drugs or prophylactic vaccines are available. developing effective viral inhibitors and antibody-based therapeutics to prevent or treat covid-19 infection is a high global priority. the sars-cov-2 rna genome encodes 29 structural and non-structural proteins, including spike (s), envelope (e), membrane (m), nucleocapsid (n) proteins, and the orf1a/b polyprotein. 3 the s glycoprotein is responsible for sars-cov-2 attachment and entry into target host cells via its binding to the angiotensin-converting enzyme 2 (ace-2) receptor. 4 virus-specific neutralizing antibodies (nabs) play a key role in reducing viral replication and increasing viral clearance. 5, 6 nabs mainly act against the receptor-binding domain (rbd) of the sars-cov-2 s protein, [7] [8] [9] effectively blocking viral entry. thus, serological testing, especially to detect nabs, is essential in determining the onset of the serological immune response, evaluating the potential capacity of the host body for viral clearance, and identifying donors for passive antibody therapy trials. in covid-19 patients, nabs can be detected within 2 weeks of symptom onset. 10, 11 the serological antibody response continues for at least 3 weeks and, in some cases, substantially longer. 12, 13 however, the dynamics and roles of sars-cov-2-specific nabs and their correlation with antibody responses have not been explored in covid-19 patients more than two months after symptom onset. a c c e p t e d m a n u s c r i p t 6 previous studies of sars-cov and mers-cov demonstrated that the most immunogenic antigens are the s-and n-proteins, and development of serological tests (such as enzymelinked immunosorbent assay and magnetic chemiluminescence enzyme immunoassay) for sars-cov-2 igg or igm antibodies have focused on these viral proteins. however, it is still unknown whether serological antibodies predict neutralizing activities or protection against viral re-infection. 14 a c c e p t e d m a n u s c r i p t 7 a total of 30 covid-19 patients who had recovered and were discharged from the yongchuan hospital of chongqing medical university were included in our cohort. a confirmed case of covid-19 was defined as an individual with nasopharyngeal swabs that were positive for laboratory-based pcr testing. covid-19 patients who meet following criteria can be discharged: with two consecutive negative rt-pcr results on respiratory tract samples, body temperature is back to normal for more than 3 days, respiratory symptoms improve obviously, pulmonary imaging shows obvious absorption of inflammation, can be discharged. on april 2nd and may 8th, 2020 (follow-up point 1 and follow-up point 2, respectively) two follow-up visits were conducted. sequential serum samples were collected from patients in the acute phase (3 or 4 samples per patient) and the convalescent phase the sars-cov and sars-cov-2 pseudoviruses were generated as previously described, with some modifications. 18 briefly, hek293t cells (5 × 10 6 ) were co-transfected with pnl4relative luminescence units of luc activity were determined using the luciferase assay kit (promega). the titers of nabs were calculated as 50% inhibitory dose (id 50 ), expressed as the highest dilution of plasma which resulted in a 50% reduction of luciferase luminescence compared with virus control, using a cut-off titer 1:20. all serum samples were inactivated at 56 °c for 30 min and stored at −20 °c before testing. continuous variables were expressed as median (inter-quartile range, iqr) and categorical variables were expressed as number (percentage, %). comparisons between two groups were performed using the mann-whitney u test or fisher's exact test. a two-sided α of <0·05 was considered statistically significant. statistical analyses were performed using r software, v3.6.0. two-tailed pearson correlation test was used to calculate the correlation coefficient of nab to igg levels or cytokines. of the total 30 patients in the cohort, 60·0% (18/30) were female, and 10·0% (3/30) were categorized as severe based on the covid-19 treatment guidelines (national health commission of the people's republic of china) ( table 1) , who meeting any of the following criteria: 1) respiratory distress (≥30 times/minutes), 2) the oxygen saturation≤93% at rest, 3) the arterial partial pressure of oxygen (pao 2 ) / the fraction of inspired oxygen (fio 2 ) ≤ 300 mmhg. the median length of the hospital stay was 22 d (iqr 15-26). patients during hospitalization ( figure 2b ). the peak nab levels varied among the patients; 6·7%, 73·3%, and 20% patients showed low (id 50 < 500), medium-low (id 50 500-999), and medium-high (id 50 1000-2500) nab titers, respectively ( figure 2c ). there was no statistical difference among peak nab titers that occurred during hospitalization and convalescence ( figure 2d ). the duration and maintenance of peak of nab levels in covid-19 patients is of great concern. thus, we compared nab levels between the peak time point and the final follow-up time point. a decline in nab levels was observed in 93·3% (28/30) of sars-cov-2 infected patients, with a median decrease of 34·8% (iqr 19.6-42.4%) ( figure 3a ). patients were also grouped according to their rate of decrease in nab levels; more than 20% of the patients showed a >70% decrease in nab levels during this time period (21/30) ( figure 3b) . the kinetic levels of nabs and virus-specific igg over time in covid-19 patients are still unknown. to address this, we first determined the relationship between the nab levels and virus-specific igg levels in individual patients ( figure 4a and supplementary figure 1) ; similar dynamic changes were observed for the nabs and virus-specific igg levels in some patients. furthermore, to determine if there was a statistical correlation between nab levels and virus-specific igg levels in covid-19 patients, serum samples were grouped by time (weeks) after symptom onset. a statistically significant positive correlation was only observed in samples obtained 3 weeks after symptom onset (p = 0·027, r = 0·410) ( figure 4b ). we analyzed the correlation between cytokine and chemokine levels and nab levels in covid-19 patients during the acute phase. interestingly, we observed that nab levels were positively correlated with stem cell factor (scf) (r = 0·616, p = 0·001), tnf-related apoptosis-inducing ligand (trail) (r = 0·514, p = 0·008), and macrophage colonystimulating factor (m-csf) (r = 0·454, p = 0·017) levels ( figure 5 ). virus-specific nabs have been considered an important determinant for viral clearance. the pseudovirus-based assay is suitable for the high-throughput screening of sars-cov-2 nabs in plasma donors without the requirement of bsl-3 laboratories. the assay has been widely used for evaluating nabs in highly pathogenic viruses, such as ebola, sars-cov, mers-cov, and highly pathogenic influenza viruses. 20 herein, we described the dynamics of sars-cov-2-specific nabs generated during both the acute and convalescent phases of a c c e p t e d m a n u s c r i p t 13 sars-cov-2 infection using a pseudovirus-based neutralization assay. we found that sars-cov-2-specific nab titers were low before day 7-10, peaked at approximately day 33 after symptom onset, and then gradually declined over a 3-month period. meanwhile, sars-cov-2-specific nabs were detected concurrently with and positively correlated with igg antibodies in our cohort, indicating that the nab response may play an important role in viral clearance. our understanding of the duration and nature of protective immunity to sars-cov-2 is currently very limited. the kinetics of antibody-mediated immunity to sars-cov-2 infection and how long this immunity lasts are unknown. our data suggest that nab titers in patients were variable, and the protective humoral immune response to sars-cov-2 may abate over time, which is in accordance with findings in patients infected with other human coronaviruses, such as hcov-229e. 21 recently, in a rhesus macaques model, sars-cov-2 infection evoked a robust protective immune response when the animals were re-exposed to sars-cov-2 one month after the initial viral infection. 25 however, natural infection and volunteer challenge studies hint that coronavirus infections, including those with hcov-229e and hcov-oe43, cannot induce stable protective immunity; thus, reinfection occurs frequently. moreover, a sars-cov a c c e p t e d m a n u s c r i p t 14 antigen-specific memory b cell response was not detectable in recovered sars patients at 6-years after disease onset, whereas sars-cov-specific memory t cells persisted in recovered sars patients. 26, 27 although the role of memory t cells in the protective immune response to sars-cov-2 needs further evaluation, a robust t cell response is required for viral clearance. we also described here, the dynamic correlation between sars-cov-2-specific nabs and serological total igg levels. nab titers appeared concomitantly and correlated moderately with igg levels at week 3 after symptom onset, which is consistent with other reports regarding covid-19 recovered patients 13,28 . the antigen epitope used for igg detection in our study contained the nucleoprotein peptide, as well as the rbd domain of the spike protein, which partially explains the discrepancy in nab titers and igg levels at weeks 4, 9, and 14 after symptom onset. the nucleoprotein is one of the major antigens of the sars-cov-2. 29 the binding antibodies detected by the total igg assay may also be involved in viral clearance through antibody-dependent cytotoxicity, therefore, the roles of binding antibodies and nabs in disease progression need further evaluation. currently, adaptive immunotherapy using convalescent plasma (cp) from recovered covid-19 patients is being employed as a potential therapeutic approach to confer antiviral protection. 30 several preliminary clinical trials have proven its effectiveness in treating sars-cov-2. 5, 6 the efficacy of cp transfusion is attributed to the neutralizing effect of antibodies; thus, the nab titer is the major determinant for cp therapy. monitoring nab levels and their duration will provide valuable data for evaluating the effectiveness of cp therapy. in our study, the levels of nabs declined gradually over the 3-month follow-up period, with a median decrease of 34.8%. thus, cp samples with high titers of nabs from patients in the early stage of convalescence will be more suitable for clinical use. there are some limitations to this study, which should be addressed. due to the small sample size, we could not find any correlation between the dynamics of nab titers and clinical characteristics contributing to different clinical outcomes. serological blood samples were collected up to 3 months after symptom onset; data collected over longer follow-up times should be obtained to demonstrate the duration of humoral immunity after sars-cov-2 infection. the lack of data to determine an anamnestic immune response, such as tests for sars-cov-2-specific memory b cells, memory t cells, and specific cytokine-dependent memory cells, hampered the evaluation of the immune response, especially protective immunity against viral reinfection. these are major issues that should be investigated in future studies. in summary, we determined the dynamics of nab titers within 3 months after symptom onset in 30 sars-cov-2-infected patients and found a positive correlation between nab titers and igg antibodies. our work provides valuable insight into the humoral immunity against sars-cov-2 infection. we also described a pseudotype system for measuring nab titers, which could be expanded to antiviral drug screening and vaccine development. a pneumonia outbreak associated with a new coronavirus of probable bat origin coronavirus disease (covid-19) situation reports covid-19: epidemiology, evolution, and cross-disciplinary perspectives the potential danger of suboptimal antibody responses in covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells a human neutralizing antibody targets the receptor-binding site of sars-cov-2 antibody responses to sars-cov-2 in patients with covid-19 neutralizing antibody response in mild covid-19 convalescent plasma therapy for the treatment of patients with covid-19: assessment of methods available for antibody detection and their correlation with neutralising antibody levels sars-cov-2 seroprevalence and neutralizing activity in donor and patient blood from the san francisco bay area serological analysis of new york city covid19 convalescent plasma donors kinetics of viral load and antibody response in relation to covid-19 severity an mrna vaccine against sars-cov-2 -preliminary report vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov comparison of lentiviral vector titration methods pseudotyping viral vectors with emerging virus envelope proteins the time course of the immune response to experimental coronavirus infection of man disappearance of antibodies to sars-associated coronavirus after recovery a serological survey on neutralizing antibody titer of sars convalescent sera challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience sars-cov-2 infection protects against rechallenge in rhesus macaques lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications the nucleocapsid protein of sars-cov-2: a target for vaccine development convalescent plasma as a potential therapy for covid-19 we would like to thank prof. cheguo cai (wuhan university, wuhan, china) for providing the pnl4-3.luc.r-e-plasmid. the authors declare no competing interests. a c c e p t e d m a n u s c r i p t 17 a c c e p t e d m a n u s c r i p t 18 key: cord-007296-q9rn75qb authors: muether, philipp s.; gwaltney, jack m. title: variant effect of firstand second-generation antihistamines as clues to their mechanism of action on the sneeze reflex in the common cold date: 2001-11-01 journal: clin infect dis doi: 10.1086/322518 sha: doc_id: 7296 cord_uid: q9rn75qb treatment with first-generation antihistamines reduces sneezing, rhinorrhea, nasal mucus weight, and, in some instances, cough in subjects with experimental or natural colds; however, treatment with second-generation antihistamines has not been effective for these complaints in trials in subjects with natural colds. this article reports the negative results of a clinical trial with loratadine, a second-generation antihistamine, in adults in the rhinovirus challenge model. this finding in the highly controlled setting of the challenge model confirms the earlier negative studies with second-generation antihistamines in natural colds. first-generation antihistamines block both histaminic and muscarinic receptors as well as passing the blood-brain barrier. second-generation antihistamines mainly block histaminic receptors and do not pass the blood-brain barrier. the effectiveness of first-generation antihistamines in blocking sneezing in colds may be due primarily to neuropharmacological manipulation of histaminic and muscarinic receptors in the medulla. mediators did not [2] . also, treatment with first-generation antihistamines is highly effective in reducing sneezing in subjects with experimental and natural colds [3] [4] [5] . it is, therefore, surprising that, unlike with allergic rhinitis, histamine levels are not elevated in nasal secretions of patients with colds [6] [7] [8] [9] , although nasal mucosal sensitivity to histamine has been reported to be increased [10] [11] [12] [13] . both first-and second-generation antihistamines are competitive antagonists to histamine at the h1-receptor site [14] . an additional pharmacological activity of first-generation, but not second-generation antihistamines is the competitive antagonism of acetylcholine at neuronal and neuromuscular muscarinic receptors. first-generation antihistamines also pass the bloodbrain barrier and thus have a potential for activity in the brain; second-generation antihistamines do not. in limited testing, and despite their h1-blocking activity, second-generation antihistamines have been ineffective in suppressing sneezing in patients with natural colds [15] [16] [17] [18] . these findings raise an interesting question about the mechanism of action of first-generation antihistamines in reducing sneezing in patients with colds and about the ineffectiveness of second-generation antihistamines in this setting. also, natural cold studies have certain technical problems, such as difficulty in enrolling patients in the early stages of a cold, when treatment effects are most accurately measured [19] . therefore, it would be desirable to confirm the results of the natural cold studies by testing a second-generation antihistamine in the rhinovirus challenge model, which provides more precision in the measurement of sneezing. this article reports a clinical trial using a second-generation antihistamine, loratadine, in adults with experimental rhinovirus colds and reviews the possible sites of action of first-generation and second-generation antihistamines. although the study was originally designed to determine whether loratadine by down-regulating expression of intercellular adhesion molecule-1 (icam-1) on nasal epithelial cells reduces rhinovirus infection rates, it provides heretoforemissing information on the results of testing a second-generation antihistamine in the virus challenge mode. a total of 66 adult volunteers from the charlottesville, virginia, area with neutralizing antibody titers of р2 to rhinovirus type 16 were enrolled in the study. subjects were required to have been free of cold symptoms and fever (137.8њc) for 1 week prior to entering the trial and to have no history of hypersensitivity to antihistamines. in addition, subjects were excluded if they had (or had a history of) allergic rhinitis, bronchial asthma, or other lower respiratory tract diseases such as chronic obstructive lung disease or emphysema. subjects with a history of alcohol and drug abuse were excluded, as were volunteers who had used investigational drugs within 30 days, antihistamines and/or cold preparations within 14 days, monoamine oxidase inhibitors within 7 days, astemizole within 90 days, or any other medication thought to interfere with the study drug. other exclusion criteria included pregnancy or lactation, glaucoma, and renal, hepatic, endocrine, digestive, genitourinary, neurologic, or psychologic disease. the protocol was reviewed by the human investigation committee of the university of virginia. loratadine was administered in 10-mg tablets. the placebo tablets were identical to the loratadine tablets but contained pharmacologically inert ingredients. virus challenge. intranasal challenge with rhinovirus type 16 was performed by coarse drops by use of 0.5 ml (0.25 ml per nostril) an inoculum pool containing 100 tissue culture infection dose 50/ml (tcid 50 /ml) of virus. the challenge was performed twice with a 20-min interval between challenges. the inoculum pool was safety tested for extraneous agents [20] . measures of infection. nasal washings were collected 8 days prior to and immediately before the inoculation of the challenge virus to determine whether subjects were infected with a wild-type virus. after virus challenge, nasal washings were collected each morning before administration of medication. washings were cultured for rhinovirus for 5 days after challenge in human embryonic lung cells (wi-38). isolates were identified as rhinovirus type 16 by neutralization with typespecific antibody. venous blood was obtained 7 days prior to treatment and 14-21 days after intranasal inoculation for measurement of homotypic neutralizing antibody [21] . measures of illness. the presence and severity of symptoms were determined daily beginning on the first day of treatment, 7 days prior to viral challenge, for a total of 13 days. data on symptoms were collected immediately before administration of loratadine or placebo by a nurse who recorded the subject's assessment of the severity of symptoms over the prior 24 h on a 5-point scale (0, "none"; 1, "mild"; 2, "moderate"; 3, "severe"; 4, "very severe") [22] . the symptoms assessed were sneezing, runny nose, nasal obstruction, sore throat, cough, headache, malaise, and chilliness. the total symptom score was determined by adding severity scores for the symptoms over the 5-day period after viral challenge. the score for each symptom present immediately before challenge was subtracted from each of the daily scores for that symptom. evaluation of illness severity also included daily measurements of nasal secretion weights [23] . each subject kept a daily log of the number of coughs and sneezes. nasal secretion measurements and cough and sneeze counts were started after the subjects were cloistered in a hotel after challenge had occurred. before leaving the hotel on the last day, subjects were asked whether, in their opinion, they experienced a cold. information on the occurrence and severity of any adverse effects was collected daily, graded as "none," "mild," "moderate," or "severe." experimental design. the trial was a 13-day, single-center, randomized double-blind, placebo-controlled, parallel group study in healthy volunteers 18-40 years of age, of either sex. loratadine or placebo was administered each morning between 6 a.m. and 8 a.m., on days 1-13 of the study, with viral challenge given on day 8. the subjects were randomly assigned to receive either treatment or placebo and were blinded as to their treatment status, as were the observers recording clinical information. data analysis. for comparing proportions, fisher's exact test was used. the t test was used for comparing ordinal and interval data. the results of probability testing were 2-tailed. of the 66 subjects enrolled, 34 received loratadine and 32 received placebo. four subjects in the loratadine group and 1 in the placebo group were infected with a wild-strain rhinovirus at the time of entry into the study. they were excluded from evaluation, as was 1 subject on placebo who discontinued medication after developing a migraine headache on day 10, leaving 60 evaluable subjects for analysis. occurrence of illness. twenty-three (79%) of 29 infected subjects on loratadine and 14 (58%) of 24 infected subjects on placebo met the modified jackson criteria for illness ( ). p p .2 symptom scores. mean (‫ע‬se) sneezing severity scores were similar for the first 3 days and tended to be lower in the placebo group on the fourth day (figure 1). mean (‫ע‬se) total symptom scores were similar in the 2 groups (figure 2b). rhinorrhea scores tended to be lower in the placebo group on days 2 and 3, but the reverse was seen on days 4 and 5 ( figure 2d ). mean (‫ע‬se) nasal obstruction, sore throat, and cough scores were similar in the 2 groups (figures 2c, 2f, 2g). mean (‫ע‬se) headache and malaise scores tended to be lower in the loratadine group for the latter days of illness (figures 2h, 2i). nasal mucus weights. there was a consistent trend for nasal secretion weights to be lower in the placebo group (figure 2c). total mean ‫ע‬ se nasal mucus weights for 5 days were g for the loratadine group and g for the 27. adverse events. one subject in the placebo group had a migraine headache and vomiting and another had vomiting. otherwise, no adverse events were reported. in regard to the original purpose of the study, no differences were observed between the groups receiving loratadine and groups receiving placebo for viral shedding rates, viral titers, overall infection rates, illness rates, or symptom scores. icam-1 levels in nasal secretions in the 2 groups were also similar. the results also showed no therapeutic effect of loratadine on sneezing. this supports earlier work in patients with natural colds in whom second-generation antihistamines were ineffective in reducing sneezing [15] [16] [17] [18] . why first-generation antihistamines are effective in reducing sneezing in colds [3, 5] and second-generation antihistamines are not is of interest. firstgeneration antihistamines, beside their ability to block h1-receptors, also block muscarinic receptors and pass the bloodbrain barrier [14] . second-generation antihistamines are specific h1-receptor blockers without other recognized pharmacological properties and do not pass the blood-brain barrier. information on the neurologic pathways of the sneeze reflex comes mainly from work in animals [24, 25] . the sneeze reflex travels along peripheral nerves and through the medulla oblongata. the neuropharmacology of the sneeze involves h1, muscarinic, and nicotinic receptors. with colds, the sneeze reflex begins in the nose with the infection of nasal cells by a cold virus ( figure 4 ). there is no evidence that activation of basophils and mast cells with release of histamine occurs in colds; however, there is presumed stimulation of free nerve endings of the ethmoidal branch of the trigeminal nerve by inflammatory mediators known to be present, such as bradykinin [1] . the nerve impulse then travels along the afferent nerve fibers to the sensory trigeminal nucleus in the medulla oblongata and arrives at the adjacent sneeze center [26] [27] [28] . via another synapse, the impulse then arrives at the parasympathetic superior salivary nucleus of the facial nerve. here, it crosses a synapse and travels via the preganglionic fibers of the greater petrosal nerve to the sphenopalatine ganglion. supporting this route of transmission in sneezing is the finding that injection of alcohol into the sphenopalatine ganglion blocks the sneeze reflex [29] . the impulse then travels across another synapse (mainly nicotinic, some muscarinic) and proceeds via postganglionic fibers to the synapses (muscarinic) of mucus glands and blood vessels, which are then stimulated. the resultant glandular secretion and vascular transudation restimulate the free nerve endings of the trigeminal nerve by which pathway the impulse is redirected to the trigeminal nucleus and, subsequently, to the sneeze center in the medulla. when the restimulation of the sneeze center is sufficient, the impulse is then directed by intramedullary fibers to the synapses of multiple respiratory centers in the reticular formation, and from there to the synapses of respiratory neurons of the vagus, phrenic, and intercostal nerves. then, via nicotinic synapses, the nerve impulse stimulates the muscular contractions responsible for a sneeze. the nose is the first site at which histamine may be involved in the pathogenesis of sneezing and where an antihistamine might inhibit the sneeze reflex. h1-receptors are present on the free nerve endings of the trigeminal nerve [30] . against the possibility that the nasal mucosa is the primary site of action of first-generation antihistamines is the finding that histamine levels have not been found to be elevated in nasal secretions during colds [6] [7] [8] [9] . also, in earlier natural cold studies and in the current study, second-generation antihistamines, despite reaching the nasal mucosa and despite having h1-blocking activity, have been ineffective in sneeze reduction [15] [16] [17] [18] ; however, because the nasal mucosa appears to have enhanced sensitivity to histamine during colds [10] [11] [12] [13] , the role of the nasal sites cannot be entirely excluded. the next possible site for a first-generation antihistamine to block sneezing is in the medulla oblongata, where synaptic junctions are present at several locations. both h1 and muscarinic receptors have been identified in certain areas of the brain [14, 31] . such receptors would be potential targets for the action of first-generation, but not second-generation antihistamines; however, the synaptic mediators that are involved in the sneeze reflex in the medulla have not been characterized. the observed failure of the second-generation antihistamines to reduce sneezing in colds supports the possibility of these sites being important. muscarinic activity is exclusively responsible for parasympathetic stimulation of the glandular secretion and vascular dilatation with transudation that occurs next. the anticholinergic activity of first-generation antihistamines would be expected to operate at these sites. this is supported by the welldocumented effect of first-generation antihistamines in reducing the volume of nasal fluid production during colds [3, 4] . the second afferent impulse to the medulla is again initiated by stimulation of the free nerve endings of the trigeminal nerve. vascular transudation leads to release of kininogen with resultant generation of kinin [14] . this event provides a means for direct stimulation of free nerve endings by kinins as well as by histamine released from mast cells by kinin stimulation. this provides another possible target for the action of an antihistamine. also, when the nerve impulse is redirected to the medulla, h1 and muscarinic synaptic sites may be blocked by first-generation antihistamines as described above. from that point on, when the motor neurons become involved, nerve transmission depends on nicotinergic receptors and thus would not be susceptible to the action of an antihistamine. this analysis suggests that an important site for the therapeutic effect of first-generation antihistamines on sneezing is in the medulla oblongata, where both h1 and muscarinic receptors may be involved. h1 receptors are known to be present in high concentrations in the hypothalamus, where histamine acts as a neurotransmitter to help regulate the level of wakefulness [14] . this accounts for the drowsiness associated with the use of first-generation antihistamines. because of the dense concentration of h1 receptors in the hypothalamus, and because parasympathetic nerve fibers arise from and are activated by the hypothalamus, it is probable that histamine plays a role in signal transmission in this region. first-generation antihistamines are also known to be helpful in reducing the nausea associated with motion sickness [31] . scopolamine, an anticholinergic drug, which passes the bloodbrain barrier, is an effective treatment for motion sickness, whereas atropine, which does not pass the blood-brain barrier as readily, is not effective. the antinausea effect of scopolamine depends partially on blocking of muscarinic receptors of the vestibular nuclei and the area postrema of the brain. other nuclei in the brainstem may use the muscarinic receptor system as well, which supports the idea of muscarinic receptors in the cns being part of the sneeze reflex. at present, information is not complete on the medullary synapses and neurotransmitters involved in the sneeze reflex, and further work is needed in this area; however, available information suggests that to be effective, a treatment for the sneezing of colds requires compounds that pass the blood-brain barrier and possess both h1 and muscarinic-blocking activity. clinical virology. new york: churchill livingstone physiologic responses to intranasal dose-response challenges with histamine, methacholine, bradykinin, and prostaglandin in adult volunteers with and without nasal allergy efficacy of brompheniramine maleate treatment for rhinovirus colds randomized controlled trial of clemastine fumarate for treatment of experimental rhinovirus colds effectiveness of clemastine fumarate for treatment of rhinorrhea and sneezing associated with the common cold histamine in nasal secretions is histamine responsible for the symptoms of rhinovirus colds: a look at the inflammatory mediators following infection analysis of nasal secretions during experimental rhinovirus upper respiratory infections histamine in nasal secretions and serum may be elevated during viral respiratory tract infection experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects mircovascular exudative hyperresponsiveness in human coronavirus-induced common cold effect of experimental rhinovirus 39 infection on the nasal response to histamine and cold air challenges in allergic and nonallergic subjects viral infections and allergic disease goodman & gilman's the pharmacological basis of therapeutics ineffectiveness of oral terfenadine in natural colds: evidence against histamine as a mediator of common cold symptoms evaluation of oral terfenadine for treatment of the common cold efficacy of terfenadine in the treatment of common cold: a double-blind comparison with placebo the antihistamines of the nineties the influence of signal variation, bias, noise, and effect size on statistical significance in treatment studies of the common cold updated recommendations for safety-testing of viral inocula used in volunteer experiments on rhinovirus colds diagnostic procedures for viral, rickettsial, and chlamydial infections transmission of the common cold to volunteers under controlled conditions. i. the common cold as a clinical entity a double-blind, placebo-controlled clinical trial of the effect of chlorpheniramine on the response of the nasal airway, middle ear and eustachian tube to provocative rhinovirus challenge neural mechanisms of sneeze sneeze-evoking region within the brainstem inability to sneeze as a manifestation of medullary neoplasm cough and other respiratory reflexes the photic sneeze reflex: literature review and discussion sneezing: its physiology and management the nose: upper airway physiology and the atmospheric environment goodman & gilman's the pharmacological basis of therapeutics key: cord-280005-i9fp5rys authors: wang, mengmei; zhao, yang; hu, weihua; zhao, dong; zhang, yunting; wang, tao; zheng, zhishui; li, xiaochen; zeng, shaolin; liu, zhenlian; lu, li; wan, zhihui; hu, ke title: treatment of covid-19 patients with prolonged post-symptomatic viral shedding with leflunomide -a single-center, randomized, controlled clinical trial date: 2020-09-21 journal: clin infect dis doi: 10.1093/cid/ciaa1417 sha: doc_id: 280005 cord_uid: i9fp5rys objective: to evaluate the efficacy and safety of leflunomide, an approved dihydroorotate dehydrogenase inhibitor, to treat covid-19 patients with prolonged post-symptomatic viral shedding. methods: we conducted a prospective, randomized, controlled, open-label trial involving hospitalized adult covid-19 patients with prolonged pcr positivity. patients were randomly assigned to receive either leflunomide (50 mg, q12h, three consecutive times, orally; then 20 mg, once daily for 8 days), in addition to nebulized interferon alpha 2a (ifn α-2a, 3 million iu each time, twice daily for 10 days), or nebulized ifn α-2a alone for 10 days. the primary end point was the duration of viral shedding. results: a total of 50 covid-19 patients with prolonged pcr positivity were randomized into 2 groups; 26 were assigned to the leflunomide group, and 24 were assigned to the interferon alone group. treatment with leflunomide was not associated with a difference from the interferon alone group in the duration of viral shedding (hazard ratio for negative rt-pcr, 0.70; 95% confidence interval, 0.391-1.256; p=0.186). in addition, the patients given leflunomide did not have a substantially shorter length of hospital stay than patients treated with interferon alone, with median (iqrs) durations of 29.0 (19.3-47.3) days and 33.0 (29.3-42.8) days, respectively, p=0.170. two leflunomide recipients were unable to complete the full 10-day course of administration due to adverse events. conclusions: in covid-19 patients with prolonged pcr positivity, no benefit in terms of the duration of viral shedding was observed with the combined treatment of leflunomide and ifn α-2a beyond ifn α-2a alone. although clinical trials of compassionate or off-label uses of several drugs have been conducted, there is no specific and effective medication to treat patients with covid-19 [1, 2, 3] . partial clinical trial results of lopinavir-ritonavir, remdesivir, chloroquine and hydroxychloroquine have already been performed in different countries, but have shown only moderate and controversial effects [2, 4] . therefore, it is still necessary to seek safe and solid strategies to treat covid-19 when facing the increasing number of patients worldwide [5] . the pandemic of covid-19 has been under control in wuhan, china since march, 2020, but some patients remained viral rna-positive after their symptoms had resolved and their abnormal ct imaging had improved significantly [6, 7, 8] . long-term covid-19 positive patients cause many problems [9] , for example, they have to stay in the hospital for a long time and require more medical resources. in addition, they often had psychological disorders. moreover, no specific therapeutic agents have been recommended for covid-19 patients with prolonged post-symptomatic shedding [10] , which has become a great concern [11] . acute rna virus replication, including sars-cov-2, largely depends on intracellular pyrimidine resources, and antagonists of dihydroorotate dehydrogenase (dhodh), a rate-limiting enzyme in the fourth step of the de novo pyrimidine biosynthesis pathway, can efficiently prohibit viral genome replication in infected cells [12] . leflunomide, an approved dhodh inhibitor, has been widely used to treat patients with autoimmune diseases [13] , but whether leflunomide can be used to treat covid-19 patients is unknown. as covid-19 patients also suffer from excessive inflammations similar to autoimmune patients [14] , leflunomide may benefit covid-19 patients through its antiviral and antiinflammation effects. a small-scale study of leflunomide treatment for confirmed patients with covid-19 was conducted by our team, in which, leflunomide resulted in beneficial virologic clearance and length of hospital stay [15] . based on that background, we conducted a prospective randomized, controlled, open-label trial, to evaluate the efficacy and safety of oral leflunomide to treat hospitalized covid-19 patients with prolonged post-symptomatic viral shedding. from march 10, 2020 to april 12, 2020, a total of 50 consecutive patients with confirmed covid-19 with prolonged viral shedding were enrolled as study candidates. all patients were referred from other covid19 the inclusion criteria were as follows: (1) aged 18 -70 years with a diagnosis of covid-19 conforming to the chinese guidelines [16] ; (2) hospitalized for prolonged post-symptomatic viral shedding; (3) able to orally take medication; (4) non-pregnant women; (5) effective contraception for 7 days after taking the last medication. candidates were excluded based on the following: (1) presence of any condition that would not allow the protocol to be followed, including known allergy to leflunomide, use of medications that are contraindicated with leflunomide and that could not be replaced or stopped during the trial period; (2) pregnant or breast-feeding; (3) known other serious comorbidities, such as liver, cardiovascular, cerebrovascular diseases, severe renal insufficiency or advanced cancer; (5) had received interferon before enrollment; (6) unwilling to participate in the study. this clinical trial received approval from the ethics committee of the renmin hospital of wuhan university (no.wdry2020-k063) and written informed consent was obtained from each participant. the study was registered at the chinese clinical trial registry (chictr 2000030058). patients were assessed for eligibility on the basis of the inclusion and exclusion criteria (figure 1 ). at the first interview, each candidate completed a comprehensive questionnaire including demographics, comorbidities, initial-episode syndromes and disease severity at the first admission, length of virus shedding from onset to enrollment, duration of post-symptomatic viral shedding, antiviral medication before enrollment,etc.however,the original protocol had been amended,which was for a multicenter, randomized, double-blind, controlled clinical trial. due to few new covid-19 patients in wuhan, china since early march 2020, only convalescing patients with prolonged post-symptomatic viral shedding rather than those in the acute stage were enrolled in single center,with a small sample size. fifty eligible patients were randomly assigned to a combination treatment group that received leflunomide (50 mg, q12h, three consecutive times, orally; then 20 mg, once a day for 8 days; a total course of 10 days) plus nebulized ifn -2a (3 million iu each time, adding 2 ml of sterilized water, atomization inhalation twice daily for 10 days), or to a control group that received nebulized ifn -2a this was an open-label, prospective randomized, controlled trial, which was conducted at east campus, renmin hospital of wuhan university. the enrollment was initiated on march 10, 2020 and ended on april 12, 2020. the last patient studied was discharged on april 26, 2020 and was followedup until may 25, 2020. m a n u s c r i p t since there is no standard definition, we adopted the following definition of covid-19 patients with prolonged post-symptomatic viral shedding, which refers to laboratory confirmed patients with covid-19 who continued to have nasopharyngeal rt-pcr positivity at least two weeks after symptom resolution and after their abnormal ct imaging improved significantly. after enrollment, serial nasopharyngeal swab specimens were obtained at the baseline (before leflunomide or ifn -2a was administered) and once every two days until nucleic acid tests were negative twice consecutively with an interval of ≥24 hours. rt-pcr for sars-cov-2 was performed using a commercial kit (geneodx biotech co., ltd, shanghai, china). clinical symptoms of patients were assessed once daily by trained nurses using diary cards, analysis of peripheral blood cells, biochemical indicators and chest imaging studies performed at the baseline, on day 3, one day after treatment and or one day before discharge for patients meeting discharge criteria within ten days of enrollment. data were recorded on paper case record forms, then were entered into an electronic database and validated by the clinical trial staff. discharge criteria were as follows [16] : having a normal temperature for >3 days, significant improvements of respiratory symptoms and ct imaging, nucleic acid tests negative twice consecutively with an interval of ≥24 hours. after discharge, the patients were isolated at a designated place for 14 days as recommended [16] , which was arranged by community committees where the patients resided. they were followed-up by primary health-care facilities and were retested for viral nucleic acid on days 7 and 14. after that, they stayed in their homes for a second isolation period of 14 days, and were then retested for viral nucleic acid by the end of this quarantine period. we collected each patient's medical information during the isolation, which was shared with permission. in our study, enrolled patients with five consecutively negative nucleic acid tests were considered as having "true negative" results (two times during hospitalization, two times during the first isolation, and one time at the end of the second quarantine). if any patient at any time-point had a positive test for sars-cov-2, they were sent to a designated site for isolation and medical observation. the primary end point was the duration of viral shedding, which was defined as the time from randomization to the first negative nucleic acid test of five consecutive rt-pcr results. other clinical outcomes included clinical status, i.e. progressive rate to severe illness, syndromes, peripheral blood cells and biochemical parameters, c-reactive protein and inflammatory cytokines, length of hospital stay, etc. safety outcomes included adverse events that occurred during treatment, serious adverse events, and premature discontinuation of treatment. continuous variables are presented as medians (iqr). the normality of the distribution of variables was performed using the kolmogorov-smirnov test and statistical comparisons using a t-test. categorical variables are expressed as absolute numbers or percentages and are compared by the χ² test, fisher's exact test or one-way anova. the time to negative rt-pcr test was developed using the kaplan-meier method and was compared with a log-rank test. a p˂0.05 was considered statistically significant. all statistical analyses were performed using sas 9.4 software (sas institute inc, cary, nc). the characteristics of the patients in this study are summarized in tables 1 and 2 . of the 50 patients who underwent randomization and treatment assignment, 26 were assigned to the combination treatment group that orally received leflunomide plus nebulized ifn -2a, and 24 were assigned to the control group that received nebulized ifn -2a alone. in the combination treatment group, 24 patients (92.3%) received all treatments as assigned, but two patients did not complete the 10 day treatment regimen, one due to serious diarrhea 2 days after taking the drug, and the other due to impaired liver function. there were no significant differences in age (table 1) . there were no important between-group differences in baseline laboratory test results at enrollment, except for the level of creatine kinase in the control group, the level of tumor necrosis factor in the combination group was slightly higher, although both were within the normal range (table 2) . twenty-four of the 26 patients in the combination treatment group and all 24 patients in the control group completed this study and were discharged. no deaths or severe illness occurred and the illness severity was not worse in either group. in terms of the duration of viral shedding after treatment, patients assigned to the combination treatment group had a time to negative rt-pcr results that was not different from patients assigned to the control group (figure 2 ), the median time laboratory examinations were conducted before and after treatment for all patients ( table 2) . of the post-treatment test results, there were no differences between the two groups except that the lymphocyte count in the control group was slightly higher than in the combination treatment group for safety, a total of 10 patients in the combination treatment group and 4 in the control group reported adverse events (table 3) but that was not significantly different between the two groups (41.7% vs. 16.7%, p=0.057). there was one serious gastrointestinal adverse event that caused the discontinuation of treatment in the combination treatment group but none occurred in the control group, which was judged by the investigators to be related to the trial medication. for laboratory results, the absolute number of increased liver enzymes in the combination treatment group was higher than in the control group but was not statistically different (table 2) persistent viral shedding is a serious problem [17] . cao and colleagues reported that sars-cov-2 rna was detected in 40.7% of their patients on day 28 after a 14-day treatment regimen with lopinavir-ritonavir [18] . another report showed that the median duration of viral shedding was 20 days in patients with covid-19 and could be as long as 37 days [19] . an analysis of the transmission of covid-19 revealed that 86% of subjects in china in january-february 2020 potentially contracted the virus from patients with no or minimal symptoms [20] . the prolonged existence of virus presents difficulties in attempts to control the community spread of sars-cov-2. partial in vitro studies or clinical trials have suggested the potential therapeutic activity of several compounds against coronaviruses [21] , however, there are no specific antiviral pharmaceutical treatments available for patients with covid-19 [22] . the results of those studies did not show clinical improvement or the clinical trial results were controversial, including lopinavir-ritonavir [18] , remdesivir [23] , favipiravir [24] and chloroquine or hydroxychloroquine [25] . we evaluated the efficacy and safety of leflunomide on sars-cov-2 infection in this study and compared it with the roles of interferon treatment alone. interferon is recommended to be used for patients with covid-19 by the chinese guidelines [16] , for it has broad-spectrum antiviral activity [26] , has been widely used for the treatment of virus infections [26, 27, 28] , and is also effective for treating patients with covid-19 [29, 30] . leflunomide is capable of inhibiting viral rna genome replication and rescues mice from advanced influenza infections [12] . leflunomide directly targets dhodh, the host's de-novo pyrimidine synthesis enzyme, to cut off intercellular pyrimidine resources required as the starting step of building the viral rna genome [12] . like chloroquine and hydroxychloroquine, leflunomide has a dual mechanism of antiviral and immunoregulation and has been approved to treat arthritis for many years [31, 32]. leflunomide has a clear-cut drug target of dhodh and has few off-target effects [33], whereas chloroquine and hydroxychloroquine are multitargeted and have more severe adverse effects [34] . therefore, dhodh inhibitors may be attractive drugs for treating acute and severe virus infection diseases [35] . in a preliminary trial, we found that leflunomide resulted in beneficial virologic clearance and length of hospital stay for patients with covid-19 [15] . a c c e p t e d m a n u s c r i p t 9 in the present study, the baseline characteristics of the patients at enrollment were generally balanced across the two groups that did not differ with regard to duration, severity of illness and majority baseline laboratory results. however, differences in the negative conversion of virus nucleic acid between the combination treatment group and the control group were not observed. as compared to treatment with nebulized ifn -2a only, the combination of oral leflunomide and nebulized ifn for safety, two leflunomide recipients discontinued treatment due to gastrointestinal adverse events or abnormal liver function, however, there was no statistical difference in the total number of adverse events between the two groups. the side-effect profile observed in the current trial arouses concern about the use of higher or more prolonged leflunomide dose regimens in efforts to improve outcomes. ＊ length of virus shedding from onset to enrollment in patients with initial cough and expectoration. ※ patients with initial cough and expectoration in patients with initial cough and expectoration. # lianhua qingwen capsule is a kind of chinese traditional medicine and is recommended for patients with covid-19 [16] . a c c e p t e d m a n u s c r i p t 17 who launches global megatrial of the four most promising coronavirus treatments potential antiviral drugs for sars-cov-2 treatment: preclinical findings and ongoing clinical research the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future treatment options for covid-19: the reality and challenges race to find covid-19 treatments accelerates clinical features of covid-19 convalescent patients with re-positive nucleic acid detection probable causes and risk factors for positive sars-cov-2 test in recovered patients: evidence from brunei darussalam south korea's covid-19 infection status: from the perspective of re-positive test results after viral clearance evidenced by negative test results recurrence of positive sars-cov-2 in patients recovered from covid-19 prolonged viral rna shedding duration in covid-19 pcr assays turned positive in 25 discharged covid-19 patients novel and potent inhibitors targeting dhodh are broad-spectrum antiviral against rna viruses including newly emerged coronavirus sars-cov-2 leflunomide and teriflunomide: altering the metabolism of pyrimidines for the treatment of autoimmune diseases clinical features of patients infected with 2019 novel coronavirus in wuhan, china a small-scale medication of leflunomide as a treatment of covid-19 in an open-label blank-controlled clinical trial released by national health commission & national administration of traditional chinese medicine persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro characteristics of and public health responses to the coronavirus disease 2019 outbreak in china remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial experimental treatment with favipiravir for covid-19: an open-label control study. engineering (beijing) 2020 a rapid systematic review of clinical trials utilizing chloroquine and hydroxychloroquine as a treatment for covid-19 treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset bc hepatitis testers cohort team. sustained virological response from interferon-based hepatitis c regimens is associated with reduced risk of extrahepatic manifestations combination group: leflunomide plus ifn -2a; control group: ifn -2a alone ldh = lactate dehydrogenase; ultra-tni = ultratroponin i; aptt = activated partial thromboplastin time tnf = tumor necrosis factor. a: comparison of baseline data between the two groups. b: data comparison between after treatment the two groups we respectfully thank all patients enrolled in this study. this work was supported by the national key the funding agencies had no role in the study design and clinical medications; collection, analysis, and interpretation of the data; preparation, written, review, or approval of the manuscript. zhao y and hu wh contributed equally with wang mm. the authors have no competing interest to declare for this study. a c c e p t e d m a n u s c r i p t 19 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 21 figure 2 key: cord-283780-h4lwzpl9 authors: zhang, john j y; lee, keng siang; ang, li wei; leo, yee sin; young, barnaby edward title: risk factors of severe disease and efficacy of treatment in patients infected with covid-19: a systematic review, meta-analysis and meta-regression analysis date: 2020-05-14 journal: clin infect dis doi: 10.1093/cid/ciaa576 sha: doc_id: 283780 cord_uid: h4lwzpl9 the coronavirus disease 2019 (covid-19) pandemic spread globally in the beginning of 2020. at present, predictors of severe disease and the efficacy of different treatments are not well-understood. we conducted a systematic review and meta-analysis of all published studies up to march 15, 2020 which reported covid-19 clinical features and/or treatment outcomes. 45 studies reporting 4203 patients were included. pooled rates of intensive care unit (icu) admission, mortality and acute respiratory distress syndrome (ards) were 10.9%, 4.3% and 18.4%, respectively. on meta-regression, icu admission was predicted by raised leukocyte count (p<0.0001), raised alanine aminotransferase (p=0.024), raised aspartate transaminase (p=0.0040), elevated lactate dehydrogenase (ldh) (p<0.0001) and increased procalcitonin (p<0.0001). ards was predicted by elevated ldh (p<0.0001), while mortality was predicted by raised leukocyte count (p=0.0005) and elevated ldh (p<0.0001). treatment with lopinavir-ritonavir showed no significant benefit in mortality and ards rates. corticosteroids were associated with a higher rate of ards (p=0.0003). a pandemic of coronavirus disease caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spread from asia to the rest of the world in the first three months of 2020. the consequences for human health, the global economy and normal functioning of society have been unprecedented. covid-19 causes infection in any age group, though severe disease is more common in older adults [1] . the clinical spectrum of disease ranges from asymptomatic or subclinical infections to organ dysfunctionshock, acute respiratory distress syndrome (ards), acute cardiac injury, and acute kidney injury (aki)and death [2] . as of may 2, 2020, there was a total of 3,421,226 confirmed cases globally. of the 1,333,313 cases that had an outcome reached, 240,222 had resulted in mortality [3] . the growth curve of covid-19 academic literature since the first report of this outbreak from wuhan, hubei province, china in december 2019 has been exponential [13, 131 publications found on the national institutes of health covid-19 portfolio and 8502 publications found on pubmed on may 2, 2020 using the search string 'coronavirus disease 2019 or sars-cov-2']. however, systematic reviews which consolidate these findings remain scarce, with none focused on understanding the predictors for severe disease including the effects of different experimental antiviral and immune-modulatory treatments [4] . to address this gap in the literature, we conducted a systematic review, meta-analysis and meta-regression to 1) investigate the predictive value of laboratory investigations for severe disease and adverse outcomes, and 2) evaluate the efficacy of antivirals and corticosteroids for covid-19. m a n u s c r i p t 5 reviews and meta-analyses (prisma) guidelines [5] . all titles and abstracts were screened independently by two reviewers (jjyz and ksl) against a set of pre-defined eligibility criteria. potentially eligible studies were selected for full-text analysis. disagreements were resolved by consensus or appeal to a third senior reviewer (bey). agreement among the reviewers on study inclusion was evaluated using cohen's kappa [6] . all original studies reporting the clinical characteristics (symptoms and signs, laboratory investigations and radiological findings) and treatment outcomes of patients with covid-19 were included in our meta-analysis. case reports and series with a sample size of <5 were excluded per recommendations by the cochrane statistical methods group and in accordance with methodologies of previously published meta-analyses [7] [8] [9] . other exclusion a c c e p t e d m a n u s c r i p t 6 criteria included non-english articles, non-original research papers, laboratory-based and epidemiological studies with no clinical characteristics reported, as well as non-human research subjects [see supplementary table 2] . the quality of included studies was assessed using the joanna briggs institute (jbi) checklist for prevalence studies and the jbi checklist for case series [10] . full details are in supplementary tables 3 and 4 . in summary, these tools rated the quality of selection, measurement and comparability for all studies and gave a score for prevalence studies (maximum of 9) and case series (maximum of 10). two researchers (jjyz and ksl) assessed the quality of all included studies and discussed discrepancies until consensus was reached. data were extracted on the following variables: study details, sample size of study, method of diagnosis, age, gender, coexisting medical conditions, clinical symptoms, laboratory investigations, radiological findings, treatment details and patient outcomes. primary outcome measures were intensive care unit (icu) admission rate, mortality rate and the event rate of ards. icu admission was used as a surrogate marker for severe infection. secondary outcome measures included other morbidities such as respiratory failure, septic shock, coagulopathy, acute cardiac injury, aki and secondary infection, as well as length of hospital stay (los) and discharge rate at the point of study completion. a c c e p t e d m a n u s c r i p t 7 to account for intra-study and inter-study variance, random effects models were used for meta-analyses of variables and end points [11] . pooled proportions were computed with the inverse variance method using the variance-stabilizing freeman-tukey double arcsine transformation [12] . confidence intervals (ci) for individual studies were calculated using the wilson score confidence interval method with continuity correction. the i 2 statistic was used to present between-study heterogeneity, where i 2 ≤ 30%, between 30% and 50%, between 50% and 75%, and ≥ 75% were considered to indicate low, moderate, substantial, and considerable heterogeneity, respectively [13] . p values for the i 2 statistic were derived from the chi-square distribution of cochran q test. for pooling of means of numerical variables, we computed missing means and standard deviations (sds) from medians, ranges (minimum to maximum) and interquartile ranges (iqrs) using the methods proposed by hozo et al. and wan et al [14, 15] . summary-level meta-regression was performed using the mixed-effects model after computation of the sd of freeman-tukey double arcsine transformed proportions. publication bias of studies was assessed using funnel plots, where an asymmetrical distribution of studies was suggestive of bias [16] . quantitative analysis of funnel plot asymmetry was done using egger's regression test, based on a weighted linear regression of the treatment effect (expressed as a freeman-tukey double arcsine transformed proportion) on its standard error [17] . the grade approach was used to evaluate the quality of evidence for each outcome [18] . all statistical analyses were performed using r software version 3.4.3 (r foundation for statistical computing, 2016), with the package meta [19] . p-values less than 0.05 were considered statistically significant. all included studies were non-randomized, retrospective observational studies. 42 studies reported data from china, with one each from singapore, south korea and hong kong. details of included studies are reported in supplementary table 5 . of the 36 prevalence studies, 33 studies attained a full score of 9 on the jbi checklist for prevalence studies, two studies attained a score of 8 and one study attained a score of 7 [see supplementary table 3 ]. of the nine case series, 7 studies attained a full score of 10, one study attained a score of 8 and one study attained a score of 7 [see supplementary table 4 ]. of the total 4203 patients, 2797 were male (66.5%) and 1406 were female (33.5%). a c c e p t e d m a n u s c r i p t 9 the most common blood abnormalities observed were elevated c-reactive protein (crp) (59.4%), decreased albumin (58.6%), elevated lactate dehydrogenase (ldh) (51.7%) and lymphopenia (47.7%). the most common radiological abnormalities seen on chest computed tomography (ct) scan were bilateral infiltrates (80.8%), ground glass opacities (73.0%), interlobular septal thickening (46.3%), subpleural lines (45.5%) and consolidation (41.6%). in terms of treatment, type of antivirals used included combinations of oseltamivir, ganciclovir, lopinavir, ritonavir, ribavirin and arbidol. type of antibiotics used comprised moxifloxacin, ceftriaxone and azithromycin. table 1 funnel plots and egger's regression test were done to assess for publication bias for icu admission, mortality and ards rates. there was no evidence of publication bias for icu admission (p = 0.42), mortality (p = 0.41) and ards (p = 0.14) [see supplementary figure 4 ]. at baseline, the quality of evidence derived from a review of covid-19 studies was assessed as low, owing to their observational nature. the quality of evidence for respiratory failure was rated down to very low for imprecision, due to the large confidence interval range and the relatively small sample size analyzed. despite considerable study heterogeneity demonstrated by the i 2 values for most outcome measures, there was no rating down due to inconsistency, as the heterogeneity could likely be explained by differences in patient demographics, diagnostic criteria, treatment methods and management protocols given that covid-19 is a newly emergent disease. meta-regression was performed to identify risk factors of icu admission, ards and mortality [ table 2 ]. fourteen studies with a total of 2153 patients reported icu admission rates. subgroup analysis was performed for studies with the use of corticosteroids reported. sixteen studies with a total of 2407 patients reported the use of corticosteroids. pooled mortality rate in these patients was 7.2% (95% ci: 1.7 -15.4) and pooled ards rate was 22 .7% (95% ci: 9.9 -38.6). meta-regression demonstrated a significant association between corticosteroids use and higher rate of ards (p = 0.0003) [fig. 5 a c c e p t e d m a n u s c r i p t 12 our meta-analysis provides an in-depth analysis of the key epidemiological features, clinical characteristics, laboratory investigations, radiological findings, treatment details and outcomes of covid-19 from published literature. we identified elevated ldh as a significant predictive marker of ards, and found that both elevated leukocyte count and elevated ldh predict mortality. treatment with the anti-retroviral drug lopinavir-ritonavir was not associated with significant benefit, while corticosteroids were associated with possible harm. early recognition of severe infection may allow early intervention with supportive measures and therapeutics and improve outcomes [20] . our meta-regression identified five significant markers of icu admission: raised leukocyte count, raised alt and ast, in addition to elevated ldh and finally increased procalcitonin. while 10.7% of patients had a raised leukocyte count in our meta-analysis, the degree of leukocytosis was modest (pooled mean leukocyte count was 6.0 x10 9 /l). raised alt and ast in severe covid-19 disease may be a result of liver damage caused by the direct binding of sars-cov-2 to angiotensinconverting enzyme 2 positive cholangiocytes [21] . in our analysis, ldh was the only marker that significantly predicted all three measured outcomes: icu admission, ards and mortality. ldh is released from cells upon damage to their cytoplasmic membrane, and is not only a metabolic but also an immune surveillance prognostic biomarker [22, 23] . ldh increases the production of lactate, which leads to enhancement of immune-suppressive cells and inhibition of cytolytic cells [24] . these changes could weaken the immune response mounted against the viral infection, resulting in more severe disease in patients with elevated ldh. increased procalcitonin may have been a marker of bacterial co-infection, thereby resulting in complications of covid-19 disease and hence a higher rate of icu admission in these patients [25] . interestingly, lymphopenia was not found to be a significant predictor of icu admission, mortality and ards in our meta-analysis. a possible explanation may be that we analyzed lymphopenia as a dichotomous variable without taking into account a c c e p t e d m a n u s c r i p t 13 the degree of lymphopenia i.e. the numerical value of lymphocyte count, which lies on a spectrum and could affect disease severity among patients with lymphopenia. the results of randomized clinical trials of covid-19 interventions are of critical importance as only weak evidence supports the currently available repurposed and novel antivirals [26] . among the patients with antiviral use reported in our meta-analysis, overall rates of mortality, icu admission and ards were 5.7%, 11.8% and 20.2%, respectively. we found no overall benefit from treatment with lopinavir-ritonavir, in line with a recent randomized controlled trial however, this trial demonstrated that lopinavir-ritonavir treatment granted a significant reduction in icu length of stay in survivors. further trials (nct04252885 and nct04307693) are in progress to assess the efficacy of both lopinavir and ritonavir in reducing the covid-19 viral load, and we look forward to future developments to provide recommendations on the use of antiviral therapy [28, 29] . severe covid-19 is associated with a dysregulated host inflammatory response, suggesting immune modulators as an attractive treatment modality [30] . corticosteroids were used during the sars-cov outbreak, however, in a meta-analysis only four studies provided conclusive data, and all four indicated possible harm [31, 32] . these harms included risks of prolonged viremia, corticosteroid-induced diabetes, avascular necrosis and psychosis [31, 33, 34] . our meta-analysis suggested that the use of corticosteroids is associated with disease severity (icu admission) and higher ards rates. it is not clear if this effect is a consequence of corticosteroid treatment, or confounding by indication bias where sicker patients are more likely to receive corticosteroids. an rct of corticosteroids in severe respiratory viral infections has long been called for, and at least one clinical trial in covid-19 (nct04244591) is ongoing [35] . a c c e p t e d m a n u s c r i p t 14 sars-cov-2-induced pneumonia is marked by a cytokine stormhyperactivation of effector t cells and excessive production of inflammatory cytokines, particularly interleukin-6 (il-6) [36] . blockade of il-6 function using tocilizumab, a specific monoclonal antibody against its receptor appears to be useful in alleviating hyperinflammation symptoms in severe cases [37, 38] . selective janus kinase-signal transducer and activator of transcription (jak-stat) inhibitors such as baricitinib may also be beneficial, though clinical trials are required and any benefit is likely to be greatest in combination with an effective antiviral [39] . to the best of our knowledge, this is the first systematic review and meta-analysis of covid-19 to describe specific laboratory predictors of severe disease and adverse outcomes. our study is also the first meta-analysis to evaluate the efficacy of antivirals and corticosteroids. careful attention should be given to the management of patients with raised leukocyte count, raised alt and ast, elevated ldh, increased procalcitonin and raised leukocyte count as these factors predict icu admission, mortality and ards. in terms of treatment efficacy, the use of corticosteroids in covid-19 patients is significantly associated with higher rates of ards. compared to other antivirals, the use of lopinavir and ritonavir is non-superior in terms of lowering mortality rate. further prospective studies are vital to clarify our findings. a c c e p t e d m a n u s c r i p t 16 no funding was used for the production of this work. all authors have no potential conflicts of interest to disclose. m a n u s c r i p t 20 a c c e p t e d m a n u s c r i p t 28 figure 5 sw epidemiological and clinical characteristics of 99 cases of 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community-acquired pneumonia in autoimmune disease-induced immunocompromised host: a retrospective observational study elevated lactate dehydrogenase (ldh) can be a marker of immune suppression in cancer: interplay between hematologic and solid neoplastic clones and their microenvironments. cancer biomarkers : section a of disease markers procalcitonin in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis pharmacologic treatments for coronavirus disease 2019 (covid-19): a review a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 the efficacy of lopinavir plus ritonavir and arbidol against novel coronavirus infection (elacoi) comparison of lopinavir/ritonavir or hydroxychloroquine in patients with mild coronavirus disease (covid-19) the trinity of covid-19: immunity, inflammation and intervention sars: systematic review of treatment effects corticosteroid therapy for critically ill patients with middle east respiratory syndrome factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients glucocorticoid therapy for novel coronaviruscritically ill patients with severe acute respiratory failure (steroids-sari) clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china covid-19: consider cytokine storm syndromes and immunosuppression tocilizumab treatment in covid-19: a single center experience covid-19: combining antiviral and antiinflammatory treatments a c c e p t e d m a n u s c r i p t 17 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-277788-6ls21tkr authors: nelson, brian c; laracy, justin; shoucri, sherif; dietz, donald; zucker, jason; patel, nina; sobieszczyk, magdalena e; kubin, christine j; gomez-simmonds, angela title: clinical outcomes associated with methylprednisolone in mechanically ventilated patients with covid-19 date: 2020-08-09 journal: clin infect dis doi: 10.1093/cid/ciaa1163 sha: doc_id: 277788 cord_uid: 6ls21tkr background: the efficacy and safety of methylprednisolone in mechanically ventilated patients with acute respiratory distress syndrome due to coronavirus disease 2019 (covid-19) are unclear. in this study, we evaluated the association between use of methylprednisolone and key clinical outcomes. methods: clinical outcomes associated with the use of methylprednisolone were assessed in an unmatched, case-control study; a subset of patients also underwent propensity-score matching. patients were admitted between march 1 and april 12, 2020. the primary outcome was ventilator-free days by 28 days after admission. secondary outcomes included extubation, mortality, discharge, positive cultures, and hyperglycemia. results: a total of 117 patients met inclusion criteria. propensity matching yielded a cohort of 42 well-matched pairs. groups were similar except for hydroxychloroquine and azithromycin use, which were more common in patients who did not receive methylprednisolone. mean ventilator free-days were significantly higher in patients treated with methylprednisolone (6.21±7.45 versus 3.14±6.22; p = 0.044). the probability of extubation was also increased in patients receiving methylprednisolone (45% versus 21%; p = 0.021), and there were no significant differences in mortality (19% versus 36%; p = 0.087). in a multivariable linear regression analysis, only methylprednisolone use was associated with higher number of ventilator-free days (p = 0.045). the incidence of positive cultures and hyperglycemia were similar between groups. conclusions: methylprednisolone was associated with increased ventilator-free days and higher probability of extubation in a propensity-score matched cohort. randomized, controlled studies are needed to further define methylprednisolone use in patients with covid-19. in december 2019, the outbreak of a novel respiratory virus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in wuhan, china marked the beginning of a global pandemic that has since led to over four million cases and 300,000 deaths worldwide [1] . the clinical manifestations of coronavirus disease 2019 are varied and range from asymptomatic disease to acute respiratory distress syndrome (ards) with septic shock and multiorgan failure [2, 3] . the reported mortality among critically-ill patients with covid-19 has varied significantly. an early study reported 61.5% mortality among critically-ill patients [4] , while a more recent study reported an overall mortality of 25.8% in a critically-ill cohort [5] . emerging data and analogy to other coronaviruses indicate that a dysregulated immune response to sars-cov-2 infection results in a cytokine storm-like syndrome that precipitates the severe clinical manifestations in covid-19 [6] . corticosteroids inhibit expression of the cytokines involved in the inflammatory response [7] and may improve outcomes in patients with severe ards [8, 9] . consequently, there is considerable interest in using corticosteroids to treat severe covid-19. more data are needed to further define the role of corticosteroids in emerging coronavirus infections. prior studies on the use of corticosteroids in severe acute respiratory syndrome coronavirus (sars) and middle eastern respiratory syndrome coronavirus (mers) were inconclusive. these studies failed to show improved mortality, and one study showed delayed viral clearance [10, 11] . early reports out of wuhan, china describing the effect of corticosteroids in covid19 have demonstrated variable results [12, 13] . the recently published recovery trial demonstrated improved mortality in patients that received dexamethasone; however, it did not examine other corticosteroids and included all hospitalized patients, including patients who were not critically ill [14] . we sought to further evaluate the effect of corticosteroids in cases of severe covid-19 and to assess adverse effects, such as hyperglycemia and secondary infections. in this retrospective, observational, case-control study, we investigated the effect of methylprednisolone on critically-ill, mechanically ventilated patients with a c c e p t e d m a n u s c r i p t 4 study design. this study retrospectively evaluated consecutive adult patients with covid-19 pneumonia requiring intubation and mechanical ventilation. all patients were admitted to a quaternary care medical center in new york city, comprised of a large academic hospital and a smaller community hospital, between march 1, 2020 and april 12, 2020. the study was approved by the columbia university irving medical center institutional review board with a waiver for informed consent. patients were eligible for inclusion if they tested positive for sars-cov-2 by reverse transcriptase polymerase chain reaction (rt-pcr) of nasopharyngeal and/or oropharyngeal swab specimen within 48 hours after admission and subsequently required mechanical ventilation. patients were excluded if they died less than five days after hospital admission, if they weighed more than 200 kg, or if they received steroids other than methylprednisolone for greater than 24 hours. patients more than 200 kg were excluded to account for our weight-based dosing protocol that recommended a maximum dose of 80 mg. those who died prior to hospital day five were excluded because most patients in the study period were not evaluated for steroid treatment until at least four days into their hospital course. those included in the methylprednisolone group received greater than 24 hours of methylprednisolone initiated within the first 14 days after admission. our hospital dosing protocol for methylprednisolone was one mg/kg/day with a max dose of 80 mg per day, with recommended duration of five days, although the course could be extended at the discretion of the treating physician. our protocol also recommended that steroids only be started in patients at least five to seven days after symptom onset and only in those with evidence of systemic inflammation. early in the pandemic, our hospital guidelines permitted the use of hydroxychloroquine and azithromycin for patients with vital sign abnormalities or risk factors for disease progression. this recommendation was later removed after publication of new findings showing minimal benefit with these agents [15] . the primary outcome evaluated was ventilator-free days at hospital day 28. ventilator-free days were defined as days after extubation, with days prior to intubation not included. patients who died within 28 days were assigned zero for this outcome, as were those who were not extubated during the follow-up period [16] . secondary outcomes included extubation, death, and hospital discharge at both 28 and 60 days after admission. extubation was defined as either removal of the endotracheal tube or discontinuation of mechanical ventilation without relapse in patients who had undergone tracheostomy. safety outcomes were also assessed, including hyperglycemia and all positive clinical cultures (except for coagulase-negative staphylococcus isolated from a single blood culture). hyperglycemia was defined as days with a blood glucose ≥ 180 mg/dl during the first 21 days, chosen to reflect the time period during which most patients received steroids. positive cultures from any source during the first 28 days served as a proxy for secondary infections, given the difficulty of discerning between colonization and infection in this patient population. data points collected at the time of admission included patient demographics, chronic comorbidities summarized using the charlson comorbidity index (cci) [17] , laboratory values, and the reported date of symptom onset. the sequential organ failure assessment (sofa) score [18] was calculated on the day of intubation. days from admission to intubation were also documented. the use of additional agents with antiviral and immunomodulatory effects targeting sars-cov-2 was also noted. pao 2 /fio 2 (pf) ratios, highsensitivity c-reactive protein (crp) values, and use of vasopressors were recorded during the first 48 hours after admission. statistics. patients who received methylprednisolone were analyzed as cases, and those who did not served as controls. we performed unmatched, case-control analysis of all patients meeting criteria for inclusion. we then used propensity scoring to create well-matched groups by using 1:1 nearest-neighbor matching without replacement. covariates included in the propensity score matched analysis were the following: body mass index ≥ 30 kg/m 2 , age ≥ 65 years, gender, admission crp ≥ 150 mg/l, admission d-dimer ≥ one mcg/ml, pf ratio < 200 at hospital day two, sofa score on the day of intubation, cci score, and days from symptom onset to admission. dichotomous cutoffs were chosen in order match specific populations (i.e. elderly or obese a c c e p t e d m a n u s c r i p t 6 patients) or because our system reported laboratory values with a specified upper limit that precluded continuous analysis. patients who did not have a match within 0.2 propensity score standard deviations were excluded from the analysis. categorical variables were compared using the chi-square test or fisher's exact test as appropriate. continuous variables were compared using the mann-whitney u test. variables with p values < 0.1 in univariable analysis were considered for inclusion into a multivariable linear regression model identifying factors associated with ventilator-free days. methylprednisolone use was also included a priori. statistical analyses were performed using ibm spss statistics, version 26 (spss inc., chicago, ill., usa). patient characteristics. 142 patients with covid-19 who required mechanically ventilation were identified during the study period. of these, 25 were excluded from the analysis: 13 died within five days of admission; seven received other steroids; four received methylprednisolone starting more than 14 days after admission; and one weighed > 200 kg. a total of 117 patients were included in the overall analysis. other key variables such as age, bmi, gender, cci, sofa score, and days from symptom onset to admission were similar between groups (table 1) . clinical outcomes. within the matched cohorts, the primary outcome of ventilator-free days was significantly higher in the steroid group (mean ± standard deviation, 6.21±7.45 versus 3.14±7.45; p = 0.044) ( table 2 ). the probability of extubation by day 28 was significantly higher in patients who received steroids (45% versus 21%; p = 0.021), and there was a statistically non-significant trend toward reduced mortality (19% versus 36%; p = 0.087). mortality was still numerically lower at the end of the 60-day follow-up period but the difference remained non-significant. there were also no significant differences in hospital discharge between those who received steroids and those who did not at day 28 (17% versus 19%; p = 0.776) or day 60 (45% versus 36%; p = 0.374). regarding safety outcomes, the proportion of patients with positive cultures was similar between steroid and control patients at hospital day 28 (52% versus 45%; p = 0.513). days with blood glucose values ≥ 180 mmol/l over the first 21 days were also similar (9 [iqr, 3-17] versus 8 [2, 14] ; p = 0.415). in a multivariable, linear regression model including matched pairs (table 3) , the use of methylprednisolone was found to be independently associated with a higher number of ventilator-free days (p = 0.045) after controlling for lactate dehydrogenase (ldh). the use of hydroxychloroquine, azithromycin, or tocilizumab was not significantly associated with the primary outcome. after controlling for age, sofa score, white blood cell count (wbc), ldh, and d-dimer, the use of methylprednisolone (p = 0.015) was independently associated with improved outcome within the overall cohort, while treatment with hydroxychloroquine, azithromycin, or tocilizumab was not. a c c e p t e d m a n u s c r i p t 8 our study evaluated the association of methylprednisolone treatment with duration of mechanical ventilation and mortality in intubated, critically-ill patients with covid-19. all patients had bilateral infiltrates and hypoxemic respiratory failure consistent with ards. we observed an increase in the number of ventilator-free days and the likelihood of extubation, as well as a statistically non-significant trend towards improved mortality, in the corticosteroid group when compared to control patients in a propensity-matched cohort by day 28. numerically fewer patients died by day 60 but the difference was not statistically significant. few patients were discharged by day 28, but there was a trend toward earlier discharge by hospital day 60 in those who received methylprednisolone. conflicting results on the benefit and safety of corticosteroids in other viral pneumonias led to early recommendations by the who against routine use in the management of covid-19 [19] . a systematic review found that corticosteroids used to treat influenza were associated with increased mortality [20] . in a study of patients with mers-cov, there was no effect of corticosteroids on mortality, and there was a delay in viral clearance [11] . a systematic review of the literature on corticosteroid use in sars-cov yielded inconclusive results, with some studies trending towards harm [10] . steroid dosing regimens have varied significantly among studies. for example, in one study sars-cov patients, doses as high as 160 mg of methylprednisolone per day were used [21] , while in another study of patients with ards receiving less potent corticosteroid doses, treatment duration was as long as 32 days [8] . several other studies have shown no mortality benefit associated with corticosteroids in patients with ards, and one larger study found an increase in mortality when methylprednisolone was initiated more than two weeks after the onset of ards [22, 23] . corticosteroids and other immunomodulators have also been studied as a potential treatment for ards. prior studies have shown that infection with sars-cov and mers-cov can result in a cytokine release syndrome leading to massive inflammatory cell infiltration, acute lung injury, and ards [24], and a similar process has been theorized as the cause of ards in covid-19 [6] . recent studies have shown improved outcomes in patients with ards who received corticosteroids [8, 9] . in one multicenter, randomized, controlled trial, a c c e p t e d m a n u s c r i p t 9 mechanically ventilated patients with ards who received dexamethasone had a significant increase in the number of ventilator-free days and a significant reduction in mortality compared to the control group [9] . in a small, observational study among patients with ards from severe covid-19, treatment with methylprednisolone reduced mortality from 61.8% to 46.0% [25], although these results should be interpreted with caution given the limited quality of the trial design. most recently, the recovery trial found that patients with covid-19 who received dexamethasone six mg daily for 10 days experienced a mortality benefit. although this benefit was greatest in the subset of patients that required mechanical ventilation, the trial only evaluated outcomes through hospital day 28 and did not assess other corticosteroids, such as methylprednisolone [14] . the earlier time to recovery seen in our study suggests that corticosteroids may have a favorable effect on the hyperinflammatory state in patients with covid-19. we observed a significant increase in both ventilator-free days and in the likelihood of extubation by day 28. there was also a numerical increase in earlier discharge by day 60, but this difference was not statistically significant. additional studies examining the pathophysiology of ards in covid-19 are needed to determine the interplay between the immune response and disease progression and how this is affected by different management strategies. concerns have been raised regarding the incidence of hyperglycemia in patients who are treated with corticosteroids [26, 27] . our study did not detect a difference in days with blood glucose values ≥ 180 mmol/l by day 21. an additional concern is the potential for secondary infections. this finding was noted in a review of patients with influenza pneumonia who were treated with corticosteroids [19] , although another study found that corticosteroids did not increase the type or incidence of infectious complications among patients with ards admitting to intensive care [9] . our study did not detect a difference in incidence of positive cultures at day 28 in patients who received methylprednisolone compared to those who did not. a c c e p t e d m a n u s c r i p t 10 the optimal dose and duration of methylprednisolone treatment in patients with viral pneumonia and ards remains unknown. our hospital protocol for use of methylprednisolone in treatment of covid-19 was one mg/kg/day with a maximum daily dose of 80 mg, with median duration of four to six days. the corticosteroid regimen used in our study was similar to regimens used in prior studies that found improved clinical outcomes in patient with ards [9, 13] . the recovery trial used a dexamethasone dose that was approximately half the equivalent methylprednisolone dose used in our patients with a duration of 10 days compared to the four to six days use in this study there are several limitations to our study. first, our study only included patients with covid-19 requiring mechanical ventilation, which limits the generalizability to less critically-ill patients. however, several recent studies have highlighted the benefits of early corticosteroids in patients with moderate covid-19 [14, 28, 29] . our study was conducted at a single hospital center with limited sample size, and efforts at propensity matching to reduce selection bias may not have accounted for all the variables determining which patients received corticosteroids. rapidly changing practice patterns at our hospital throughout the course of the pandemic may also have influenced our results. during the second month of the pandemic, there was a higher threshold for intubation. however, in our study the timing of intubation was similar between groups, reflecting the overall early enrollment of these patients. similarly, the threshold to admit patients was also raised later in the outbreak, which may have resulted in prolonged time to admission, although no differences were noted between groups in our study. our hospital guidelines regarding management of covid-19 were updated several times during the period of our study, as reflected in the differences seen in the use of hydroxychloroquine and azithromycin. these treatments had no significant associations the primary outcomes in multivariable analysis. finally, there were also changes to the prevention and management of venous thromboembolism during the study period. however, none of those changes were routine or protocolled at the time. our ability to distinguish true infection from colonization or contamination was limited by chart review and by diagnostic limitations in the setting of the pandemic. thus, we defined any positive culture as a possible proxy for infection (except for coagulase-negative staphylococcus isolated from a single blood culture). although we observed a trend towards improved mortality both at day 28 and day 60 in patients treated with methylprednisolone, our study may have been underpowered to detect a significant difference in a c c e p t e d m a n u s c r i p t 11 mortality between the two groups. given the limitations of our study design, a randomized-controlled trial is needed to better able to evaluate survival benefits associated with methylprednisolone. in conclusion, our study adds to a growing body of literature regarding the use of corticosteroids for treatment of covid-19. we found that treatment with methylprednisolone increased the number of ventilator-free days and probability of extubation compared with a propensity matched control group among patients with severe covid-19 requiring mechanical ventilation, but we did not detect a significant difference in mortality. a randomized-controlled trial is necessary to further define the role of methylprednisolone in this emerging disease. a c c e p t e d m a n u s c r i p t coronavirus disease 2019. who 2020. available at asymptomatic cases in a family cluster with sars-cov-2 infection presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, 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with covid-19 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-281726-s1o5l7ns authors: yu, ignatius t. s.; wong, tze wai; chiu, yuk lan; lee, nelson; li, yuguo title: temporal-spatial analysis of severe acute respiratory syndrome among hospital inpatients date: 2005-05-01 journal: clin infect dis doi: 10.1086/428735 sha: doc_id: 281726 cord_uid: s1o5l7ns background. we report the temporal-spatial spread of severe acute respiratory syndrome (sars) among inpatients in a hospital ward during a major nosocomial outbreak and discuss possible mechanisms for the outbreak. methods. all inpatients who had stayed in the same ward as the initial index case patient for any duration before isolation were recruited into a cohort and followed up to document the occurrence of sars. the normalized concentration of virus-laden aerosols at different locations of the ward was estimated by use of computational fluid dynamics modeling. the attack rates in the various subgroups stratified by bed location were calculated. multivariate cox proportional hazards regression was used to document important risk factors. results. the overall attack rate of sars was 41% (30 of 74 subjects). it was 65%, 52%, and 18% in the same bay, adjacent bay, and distant bays, respectively (p = .001). computation fluid dynamics modeling indicated that the normalized concentration of virus-laden aerosols was highest in the same bay and lowest in the distant bays. cox regression indicated that staying in the ward on 6 or 10 march entailed higher risk, as well as staying in the same or adjacent bays. the epidemic curve showed 2 peaks, and stratified analyses by bed location suggested >1 generation of spread. conclusions. the temporal-spatial spread of sars in the ward was consistent with airborne transmission, as modeled by use of computational fluid dynamics. infected health care workers likely acted as secondary sources in the latter phase of the outbreak. severe acute respiratory syndrome (sars), which originated from the guangdong province of china, was first recognized in february 2003 [1] . a medical doctor from guangzhou (canton), who stayed in a hong kong hotel in february 2003, was identified as the index case patient for the international spread of sars to at least 5 countries [2] . a young hong kong resident visited the hotel and contracted the disease, subsequently leading to a big outbreak in the prince of wales hospital, where he was treated. hong kong was one of the places hardest hit by sars, with a total of 1755 cases and a death toll of 299. in the initial phase of the outbreak in the prince of wales hospital alone, 1100 persons were affected, including doctors, nurses, medical students, other hos-pital patients, visitors, and their relatives [3] . a government investigation reported that 1 of the patients with sars subsequently spread the disease to 1300 residents in a private housing estate (amoy gardens) [4] . it is generally believed that sars is primarily transmitted by droplets or direct contacts [5] ; however, probable airborne transmission during the amoy gardens outbreak has been reported by us [6] . a detailed epidemiological study of the outbreak in the prince of wales hospital may shed light on the possible modes of transmission and help in the prevention of nosocomial spread in the future. four groups of subjects could be identified in this outbreak: the health care workers (hcws), including doctors and nurses, the medical students who were attending clinical teaching or examinations in the ward, other inpatients staying in the same ward, and visitors. the spread of sars among the medical students and hcws has been reported elsewhere [7, 8] . here, we report the temporalspatial spread of sars to other inpatients staying in the same ward and explore the possible factors and mechanisms involved in the nosocomial outbreak. figure 1 . layout of the ward where the index case patient with severe acute respiratory syndrome (sars) was hospitalized, showing the location of beds, air supply diffusers, and exhaust grilles. supply and exhaust flow rates were measured in liters per second). the index case patient stayed in bed 11 during the period 4-12 march. same bay, beds 9-16, 9x, and 16x; adjacent bay, beds 17-24, 17x, and 24x; distant bays, all other beds. a retrospective cohort study was conducted. the index case patient was first admitted into the male medical ward on 4 march 2003 and was put under isolation on 12 march. all inpatients who had stayed in the same ward as the index case patient for any duration during the period 4-12 march were recruited into the cohort. baseline information for each subject was obtained from the computerized medical record system and included age, date of admission, bed number, comorbidity, drug treatment, and date of leaving the ward (discharge or transfer). all subjects were observed until the end of march 2003 (at least 22 days) to observe whether they developed sars. sars was diagnosed on the basis of the interim case definition at the time [9] , and all cases were subsequently confirmed serologically by immunofluorescence assay [10] showing rising titer of igg to the sarsassociated coronavirus. the dates of onset of fever for all patients with sars were recorded. the attack rates of sars among all inpatients and in the subgroups were calculated. the relationships between sars and bed location, date of exposure, duration of exposure, smoking to explore the possible roles of hcws in the outbreak of infection, all nurses working on the ward during the study period were interviewed in person between 28 march and 8 april with a questionnaire to collect information on symptoms, contacts, and working practices. information about the ventilation system of the ward was collected, including the location and size of supply diffusers and exhaust grilles, supply air temperature, and the air-flow rate through each supply diffuser, exhaust grille, and exhaust fan. dispersion of the hypothetical virus-laden aerosols, which originated from the index case patient's bed, through the entire ward was analyzed by computational fluid dynamics (cfd) method. cfd allows prediction of detailed 3-dimensional air-flow pattern as well as pollutant dispersion in the ward. the industry-standard cfd package fluent [11] was used. the computational domain included the ward and the entrance. all 74 subjects who ever stayed in the ward during the study period were included into the cohort. thirty-six subjects had their first exposures on 4 march, when the index case was admitted, and 38 new admissions took place during 5-9 march. all subjects were men, with a mean age of 66 years (range, 19-90 years); patients with sars were slightly older than those without sars (mean, 69 vs. 65 years; , by student's t p 1 .05 test). sixteen (22%) were current smokers, and 25 (34%) were ex-smokers. thirty subjects developed sars during the follow-up period, giving an overall attack rate of 40.5%. twenty-three subjects died during the follow-up period (14 died of sars, and the other 9 died of other unrelated causes). two peaks were present in the overall epidemic curve-11 march and 15 march (figure 2). patients who stayed in the distant bays had, in general, a later onset of fever. spatial distribution of cases. thirteen (65.0%) of the 20 subjects in the same bay developed sars. the attack rate was 52.4% (11 of 21 subjects) in the adjacent bay and 18.2% (6 of 33) in the distant bays. the proportions infected in the 3 locations differed significantly ( ; ). 2 x p 13.03 p p .001 effects of exposure dates and duration. the date of presence in the ward was associated with sars (table 1). those note. the forward stepwise strategy was adopted in the regression models with the following covariates: age, smoking habit, steroid and antiviral treatments, and various chronic diseases. who were not present in the ward on 6 march had a significantly lower attack rate (7 [21.2%] of 33 subjects) than did those who were present (23 [56.1%] of 41; ). the duration of p p .005 stay did not have any influence on sars. preexisting medical diseases and treatment. all subjects had у1 chronic disease, including cardiovascular disease (61% of subjects), pulmonary disease (41%), liver disease (22%), diabetes mellitus (19%), blood disease (19%), and renal disease (12%). preexisting chronic diseases were not associated with sars. fifteen subjects were taking steroids for other diseases, and they had a slightly lower attack rate (33.3%), but the difference was statistically insignificant. stratified analysis by location did not support any protective effects. of the 3 subjects receiving antiviral treatment for viral hepatitis (2 receiving lamivudine and 1 receiving oseltamivir), 2 subsequently developed sars. multivariate analysis. the results of the cox regression model are given in table 2. presence in the ward on either 6 or 10 march was associated with a significantly higher risk of sars. subjects in the same bay or adjacent bay also had significantly higher risks. age, smoking, preexisting diseases, and drugs received were not significant. the sars-free survival curves in the 3 locations are shown in figure 3 . the log rank test result was highly statistically significant ( ). p ! .001 survey among nurses. all but 1 of the 15 nurses were infected with the agent of sars. the fever onset dates are given in figure 4a . all reported having direct contacts with the index case patient except the ward manager, who also developed sars during this outbreak. most subject reported washing their hands after caring for the index case patient. spatial distribution of the hypothetical virus-laden aerosols. the airflow pattern in the ward was very unsteady because of the interactions of various thermal buoyancy and momentum forces as well as movements of people. the ward was air-conditioned by a fan coil system with a separate fresh air supply. the exhaust air from this ward was discharged to the outside and not recirculated to other wards. a steady-state normalized concentration contour of virus-laden aerosols at a height of 1.1 m above ground is shown in figure 5 . the concentration was between 0.015 and 1 in the same bay, between 0.005 and 0.008 in the adjacent bay, and between 0.0015 and 0.005 in the distant bays. in this analysis of inpatients in the medical ward with the largest nosocomial outbreak during the sars epidemic in 2003, we included all eligible subjects into the cohort, and there was no selection bias. the data used for analysis were collected at the time of occurrence and did not depend on any recall and, thus, should be objective and unbiased. this cohort of subjects provided a good opportunity to explore the risk factors associated with nosocomial infections with the sars agent. the attack rate was very high, with 41% of subjects infected within a period of 8-9 days. the risk of infection was significantly associated with the bed location and the dates of presence on the ward. the duration of exposure and personal factors were not significant predictors of sars in this cohort. the spatial distribution of the infected inpatients was not random. the probability of such a distribution occurring by chance was only 1 in 1000. the division of bed location, based on proximity to the index case, into high-exposure (same bay), middle-exposure (adjacent bay), and low-exposure (distant bays) areas corresponded well with the normalized concentrations of the virus-laden aerosols predicted by cfd modeling. the attack rate was highest in the same bay (65%), slightly lower in the adjacent bay (52%), and much lower in the distant bays (18%), suggesting the possibility of a nonlinear relationship between concentration of virus and risk of infection. the sars-free survival curves also indicated clearly that those staying in the distant bays were at lower risk. the relationship between proximity to the index case patient and risk of infection suggested that airborne transmission probably played an important role in this nosocomial outbreak. were there any alternative explanations for the particular pattern of distribution? clustering of susceptible subjects in certain bays could theoretically lead to different attack rates. however, no particular high-risk personal characteristics or comorbidities have been identified thus far. movements of the index case patient around the ward could have spread the infection to the other bays through direct contacts or droplets, but he was very ill during his first week of hospitalization and was bed-bound in the same bed [8] . some inpatients in the ward were ambulatory, but it was most unlikely that many inpatients visited the index case patient and contracted the disease directly from him. they could also have acquired the infection in common areas that had been contaminated, such as the toilets, but because inpatients in the different bays were not particularly bed-bound or ambulatory, their probability of contracting the infection should not differ. we checked the bed movements (transfers) of the inpatients during the study period and found that none of the inpatients with sars were moved to other beds after the onset of fever and, thus, could not have played a role in the spread of the infection to other bays. the sars coronavirus could have been spread from the index case patient to other inpatients indirectly through fomites or hcws as mechanical carriers. a similar pattern of nosocomial infection has been reported for vancomycin-resistant enterococci and clostridium difficile, and contaminated hands of hcws were believed to be responsible for the contact spread [12, 13] . however, nursing duties in our ward were assigned by function and not by patient groups or geographical areas in the ward, as commonly practiced in other places. our interviews confirmed that all nurses serving in the ward cared for inpatients in all 4 bays during each shift. all physicians in the ward also had to visit different inpatients in all 4 bays. the hcws could potentially spread the virus to other inpatients through their unwashed hands after making contacts with the index case. because every nurse had a chance of contacting different inpatients in the different bays, the risk of infection should be distributed more evenly among the different bays. it was still possible that some hcws washed hands between servicing the next bay but did not wash hands between inpatients in the same bay. this would contribute substantially to withinbay spread but could not explain the relatively high attack rate in the adjacent bay. the sixth and tenth of march were the 2 important dates in the multivariate analysis, with hazard ratios of 4.5 and 2.4, respectively. these dates corresponded very well with the 2 peaks observed in the epidemic curve on 11 and 15 march, suggesting a modal incubation period of ∼5 days from exposure to fever onset, which was very similar to that of the largest community outbreak in amoy gardens [6] . the epidemic curve for the distant bays started later, on 12 march, and peaked on 15 march, suggesting that there might the index case patient had symptom onset on 24 february, and the virus load would be at its maximum around 6 march, which was 10 days after onset of symptoms [14] . in fact, the patient's cough was most severe during 4-7 march, and his condition deteriorated on 6 march; therefore, nebulizer use was started in that afternoon and maintained until 12 march. clinical details of the index case have been described in 2 earlier reports [8, 15] . the release of a large amount of virus into the air by the index case patient through coughing around 6 march probably caused the first wave of infections, which mainly affected inpatients staying in the same and adjacent bays. spread by direct contacts and droplets could explain only a small num-ber of the infections, because droplet spread is believed to be effective only within a distance of ∼1 m. airborne spread through virus-laden aerosols was probably responsible for spreading the infection to beds outside the same bay. the epidemic curves of nurses, all staff (including nurses, doctors, and other service workers), and visitors supported point source infection (see figure 4 ). there was much overlapping of the dates of onset of fever for all groups, including the inpatients (figure 2), suggesting that the infections likely came from a common source, at least in the first phase up to 12 march. unfortunately, proper spatial analysis of sars among the non-inpatient groups was not possible. all hcws visited and worked in all bays during their routine work in the ward, and all but 1 had direct contacts with the index case patient. we did not have a register to provide reliable denominator data for calculating the attack rates in the different locations for visitors. however, quite a number of the infected visitors only visited inpatients staying in the adjacent bay and did not get anywhere near the index case patient. a substantial number of infected visitors experienced onset of fever earlier than did the inpatients' relatives who they visited and, thus, could not have contracted the infection from them. this offers additional indirect support for a possible airborne spread of the virus from the index case patient. the second wave of infections could not be adequately explained by the spread from the index case patient alone. the analysis of sars among medical students suggested that the index case patient became less infectious on 7 march [8] . although it was possible that he became more infectious again around 10 march, this was not very likely, because his fever and chest condition gradually improved starting on 9 march [15] ; other explanations should be considered. we have discussed above that bed movements of inpatients were unlikely to be responsible for the spread. we noted that a number of hcws infected with sars during the earlier phase of the outbreak developed fever and other symptoms on 8 and 9 march but continued working in the ward until 10 march. one or several of them most likely spread their infections to other inpatients through direct contacts or droplets and caused the second wave of the outbreak. the analysis of the temporal-spatial spread of sars from the index case patient to other inpatients in the ward suggested that airborne spread through virus-laden aerosols possibly played an important role. unlike other reports of airborne outbreaks, we were unable to document the existence of the infective agent in aerosols. such documentation was simply impossible in early march 2003, when the infective agent was yet to be identified. sars was unlikely a communicable disease with obligate airborne transmission, such as tuberculosis, but there was evidence to suggest that sars could have at least opportunistic airborne transmission under special circumstances when virus-laden aerosols could be generated [16] . the virus-laden aerosols could come from the nebulizer [17] or the nuclei resulting from the rapid evaporation of the droplets produced by coughing in a relatively dry, air-conditioned environment. the use of the nebulizer appeared not to be essential for the outbreak, because medical students who were present in the ward only before the use of the nebulizer also became infected [8] . infected hcws who continued to work after the onset of symptoms were most likely responsible for propagating the infection in the later phase of the outbreak. the findings of this study have implications for future infection control inside hospitals. any patients in whom any sarslike infection is suspected should be adequately isolated, or at least segregated, to prevent the spread of infections. the bays in a large ward should be adequately partitioned structurally and provided with separate ventilation to reduce the mixing of air between bays. personal protective equipment for preventing contact and airborne spread should be provided to hcws, together with a proper program of equipment testing, training, and maintenance. hcws with infections or carrying infective agents should refrain from working in areas or situations that favor the transmission of infections from them to the patients. severe acute respiratory syndrome (sars): multi-country outbreak-update update: outbreak of severe acute respiratory syndrome-worldwide a major outbreak of severe acute respiratory syndrome in hong kong outbreak of severe acute respiratory syndrome (sars) at amoy gardens, kowloon bay, hong kong-main findings of investigation effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) evidence of airborne transmission of the severe acute respiratory syndrome virus an outbreak of sars among health care workers a cluster of severe acute respiratory syndrome among medical students exposed to a single patient in hong kong severe acute respiratory syndrome (sars) updated interim case definition severe acute respiratory syndromeassociated coronavirus infection 1 (computer software). lebanon, nh: fluent insights into the epidemiology and control of infection with vancomycin-resistant enterococci epidemiology of clostridium difficile-associated infections clinical progress and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study index patient and sars outbreak in hong kong airborne transmission of communicable infection-the elusive pathway sars: experience at prince of wales hospital, hong kong we gratefully acknowledge the assistance of xinghua huang (shanghai jiao tong university, shanghai) and hua qian (university of hong kong, hong kong sar) with cfd simulations and field measurements.financial support. research grants council of the hong kong special administrative region, china (project no. hku 7020/02e to y.l.).conflicts of interest. all authors: no conflicts. key: cord-266775-4npowkkz authors: xu, jun; zhong, shuqing; liu, jinghua; li, li; li, yong; wu, xinwei; li, zhijie; deng, peng; zhang, jingqiang; zhong, nanshan; ding, yanqing; jiang, yong title: detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis date: 2005-10-15 journal: clin infect dis doi: 10.1086/444461 sha: doc_id: 266775 cord_uid: 4npowkkz background. previous studies have shown that common human coronavirus might be neurotropic, although it was first isolated as a pathogen of the respiratory tract. we noticed that a few patients with severe acute respiratory syndrome (sars) experienced central nervous symptoms during the course of illness. in the present study, we isolated a sars coronavirus strain from a brain tissue specimen obtained from a patient with sars with significant central nervous symptoms. methods. using transmission electronic microscopy and nested reverse transcription–polymerase chain reaction, the causative pathogen was identified in cultures of a brain tissue specimen obtained from the patient with sars. histopathologic examination of the brain tissue was performed using the methods of immunohistochemistry analysis and double immunofluorescence staining. fifteen cytokines and chemokines were detected in the blood of the patient with sars by means of a bead-based multiassay system. results. a fragment specific for sars human coronavirus was amplified from cultures of the brain suspension, and transmission electronic microscopy revealed the presence of an enveloped virus morphologically compatible with a coronavirus isolated in the cultures. pathologic examination of the brain tissue revealed necrosis of neuron cells and broad hyperplasia of gliocytes. immunostaining demonstrated that monokine induced by interferon-γ (mig) was expressed in gliocytes with the infiltration of cd68(+) monocytes/macrophages and cd3(+) t lymphocytes in the brain mesenchyme. cytokine/chemokine assay revealed that levels of interferon-γ–inducible protein 10 and mig in the blood were highly elevated, although the levels of other cytokines and chemokines were close to normal. conclusions. this study provides direct evidence that sars human coronavirus is capable of infecting the central nervous system, and that mig might be involved in the brain immunopathology of sars. the causative pathogen of severe acute respiratory syndrome (sars) has been identified as a new member of the coronavirus family that exhibits a broad range of hosts, infecting many mammalian and avian species and causing upper respiratory, gastrointestinal, hepatic, and cns diseases [1, 2] . two known human coronaviruses, 229e and oc43, which cause up to one-third of all cases of common cold, were also found to infect the cns [3] . our recent study of the sars epidemic found that the spike protein, a surface antigen determining the tropism of coronavirus, had the strongest response to positive selection pressure [4] . we noticed that a few patients with sars in hospitals in guangzhou city, china, exhibited central nervous symptoms during the course of their illnesses. lau and colleagues [5] reported that a csf sample obtained from a 32-year-old woman with sars tested positive for sars coronavirus (sars-cov) by rt-pcr, indicating that sars-cov might cause an infection in the cns of patients with sars. in the present study, we isolated a sars-cov strain from a brain tissue specimen obtained during autopsy from a patient with sars who became severely sick and showed significant central nervous symptoms during the course of his illness. furthermore, we investigated the immunopathological mechanism of brain damage in the patient with sars. in this study, we detected a high level of monokine induced by ifn-g (mig), a member of cxc family [6, 7] , in the patient's brain and found that gliocytes were a major source for mig production in the brain. all results suggested that mig was involved in the immunopathology of the brain on invasion of sars-cov. a 39-year-old doctor who was in charge of treatment of patients with sars in the respiratory intensive care unit of the chest hospital (guangzhou, china) developed a sudden onset of fever, chills, malaise, headache, dizziness, and myalgia and was hospitalized on 2 april 2003. physical examination showed a temperature of 38.5њc, clear lungs, and no obvious focus of infection. his fever resolved with treatment with ribavirin and methylprednisolone (dosage, 120 mg per day), and his condition improved during the first week of treatment. a chest radiograph obtained on day 11 after the onset of symptoms revealed left lower lobe infiltrates. a complete blood count demonstrated an elevated wbc count of cells/l, an 9 16.4 ϫ 10 increased level of neutrophils ( cells/l), and lym9 15.5 ϫ 10 phopenia (lymphocyte count, cells/l). this suggests 9 0.4 ϫ 10 that the condition of the patient with sars was complicated by a secondary bacterial infection. treatment with methylprednisolone was switched to 160 mg per 12 h intravenously, coincident with the initiation of antibiotic therapy. after initiation of the treatment mentioned above, his condition gradually improved during the fourth week after onset, at which time a series of chest radiographs showed obvious resolution of the left lower lobe consolidation. his total wbc count returned to a normal level, but lymphopenia still persisted. treatment with methylprednisolone was then reduced to 40 mg per 12 h. for the patient's complaint of obscured monocular vision, eye-ground examination was performed and revealed an exudation around the visual yellow zone on day 26 after onset of illness (27 april) . two days later, the patient experienced progressive central nervous symptoms, including dysphoria, vomiting, and deliria, and was found to have progression of left lower lobe consolidation, for which he was treated with noninvasive ventilation and methylprednisolone at a dosage of 80 mg per 12 h, coincident with initiation of treatment with ceftazidime (fortum; glaxo wellcome) and immunoglobulin. despite the management described above, his condition deteriorated, and a series of chest radiographs obtained during the fifth week after onset showed progressive bibasilar infiltration with severe leucopenia (wbc count, /l), lympho-9 0.86 ϫ 10 penia (lymphocyte count, cells/l), and depressed 9 0.11 ϫ 10 marrow. after receiving intravenous sedative, the patient developed coma on day 33 of illness, after which he was transferred into the respiratory intensive care unit of the guangzhou institute of respiratory diseases (guangzhou, china). a ct scan of the brain revealed broad encephalic pathological changes of probably ischemia and necrosis and brain edema (figure 1). by day 2 after admission to the guangzhou institute of respiratory diseases, the patient developed breathness with slowing heartthrob; thus, he underwent intubation and received mechanical ventilation. his condition rapidly deteriorated; he experienced multiorgan dysfunction syndrome, and brain herniation occurred. he died on day 3 after his admission to the guangzhou institute of respiratory diseases (35 days after the onset of illness). serum specimens obtained from the patient at early and later phases of the illness were forwarded to the centers for disease control and prevention (guangzhou, china), where the diagnosis of sars was confirmed by elisa and indirect fluorescence assay [8] . full autopsy was performed on the patients with sars. a detailed pathologic examination was performed on the brain tissue obtained during autopsy. a paraffinembedded block of the brain tissue was sectioned at a thickness of 5 mm and dewaxed according to standard procedures. immunohistochemistry and immunofluorescence staining. histopathologic evaluation was performed on the specimen of brain tissue obtained during autopsy. immunostaining was performed with monoclonal antibodies to n protein of sars-cov (supplied as a gift from dr. xiaoyan che, zhujiang hospital, southern medical university, china), inf-g-inducible protein 10 (ip-10; abcam), cd3 and cd68 (dako), and a polyclonal antibody to mig (abcam). to identify the cell type with expression of mig, double immunofluoresence staining was performed with a primary polyclonal antibody to mig plus a monoclonal antibody to glial fibrillary acidic protein (chemicon international) and secondary goat antirabbit antibody conjugated with tetramethylrhodamine isothiocyanate and goat antimouse antibody conjugated with fluorescein isothiocyanate (sigma-aldrich). we inoculated the suspension of the brain tissue onto 2 cell lines: vero-e6 (c1008; atcc) and human embryo lung fibroblast. the initial cytopathic effect (cpe) was observed between days 3 and 5. the cpe was a refractive appearance of cell rounding followed by cell detachment and quickly spread to the entire cell monolayer within 24-48 h after the initiation of cpe. specimens were prepared for electronic microscopy by fixing a washed cell pellet with 2.5% glutaraldehyde and embedding it with epoxy resin. rt-pcr. nucleic acids were purified from the supernatants of cell cultures with cpe using a nucleic acid extraction kit (amplimedical spa-bioline division). the cdna synthesis from the extracted rna was performed using a reverse-transcription kit (amplimedical spa-bioline division). the procedure is based on the reverse-transcription reaction utilizing the random primer technique. a nested rt-pcr was performed in accordance with the protocol developed by dr. christian drosten with outer primers 5 -atgaattaccaagtcaatgg-ttac-3 and 5 -cataaccagtcggtacagcta-3 and innerprimers 5 -gaagctattcgtcacgttcg-3 and 5 -ctg-tagaaaatcctagctgg ag-3 [8] . cytokine/chemokine assay. quantification of multiple cytokines/chemokines was performed using a liquichip work-station (qiagen gmbh), which is a bead-based system for immunoassays that allows simultaneous assays of multiple analytes from a single sample [9] . a blood sample obtained from the patients with sars on the day before death was used for the cytokine/chemokine assay. the 15 cytokines/chemokines assayed in this study included the following: il-1b, il-2, il-4, il-6, il-8, il-10, il-12, tnf-a, ifn-g, granulocyte macrophage-colony-stimulating factor, mig, ip-10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1a, and regulated on activation, normal t cell expressed and secreted (rantes). serum samples were obtained from 10 healthy control subjects aged 22-51 years. samples were analyzed on the liquichip system (qiagen gmbh), according to the manufacturer's instruction. approval of the local ethics committees and informed consent were obtained. general laboratory examination revealed a peak c-reactive protein level of 418.0 mg/l and an increase in the lactate dehydrogenase level to 1233.0 u/l between days 1 and 35 after the onset of illness. detailed information is shown in table 1. histopathologic examination. histopathologic examination revealed invasive aspergillus pneumonia with multiple abscess, constitutional diffuse mycohemia, and fungal multiabscess in multiple organs. pathologic examination of the brain tissue specimen under microscope revealed neuron denaturation and necrosis, broad gliocytes hyperplasia with gliosome formation, and encephalic edema (figure 2). the result of the nested rt-pcr showed that a predicted cdna fragment (bni-1) of ∼108 bp specific for sars-cov [8] was amplified from vero-e6 cell culture inoculated with the brain suspension, but not from the culture that included medium only (figure 3a). examination by transmission electronic microscopy revealed the presence of enveloped virus particles with a diameter of ∼80-90 nm and a surface morphology compatible with a coronavirus (figure 3b) in the culture showing cpe [8] . immunohistochemistry and double immunofluoresence staining. immunohistochemistry staining of gliocytes and neurocytes obtained from the brain of the patient with sars revealed them to be positive for n proteins (figure 4a), but those obtained from a patient who died in a traffic accident were not (figure 4b). we found that mig, but not ip-10, expressed with infiltration of cd68 + monocytes/macrophages and cd3 + t lymphocytes in the brain mesenchyme of the patient. double immunofluoresence staining revealed that mig was mainly expressed in gliocytes (figure 5). immunohistochemistry stains for n protein of severe acute respiratory syndrome (sars) coronavirus (sars-cov) in a specimen of brain tissue obtained from the patient with sars during autopsy. a monoclonal antibody against n protein of sars-cov and the secondary antibody of horseradish peroxidase (hrp) conjugated goat antimouse were used for the immunohistochemical staining of brain tissue specimens obtained from the patient with sars (a; original magnification, ϫ200) and a patient who died in a traffic accident (b; original magnification, ϫ200). staining was performed with diaminobenzidine (brown). arrows, positive staining cells. figure 6 , the levels of il-1b, il-2, il-4, il-6, il-8, il-10, il-12, tnf-a, ifn-g, granulocyte macrophage-colony-stimulating factor, rantes, macrophage inflammatory protein 1a, and monocyte chemoattractant protein 1 were close to those of the control subjects, but the levels of ip-10 and mig in the blood of the patient with sars were highly elevated. on review of the patient's vital record, we found that his general condition was nearly normal, and a chest radiograph showed resolving left lower pulmonary infiltration when he developed initial central nervous symptoms with an exudation around the visual yellow zone, indicating that the pathologic change in the brain was independent of pulmonary superinfection in the patient. in addition to findings of ct scanning indicating brain damage, pathologic examination revealed gliocytes hyperplasia, neuron denaturation, and necrosis combined with a striated encephalomalacia, suggesting that a chronic progressive viral cerebritis was present in the patient during the course of illness. neuroinvasion by sars-cov was also directly evidenced by the viral morphology observed under electronic microscope, genetic identification, and the viral antigen (n protein) found in the brain. previous studies found that coronavirus might be neurotropic, neuroinvasive, and neurovirulent, although it was first isolated as a pathogen of the respiratory tract [1, 3] . at least 3 routes, including olfactory nerve, a hematogenous route, and lymphatic systems could be used by coronavirus to gain access to the cns [10] [11] [12] . the cell lines of astrocytoma, neuroblastoma, neuroglioma, and oligodendrocyte were all susceptible to the infection of human coronaviruses [13] [14] [15] [16] . mouse hepatitis virus, a murine coronavirus, has been found to induce demyelinating disease of the cns [17, 18] . moreover, glass and colleagues [19] recently reported a model of sars-cov infection in c57bl/6 mice that demonstrated that sars-cov was able to infect the brain. the finding that sars-cov infected the brain of the patient with sars was consistent with findings of previous reports on neuroinvasion of the coronavirus family [13] [14] [15] [16] [17] [18] [19] . genomic comparison of strains of sars-cov showed that immunological pressures influenced the evolution of sars-cov in the human population by a change of the spike protein [4] . however, dna sequence analysis showed that there was no variation with significant substitution of amino acids on the spike protein of the sars-cov isolated from this patient with the primary isolates of sars-cov [4] . it is possible that sars-cov is characterized by behavioral abnormalities similar to those in human coronaviruses oc43 and 229e, which have the capacity to infect the cns but do not necessarily yield severe pathological symptoms [13, 16] , because local immune surveillance limits viral replication on a large scale [20] . on the basis of the consideration that a progressive lymphopenia and leukopenia developed [21, 22] during the disease course of this patient, we speculate that local immune surveillance in his brain was removed by severe immunodepression [21] [22] [23] , leading to the amplification of infectious viruses. with regard to the superinfection with invasive aspergillus in the brain and other organs of the patient, we think that severe immunodepression resulting from the damage to the immune system induced by sars-cov infection, combined with high-dosage treatment with a corticosteroid, provided access for conditional pathogens, causing a superinfection with invasive aspergillus in multiple organs [24] . in this case, aspergillus infection might have synergized or deteriorated the harmful effects of the sars-cov infection in the brain [24] , which brought about a more complicated clinical process. our previous study showed that the ip-10 level, one of the non-elr cxc chemokines, was augmented markedly in the blood of patients in the early stage of sars and remained at a high level during the course of illness [25] . consistent with that finding, this study showed that ip-10 in the blood of the patient with sars was also highly increased. furthermore, we found that the level of mig, another cxcr3 chemokine, was highly elevated in the blood of the patient with sars. in contrast to our finding, wong et al. [26] reported that mig could not be detected with significant increase in the blood of patients with sars. the difference between the findings of wong and colleagues and our results provides evidence for the hypothesis figure 6 . the profile of cytokines and chemokines in the blood of the patient with severe acute respiratory syndrome (sars). each cytokine/ chemokine is expressed as a ratio of that of the patient with sars to the average among control subjects. of all the detected cytokines/chemokines, only ifn-g-inducible protein 10 (ip-10) and monokine induced by ifn-g (mig) showed highly increased production. that the high level of mig in the blood of this patient with sars was associated with the invasion of sars-cov in the brain tissues. it has been reported that both mig and ip-10 were involved in host defense and immune damage by attracting activated t cells, nk cells, and monocytes that express cxcr3 [27] . we could not detect a significant elevation in the ip-10 level, but we did detect a high expression of mig in the brain. this result is different from findings of our previous study, which revealed a high level of ip-10 in the lungs of patients with sars. the results of histopathologic examination and immunostaining indicated that the invasion of sars-cov of brain tissues brought about the induction of mig in gliocytes, which in turn attracted cd68 + monocytes/macrophages and cd3 + t lymphocytes to the sites of virus infection [27, 28] . infiltrated immune cells should be helpful to limit sars-cov infection of the brain. however, because the virus could not be eliminated effectively while the patient was immunodepressed, a high number of cytokines/chemokines released in response to sars-cov infection would contribute to the tissue damage [29, 30] . the brain damage caused by sars-cov infection might take the same immunopathological process that happened in the lung tissues of patients with sars [25] , although different chemoattractant factors were involved. in conclusion, to our knowledge, our findings are the first direct evidence that sars-cov has the ability to infect the cns and that the infection causes immunopathological damage leading to a more serious and complicated clinical syndrome. mig, but not ip-10, may be involved in the brain immunopathology by attracting immune effector cells to the site of virus infection. however, the specific mechanism for mig as a key mediator in the brain damage induced by sars-cov infection needs further investigation. the cellular and molecular pathogenesis of coronaviruses virus-receptor interactions and interspecies transfer of a mouse hepatitis virus neuroinvasion by human respiratory coronaviruses molecular evolution of the sars coronavirus during the course of the sars epidemic in china possible central nervous system infection by sars coronavirus cxcr3-binding chemokines: novel multifunctional therapeutic targets a potential role for cxcr3 chemokines in the response to ocular hsv 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lesions from rats with coronavirus-induced demyelinating encephalomyelitis experimental demyelination induced by coronavirus jhm (mhv-4): molecular identification of a viral determinant of paralytic disease mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice chemokine expression and viral infection of the central nervous system: regulation of host defense and neuropathology sars: understanding the coronavirus: apoptosis may explain lymphopenia of sars expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome fatal aspergillosis in a patient with sars who was treated with corticosteroids invasive pulmonary aspergillosis associated with influenza characterization of cytokine/chemokine profiles of severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome expression of mig (monokine induced by interferon-g) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system chemokines and chemokine receptors in leukocyte trafficking bystander cd8 t cell-mediated demyelination after viral infection of the central nervous system mig, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo potential conflicts of interest. all authors: no conflicts. key: cord-260274-c3586tp6 authors: somers, emily c; eschenauer, gregory a; troost, jonathan p; golob, jonathan l; gandhi, tejal n; wang, lu; zhou, nina; petty, lindsay a; baang, ji hoon; dillman, nicholas o; frame, david; gregg, kevin s; kaul, dan r; nagel, jerod; patel, twisha s; zhou, shiwei; lauring, adam s; hanauer, david a; martin, emily; sharma, pratima; fung, christopher m; pogue, jason m title: tocilizumab for treatment of mechanically ventilated patients with covid-19 date: 2020-07-11 journal: clin infect dis doi: 10.1093/cid/ciaa954 sha: doc_id: 260274 cord_uid: c3586tp6 background: severe covid-19 can manifest in rapid decompensation and respiratory failure with elevated inflammatory markers, consistent with cytokine release syndrome for which il-6 blockade is approved treatment. methods: we assessed effectiveness and safety of il-6 blockade with tocilizumab in a single-center cohort of patients with covid-19 requiring mechanical ventilation. the primary endpoint was survival probability post-intubation; secondary analyses included an ordinal illness severity scale integrating superinfections. outcomes in patients who received tocilizumab compared to tocilizumab-untreated controls were evaluated using multivariable cox regression with propensity score inverse probability weighting (iptw). results: 154 patients were included, of whom 78 received tocilizumab and 76 did not. median follow-up was 47 days (range 28-67). baseline characteristics were similar between groups, although tocilizumab-treated patients were younger (mean 55 vs. 60 years), less likely to have chronic pulmonary disease (10% vs. 28%), and had lower d-dimer values at time of intubation (median 2.4 vs. 6.5 mg/dl). in iptw-adjusted models, tocilizumab was associated with a 45% reduction in hazard of death [hazard ratio 0.55 (95% ci 0.33, 0.90)] and improved status on the ordinal outcome scale [odds ratio per 1-level increase: 0.58 (0.36, 0.94)]. though tocilizumab was associated with an increased proportion of patients with superinfections (54% vs. 26%; p<0.001), there was no difference in 28-day case fatality rate among tocilizumab-treated patients with versus without superinfection [22% vs. 15%; p=0.42]. staphylococcus aureus accounted for ~50% of bacterial pneumonia. conclusions: in this cohort of mechanically ventilated covid-19 patients, tocilizumab was associated with lower mortality despite higher superinfection occurrence. sars cov-2, the virus responsible for covid-19, has caused a global pandemic with over 6.7 million infections and 390,000 deaths as of june 5, 2020. up to 20% of patients with covid-19 develop severe illness characterized by worsening dyspnea and the need for supplemental oxygen. [1] patients may further progress to respiratory failure, acute respiratory distress syndrome (ards), multi-organ dysfunction, and death. hyperinflammation may contribute to this deterioration, resulting in elevations in c-reactive protein, ferritin, lactate dehydrogenase (ldh), d-dimer, and various pro-inflammatory cytokines including interleukin-6 (il-6). [1] [2] [3] [4] [5] [6] this profile resembles that seen in cytokine release syndrome (crs) associated with chimeric antigen receptor (car) t-cell therapy and hemophagocytic lymphohistocytosis. [4, 5, 7] in crs, il-6 blockade with tocilizumab has resulted in rapid improvement in respiratory and hemodynamic parameters, [8] and the united states food and drug administration has approved its use for car t-cell associated severe or lifethreatening crs. as a result, adjunctive therapy with either il-6 receptor antagonists (tocilizumab, sarilumab), or il-6 antagonists (siltuximab) has been proposed as treatment for severe, progressive covid-19. while multiple case series have suggested a potential role for tocilizumab [9] [10] [11] [12] [13] or siltuximab (preprint), [14] these reports are hampered by incomplete reporting, short durations of follow-up, and lack of control groups. furthermore, infection is a concern with il-6 blockade and cases of viral myocarditis [15] and candidemia [16] with tocilizumab have been reported. as secondary infection has been associated with increased mortality in covid-19, [3] controlled data are necessary to evaluate the risks and benefits of these therapies. at our institution, il-6 blockade with tocilizumab is considered for patients with severe covid-19 and suspected hyperinflammation based on rapidly worsening respiratory status and elevated inflammatory markers, with the majority of usage occurring in patients requiring mechanical ventilation. using our covid-19 rapid response registry infrastructure, we performed an observational study of outcomes in patients with covid-19 requiring mechanical ventilation, comparing those treated with tocilizumab with those who were not. a c c e p t e d m a n u s c r i p t 6 response registry for clinical characterization of persons with sars-cov-2 infection. the registry includes core items from the international severe acute respiratory and emerging infection consortium (isaric) clinical characterization protocol. [17, 18] this analysis follows strobe recommendations. [19] ethics approval was obtained by the institutional review board of the university of michigan (hum00179261). patients were eligible for inclusion in this analysis if they were admitted to michigan medicine from march 9-april 20, 2020 for severe covid-19 pneumonia, had a reverse-transcriptase polymerase chain reaction positive sars-cov-2 test, and required invasive mechanical ventilation (the first covid-19 cases in michigan were identified in early march 2020). follow-up continued through may 19, 2020. patients were excluded if they were younger than 16 years, were intubated for conditions unrelated to covid-19, or were enrolled into a randomized controlled trial (rct) for sarilumab. this analysis focuses on comparative outcomes of mechanically ventilated patients who received tocilizumab and those who did not. untreated patients who died prior to the opportunity to receive tocilizumab treatment per institutional criteria (within 48 hours of intubation) were excluded to minimize immortal time bias. [20] tocilizumab exposure during the study period, preference was given to enrollment in an il-6 inhibitor (sarilumab) clinical trial. however, given strict trial eligibility criteria and protocol requirements (e.g., timed phlebotomy and repeated sars-cov-2 testing), tocilizumab was considered in patients ineligible for the trial or when trial enrollment was not feasible due to logistical constraints (e.g., outside of enrollment hours or on non-study units). criteria for tocilizumab usage were developed by the institutional antimicrobial stewardship program and division of infectious diseases. in general, tocilizumab was recommended for consideration in patients with rapid respiratory deterioration and evidence of hyperinflammation. guidance was slightly modified during the study period based on drug availability, whether the sarilumab trial was active, and experiences of the treating team. none of these changes were substantial (usage criteria as of may 19, 2020 a c c e p t e d m a n u s c r i p t 7 adherence to this guidance was not enforced or mandatory, as within our large infectious diseases division providers had varying views on the use of investigational or repurposed agents such as tocilizumab. the language in the guidance was intentionally non-prescriptive, saying that tocilizumab 'may be considered…' and cautioning that '…the evidence for benefit is weak, and a risk for potential harm exists'. ultimately, individualized decisions on tocilizumab usage were made by the attending infectious diseases physician. the standard tocilizumab dose was 8 mg/kg (maximum 800 mg) x 1; additional doses were discouraged. the primary outcome was survival probability after intubation. a secondary endpoint assessed status at day 28 on a 6-level ordinal scale of illness severity, including bloodstream infection and pneumonia: (1) discharged alive, (2) hospitalized/off ventilator without superinfection, (3) hospitalized/off ventilator with superinfection, (4) hospitalized/mechanically ventilated without superinfection, (5) hospitalized/mechanically ventilated with superinfection, (6) deceased. data were obtained via electronic health record queries and manual abstraction, and included demographics, comorbidities, hospitalization dates, transfer status, laboratory values, microbiology results, concomitant medications, mechanical ventilation dates, oxygenation variables, and discharge status. spo2/fio2 was substituted for pao2/fio2, which has been validated in patients with ards [21] . all positive blood and respiratory cultures were assessed by an infectious diseases physician to adjudicate infection versus colonization. infections were included if they occurred after intubation and >48 hours after hospitalization. additionally, only infections occurring after administration of tocilizumab were considered in the treatment group. for patients who transferred from an outside hospital, length of stay, intubation date, and tocilizumab administration characteristics at that facility were manually abstracted from admission notes. for those intubated at michigan medicine, the lowest pao2/fio2 ratio in first twelve hours after intubation was also recorded. relevant laboratory values at times of presentation and intubation were abstracted. based on available evidence and lack of enrolling clinical trials at local onset of the pandemic, hydroxychloroquine 600 mg every twelve hours x2 doses, then 200 mg every 8 hours was recommended as standard management at the beginning of the study period. once remdesivir studies were activated, hydroxychloroquine was formally removed from our guidelines on march 26, 2020, and treatment with hydroxychloroquine was rare after these changes. adjunctive corticosteroid use was generally not recommended, but use in patients with acute respiratory distress syndrome was at the discretion of the critical care physician. descriptive characteristics were provided using means and standard deviations or median and interquartile range for continuous variables, and frequencies and percentages for categorical variables. kaplan-meier survival curves were used to describe post-ventilator onset outcomes and time-varying stacked bar plots were applied to demonstrate the 6-level ordinal outcome by elapsed day. univariate prediction ability of each covariate on the time to death and ordinal outcome at day 28 were explored using cox proportional hazards models and proportional odds models, respectively. proportional odds assumption was assessed via score test. multiple imputation [22] was used to impute missing laboratory values for inclusion in sensitivity analyses: twenty-five imputations by fully conditional specification were made based on age, sex, race, ethnicity, transfer status, history of hypertension, congestive heart failure, chronic pulmonary disease, and chronic renal disease. to address non-randomized treatment allocation, we calculated propensity scores by multivariable logistic regression with tocilizumab treatment as the binary outcome and potential confounding factors associated with both outcome and treatment assignment. using such propensity scores, we applied the inverse probability of treatment weights (iptw) to create a pseudo study cohort, where the weighted version can balance off the covariate bias and mimic a randomized treatment assignment situation: the ipt weights for tocilizumab treated patients=1/p(treated); for untreated patients=1/[1-p(treated)]. [23] [24] [25] all analyses were conducted in univariate and multivariable fashion, and before and after inverse probability of treatment of 484 cases admitted during the study period for covid-19, 34 were excluded based on enrollment in a sarilumab rct (nct04315298). also excluded were 293 who did not require mechanical ventilation, 2 untreated patients who died within 24 hours of intubation, and 1 infant. thus, this study included 154 patients requiring mechanical ventilation: 78 treated with tocilizumab and 76 untreated (figure 1) . median follow-up time was 47 days (range 28-67). patient characteristics as a function of treatment are shown in table 1 . in general, the two groups were well-balanced, and patients were similar with regards to sex, race, most comorbidities, and concomitant therapies. tocilizumab-treated patients were younger (mean 55 vs. 60 years; p=0.05) and less likely to have either chronic pulmonary disease (10% vs. 28%; p=0.006) or chronic kidney disease (35% vs. 49%; p=0.08). the majority of patients in both groups were transfers from an outside facility, with a higher number of transfers (74% vs. 58%; p=0.04) in the untreated group. laboratory values at time of intubation are shown in table 1 patients who received tocilizumab were more than twice as likely to develop a superinfection than untreated controls (54% vs. 26%; p<0.001), driven primarily by a large increase in ventilatorassociated pneumonia (45% vs. 20%; p<0.001); table 2 . there was no difference between groups with regards to timing of infection, incidence of bloodstream infections, or development of more than one infection. the causative microbiology of superinfections was similar between groups. staphylococcus aureus accounted for ~50% of the bacterial pneumonias in both groups. case fatality a c c e p t e d m a n u s c r i p t 11 rates at day 28 were similar among tocilizumab-treated patients who had a superinfection and those who did not [8/37 (22%) vs. 6/41 (15%); p=0.42]. stratified by treatment group, figure 3a in this observational, controlled study of patients with severe covid-19 necessitating mechanical ventilation, receipt of tocilizumab was independently associated with improved survival. importantly however, tocilizumab was also associated with increased incidence of secondary bacterial pneumonia. while this did not appear to negatively influence ultimate outcome, and case fatality rates were similar in infected and uninfected tocilizumab-treated patients, this finding highlights the need for adequately powered randomized controlled trials further evaluating efficacy and safety of tocilizumab in covid-19. respiratory failure in severe covid-19 is frequently characterized by high serum il-6 concentrations. [26] excessive il-6 can induce lung epithelial cells to increase inflammatory responses, leading to increased macrophage response and ultimately pulmonary damage. il-6 may also be a significant contributor to thrombosis, having been associated with both tissue and vascular a c c e p t e d m a n u s c r i p t 12 endothelial cell injury, and contributing to platelet aggregation and angiotensin ii microvascular dysfunction. [27, 28] conversely, as a critical cytokine in organizing t-cell responses to infections, il-6 may play a beneficial role in covid-19. it may suppress viral reactivation, [29] protect against superinfection, and facilitate lung repair and remodeling after viral injury. [30] thus, our approach was to administer tocilizumab in patients who were rapidly desaturating or recently intubated, in an attempt to optimize the timing of administration for maximal benefit. our dosing strategy (single, high dose of 8 mg/kg) was an attempt to saturate receptors to rapidly inhibit il-6 signaling but also allow more rapid clearance in order to not interfere with tissue remodeling and limit immunosuppression. our results support these hypotheses. given the heterogeneity in tocilizumab treatment decisions between providers at our institution, the two groups in this analysis were largely comparable with regard to factors impacting covid-19 outcomes. although there were slight imbalances with regards to age, baseline d-dimer, crp, comorbid chronic pulmonary disease, and transfer status, we utilized rigorous methods for observational data accounting for these factors and treatment propensity. tocilizumab remained associated with better outcomes across modeling strategies. furthermore, results remained consistent across various sensitivity analyses, including when patients were stratified according to d-dimer and crp thresholds previously associated with mortality [6] , by outside hospital transfer/direct admission status, and when restricted to various age groups. in addition to the survival advantage, receipt of tocilizumab was associated with improvement on a six-point ordinal scale that incorporated mechanical ventilation, development of superinfection, and discharge from the hospital (or ~0.6; p≤0.03 for ipt-weighted models). this improvement in illness severity level with receipt of tocilizumab is reflected in the statistically significant increase in patients discharged home over the study period (56% vs. 40%; p=0.04); while 17 patients in each group remained in the hospital at the end of follow-up, only 3 of 17 (18%) of tocilizumab-treated patients remained on the ventilator, compared to 8/17 (47%) of untreated controls (figure 3b ). this consistent advantage across the ordinal scale provides support for the observed benefit associated with tocilizumab in this cohort and furthermore has significant resource conservation implications. a c c e p t e d m a n u s c r i p t 13 importantly, these data also reinforce concerns with superinfection risk due to il-6 inhibition. to date, the risk of superinfection in mechanically ventilated patients with severe covid-19 remains poorly described and the incremental risk associated with a single dose of tocilizumab is not well characterized. we demonstrated that superinfection was common in this population, with 39% developing a pneumonia or bloodstream infection. furthermore, tocilizumab was associated with higher occurrence of infection (54% vs. 26%; p<0.001), driven primarily by the development of ventilator-associated bacterial pneumonia in 45% of patients receiving tocilizumab. interestingly, we also identified an association between severe covid-19 infection and staphylococcal pneumonia, as roughly half of the cases in both the tocilizumab and control group were due to s. aureus. although these data are observational, several strengths of the study warrant comment. first, this analysis utilizing a rapid response registry informed by an internationally-designed clinical characterization protocol, [18] represents the first well-controlled, comparative analysis assessing safety and effectiveness of tocilizumab for severe covid-19. in order to address potential confounding by indication or other imbalances between groups, propensity scoring and multivariable models were utilized, as well as sensitivity analyses. across various analytic strategies, results consistently indicated benefit associated with tocilizumab. additionally, median follow-up time for the cohort was 47 days (range 28-67), with all patients followed for at least 28 days, representing a substantially longer observation period than many covid-19 treatment studies to date, and indicative of sustained benefit. furthermore, all secondary infections were reviewed by an infectious diseases physician to ensure accurate reporting. however, this analysis is not without limitation. first and foremost, randomized controlled trial data will be critical for confirming the perceived benefits from this observational study and better quantify risks. second, there were incomplete data for laboratory variables, though we used contemporary methods for imputing missing data. third, for patients transferred from outside hospitals, variations in the initial period of care (prior to transfer) cannot be fully or consistently characterized; however, the vast majority of transfers occurred directly from outside emergency departments that were over capacity. it should also be noted that for transfer patients, we do not have information on tocilizumab usage criteria at the outside hospitals, though only three patients received tocilizumab outside of our institution. fourth, we focused on the impact of tocilizumab 8 mg/kg x 1 in mechanically ventilated patients. this study does not address the potential role of a c c e p t e d m a n u s c r i p t 14 tocilizumab earlier in illness for preventing mechanical ventilation, the optimal dose of tocilizumab, the potential utility of multiple doses, or the role of il-6 serum concentrations (which were not routinely available) in predicting tocilizumab response, all of which are important questions that warrant further investigation. further, though tocilizumab administration was guided by institutional criteria, usage in this clinical care setting was not dictated by a firm study protocol, and therefore not completely standardized. finally, while all patients in our cohort had a minimum follow-up time of 28 days, additional follow-up will be valuable to determine the full course of hospitalization for the 34 remaining inpatients, and to characterize long-term sequelae for survivors in this cohort. in conclusion, tocilizumab was associated with improved survival, despite higher occurrence of each row represents changes in individual patient status from time of onset of mechanical ventilation until event (death) or end of the study period (may 19, 2020). horizontal lines correspond to elapsed time, with colors corresponding to clinical status of the patient. solid circles represent death, and hollow circles represent discharge from hospital (alive). the middle panel indicates the most recent patient status. grey vertical lines mark 28-day follow-up. m a n u s c r i p t 19 clinical characteristics of coronavirus disease 2019 in china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china cytokine release syndrome in severe covid-19 covid-19: consider cytokine storm syndromes and immunosuppression factors associated with hospital admission and 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in covid-19 induced pneumonia and macrophage activation syndrome-like disease novel role of t cells and il-6 (interleukin-6) in angiotensin ii-induced microvascular dysfunction effects of il-1β, il-6 and il-8 on erythrocytes, platelets and clot viscoelasticity the role of interleukin 6 during viral infections il-6 ameliorates acute lung injury in influenza virus infection a c c e p t e d m a n u s c r i p t 16 m a n u s c r i p t 21 b median (interquartile range), otherwise listed as n (%) c for patients intubated at michigan medicine, value represents the worst pao2/fio2 value within 12 hours of intubation d chronic pulmonary includes: chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, bronchiectasis, interstitial lung disease, pre-existing requirement for long term oxygen therapy e news score calculated in subset of patients who were not on mechanical ventilation on transfer or intubated in the emergency department at michigan medicine (n = 37 tocilizumab, 24 key: cord-278259-pbnnp9i1 authors: choi, eun hwa; lee, hoan jong; kim, sun jung; eun, byung wook; kim, nam hee; lee, jin a; lee, jun ho; song, eun kyung; park, so hee kim1 ji yong; sung, ji yeon title: the association of newly identified respiratory viruses with lower respiratory tract infections in korean children, 2000–2005 date: 2006-09-01 journal: clin infect dis doi: 10.1086/506350 sha: doc_id: 278259 cord_uid: pbnnp9i1 background. this study was performed to evaluate the associations of newly recognized viruses, namely, human metapneumovirus (hmpv), human coronavirus (hcov)–nl63, and human bocavirus (hbov) with lower respiratory tract infections (lrtis) in previously healthy children. methods. to determine the prevalences of 11 viruses—respiratory syncytial virus (rsv), adenovirus, rhinovirus, parainfluenza viruses (pivs) 1 and 3, influenza viruses a and b, hmpv, hcov, hcov-nl63, and hbov—among infants or children with lrtis, in association with their epidemiologic characteristics, we performed multiplex reverse-transcriptase polymerase chain reaction on nasopharyngeal aspirates obtained from 515 children 5 years old with lrtis during the period 2000–2005. results. viruses were identified in 312 (60.6%) of the 515 patients. rsv was detected in 122 (23.7%), hbov in 58 (11.3%), adenovirus in 35 (6.8%), piv-3 in 32 (6.2%), rhinovirus in 30 (5.8%), hmpv in 24 (4.7%), influenza a in 24 (4.7%), piv-1 in 9 (1.7%), influenza b in 9 (1.7%), and hcov-nl63 in 8 (1.6%). coinfections with 2 viruses were observed in 36 patients (11.5%). twenty-two patients (37.9%) infected with hbov had a coinfection. bronchiolitis was frequently diagnosed in patients who tested positive for rsv, piv-3, or rhinovirus, whereas influenza a, piv-1, and hcov-nl63 were commonly found in patients with croup. the age distributions of patients with viral infections differed; notably, rsv was responsible for 77% of lrtis that occurred in infants 3 months old. the number of hmpv infections peaked between february and april, whereas the number of hcov-nl63 infections peaked between april and may. conclusions. this study describes the features of lrtis associated with newly identified viruses in children, compared with those associated with known viruses. additional investigations are required to define the role of hbov in lrti. dren. rhinovirus, which generally has been considered a cause of mild upper respiratory illnesses in children and adults, is now considered to be a major cause of acute lrtis and asthma exacerbations [5] [6] [7] . however, the extent to which rhinoviruses contribute to lrtis in otherwise healthy children is unclear. more recently, new viruses, such as human metapneumovirus (hmpv) [8] and human coronavirus (hcov)-nl63 [9, 10] , have been suggested to cause lrtis in children. moreover, human bocavirus (hbov) has been identified in the respiratory tracts by molecular screening [11] , but its role as a causative agent of lrti remains to be proven. to determine the relative contributions made by these newly recognized respiratory viruses to lrtis in childhood and to characterize their epidemiologic and clinical features, over a period of 5 years (2000-2005) we performed virological studies of korean infants and children with acute lrtis. specifically, we evaluated the relative prevalences and the epidemiologic characteristics of rhinovirus, hmpv, conventional hcov, hcov-nl63, and hbov and compared these findings with those of common respiratory viruses, such as rsv, piv, adenovirus, and influenza virus. patients and respiratory specimens. the study population consisted of children р5 years old with acute lrtis (i.e., bronchiolitis, pneumonia, or croup). all illnesses were diagnosed at the seoul national university children's hospital (korea) or the seoul national university bundang hospital (korea) between september 2000 and august 2005. samples obtained from children with major risk factors other than recurrent episodes of wheezing were excluded, as were those obtained from children with hospital-related infections. diagnostic definitions were as follows [12, 13] : pneumonia required rales on auscultation or demonstration of an infiltrate by chest x-ray; bronchiolitis was characterized by a cough, tachypnea, retraction, and expiratory wheezes, often accompanied by rales; and croup required a barking cough with stridor. the principal investigator determined the clinical diagnosis on the basis of a review of medical records conducted by 5 of the investigators. the predominant clinical diagnosis was determined when 11 diagnosis was present. nasopharyngeal aspirates were prospectively collected from all subjects. specimens were obtained either at the time of visiting an emergency department or immediately following hospital admission. viral rna was detected in nasopharyngeal aspirates using rt-pcr. viral diagnosis. samples of nasopharyngeal aspirates were kept frozen at ϫ70њc. viral rna in nasopharyngeal aspirates was extracted using a qiaamp viral rna mini kit (qiagen), in accordance with the manufacturer's instructions. cdna was synthesized using random hexamers and superscript rt (invitrogen). multiplex rt-pcr assays were developed to detect 11 viruses, namely, rsv and piv-3 (panel 1), hmpv and rhinovirus (panel 2), influenza viruses a and b and piv-1 (panel 3), coronaviruses oc43 and 229e and hcov-nl63 (panel 4), and hbov (panel 5). adenovirus was detected by culture in hep-2 monolayers, because this cell culture-based assay has a diagnostic sensitivity similar to that of adenovirus-specific pcr [14] . in brief, 17.5 ml of total rna was mixed with 1 ml of random hexamers at a concentration of 15 mm and then incubated at 65њc and chilled on ice. a reaction mix of 11.5 ml containing 6 ml of 5x first strand buffer, 3 ml of 100 mm dithiothreitol, 1 ml of deoxyribonucleotide triphosphate mix at a concentration of 10 mm, 1 ml of rnase inhibitor, and 100 units of superscript ii reverse transcriptase (invitrogen) was then added. the reaction was incubated at 25њc for 10 min and at 42њc for 60 min, and after a denaturation step at 94њc for 3 min, the cdna was used as a template for subsequent pcr. the 20-ml reaction mixtures consisted of 1x geneamp pcr buffer gold (applied biosystems), 2.0 mm mgcl 2 , each deoxyribonucleotide triphosphate at a concentration of 0.2 mm, 20 pmol of primers, and 2.5 units of amplitaq gold dna polymerase (applied biosystems). after incubation for 5 min at 95њc, amplication was performed at 95њc for 1 min, 50њc for 1 min, and 72њc for 1 min. the primers used were designed in this study or modified from the published methods [15] [16] [17] [18] [19] to amplify the virus-specific genomes (table 1). amplified products were then separated on agarose gels, and virus-specific pcr products were identified. the sensitivity of the multiplex rt-pcr for 5 viruses (rsv, piv-1, piv-3, hmpv, and influenza viruses a and b) was determined for each of the targets using virus suspensions in cell culture media that were quantified in terms of tcid 50 using standard virological techniques [20] . all specimens were tested by the multiplex rt-pcr for the 11 respiratory viruses and by viral culture and immunofluorescent antigen detection for 6 viruses (rsv, piv-1, piv-3, adenovirus, and influenza viruses a and b), as described elsewhere [3] . samples were considered to be positive if the 2 pcr results using separately extracted copies of viral rna were positive or if a single positive pcr result was confirmed by viral culture or immunofluorescent antigen detection methods. each pcr included distilled water as a negative control, as well as positive controls for the corresponding copies of viral cdna in each panel. clinical database. respiratory symptoms and signs were recorded on a standardized form during the emergency department visit or while the patient was hospitalized. medical records were reviewed to determine the clinical manifestations and any underlying conditions of patients. clinical data were entered into a database by a person who did not have knowledge of virus identities. statistical analysis. the x 2 test or fisher's exact test was used to compare samples with respect to percentage of each sex, symptoms, and the clinical diagnoses, and the mann-whitney u test was used to compare the mean age at onset of illness between the various groups. analyses were performed using graphpad instat software, version 3.06 (graphpad software). a total of 2198 nasopharyngeal aspirates samples associated with a diagnosis of lrti in the previously healthy children (р5 years old) were collected during the study period. tested specimens were selected by a random number assignment from the list of archived samples. a total of 515 nonconsecutive specimens (23.4%) were chosen for rtpcr after excluding unavailable samples and repeated samples collected from the same patient. the monthly proportion of selected samples ranged from 20% to 28% of the total number of archived specimens (figure 1). of the study population, 306 (59%) were male. the mean age among children was 15.4 months; 20% were infants !4 months old, 28% were 4-12 months old, 25% were 13-24 months old, and 27% were 124 months old. the remaining 1683 samples that were not selected for this study did not differ significantly from selected samples with respect to mean age at disease onset, sex, seasonal distribution, or clinical diagnosis. the detection sensitivity of the assay for 5 viruses (rsv, piv-1, piv-3, hmpv, and influenza viruses a and b) was greatest for influenza a, with a value of 10 ϫ4 tcid 50 . similarly, the assay detected tcid 50 of rsv and piv-3 and 10 ϫ3 ϫ3 5 ϫ 10 tcid 50 of piv-1, hmpv, and influenza b. detectable viral rna concentrations for rhinovirus, coronaviruses oc43 and 229e, hcov-nl63, and hbov were 5-10 ng of the total rna extracted from the cell culture supernatants or clinical specimens. of the 515 samples tested, 312 (60.6%) were positive for any of the 11 respiratory viruses (table 2). the detection rate of multiplex rt-pcr was increased because of additional viruses that can be detected by this method and because of greater sensitivity, compared with viral culture or antigen detection methods. of these 312 virus-positive samples, 36 were also positive for additional viruses, resulting in a coinfection rate of 11.5%. virus frequencies were as follows: rsv was detected in 122 children (23.7%), hbov in 58 (11.3%), adenovirus in 35 (6.8%), piv-3 in 32 (6.2%), rhinovirus in 30 (5.8%), hmpv in 24 (4.7%), influenza a in 24 (4.7%), piv-1 in 9 (1.7%), influenza b in 9 (1.7%), hcov-nl63 in 8 (1.6%), and conventional hcov in 1 (replicase 1a gene sequencing analysis revealed this to be hcov-oc43). rhinoviruses were found to be associated with acute lrti at a relatively high frequency. the prevalence of hmpv was similar to that of influenza virus a. in contrast to these, hcov-nl63 was present at a low prevalence in the study group. seasonal distribution. the monthly distributions of detected viruses over the 5-year study period are shown in figure 1. monthly variations in percentage contributions to the diagnosed viral lrtis ranged from 0% to 88%. the number of rsv infections increased during late fall and peaked between november and january. piv-3 was prevalent from april to june, and rhinovirus was detected year-round, with a peak occurring during late summer and fall. the prevalence of hmpv increased during late winter and peaked between february and april (68% of total isolates). hmpv was not detected during the 2003-2004 epidemic period, although comparable numbers of samples were tested. hcov-nl63 was identified, with peaks occurring between april and may (44% of total isolates). clinical characteristics. the clinical features of children who were tested for these viruses are summarized in 2 ). in addition, rhinovirus accounted for a larger proportion of lrtis in young infants р3 months than adenovirus or hbov ( ). in contrast, adenovirus, hmpv, and influenza a were p ! .03 more frequently detected in children 124 months old. the clinical diagnoses of the 265 patients with lrtis associated with 8 common viruses are summarized in table 3. bronchiolitis or pneumonia was frequently observed in patients with positive results for rsv, hbov, piv-3, hmpv, or rhinovirus. of these, rsv and rhinovirus were more likely to cause bronchiolitis, whereas by our definition, hmpv showed a tendency to cause pneumonia. croup was frequently associated with influenza a, piv-1, or hcov-nl63. fever was more frequently observed in hmpv-infected children (88.2%) or hcov-nl63infected children (100%) than in those infected with rsv (54%; for both comparisons) or rhinovirus (40%; for p ! .04 p ! .03 both comparisons). the frequency of rales was similar for all viruses, whereas wheezing occurred more frequently in patients infected with rsv than in patients with an lrti associated with adenovirus or hmpv. no differences were observed between children with coinfections and single infections with respect to disease severity. characteristics of hbov infection. as shown in table 2, hbov was detected in 58 children (11.3%). of these, 22 (37.9%) were found to be coinfected with other viruses (table 2) . hbov was identified in 22 (61%) of 36 patients coinfected with 11 virus. in view of the observation that adenovirus was more frequently found in patients with mixed viral infections (13 [37.1%] of 35 adenovirus-positive samples) than were the other viruses, the above findings indicate that hbov was associated with high rates of mixed infections. viruses that were frequently found to be coinfecting with hbov were as follows, in descending order: adenovirus (7 cases), hmpv (5 cases), rsv (5 cases), and piv-3 (3 cases). hbov was identified throughout each year during the 5-year study period, with a peak (57% of total isolates) occurring from may to july, and the mean age of hbov-infected children was 22.3 months. the clinical diagnoses of 36 hbov-associated lrtis were bronchiolitis for 9 patients (25%), pneumonia for 20 patients (55.6%), croup for 3 patients (8.3%), and asthma exacerbation for 4 patients (11.1%). in this study, we evaluated the overall prevalences of 11 respiratory viruses identified by multiplex rt-pcr in a cohort of previously healthy children with acute lrtis. in addition, we compared the relative contributions and clinical features between the acute lrtis associated with the recently identified viruses (rhinovirus, hmpv, hcov-nl63, and hbov) and those caused by the previously known viruses (rsv, parainfluenza viruses, adenovirus, and influenza viruses) during 5 consecutive years, 2000-2005. since both hmpv and hcov-nl63 were recognized as being the etiologic agents of lrtis [8, 9, [21] [22] [23] , comparative studies on the relative prevalences and clinical characteristics of respiratory tract infections caused by newly identified viruses in children have been hampered, primarily because of the different sensitivities of the diagnostic tests employed [24] [25] [26] . in addition, the majority of previous studies have used molecular methods to investigate the roles of hmpv and hcov-nl63 in acute respiratory diseases by using subjects that differ with respect to the number of respiratory specimens tested or results obtained by viral culture or by antigen detection methods [22, 27] . moreover, simultaneous infections by other viruses are often documented; thus, contributions made by individual viruses to respiratory diseases may be over-or underestimated. in this study, we used multiplex rt-pcr, which has been widely used to identify recently recognized viruses, such as hmpv, hcov-nl63, and hbov, to determine the prevalences of these viruses and to reassess the prevalences of older viruses, such as coronavirus and rhinovirus, among children with lrtis. eleven tested viruses were found in 312 (60.6%) of the 515 patients. the clinical characteristics of patients infected with different viruses were relatively distinct. rsv, rhinovirus, and piv-3 were frequently observed in the patients with bronchiolitis. in contrast, influenza virus, piv-1, and hcov-nl63 were found to be major viral agents of croup. although previous studies have shown that hmpv is more likely to be associated with bronchiolitis or wheezy bronchitis than pneumonia or croup [27, 28] , we found that hmpv was frequently associated with viral pneumonia in this cohort. this observation may reflect differences in diagnostic definitions or study populations between reports; nonetheless, the present study indicates that hmpv is a common virus detected in bronchiolitis patients, because it was found in an additional 3 patients among 4 who were coinfected with other viruses. previous studies of acute respiratory tract infections in children have demonstrated detection rates of 0.7%-8.8% for hcov-nl63 [18, 24, [29] [30] [31] [32] . moreover, although hcov-nl63 may be associated with mild upper respiratory tract infections, it has also been suggested to contribute to the development of bronchiolitis or croup [30, 33] . the present study indicates that hcov-nl63 is a less common respiratory pathogen in cases of acute lrtis in korean children. in fact, hcov-nl63 only accounted for 1.6% of cases, and hcov-oc 43 was rarely found. however, despite the small number of children identified with hcov-nl63 infection, when it was found, it was frequently associated with croup (3 of 6 cases). moreover, the proportion of hcov-nl63-positive croup cases was found to be similar to the proportion of cases caused by influenza virus or piv-1, which are the 2 most common causes of croup in the fall and winter seasons [3, 34] . these findings suggest that hcov-nl63 is an important etiologic agent of croup during the spring in korea. our study also has several limitations. it was based on an analysis of samples selected from a list of cases of acute lrti in a hospital database. therefore, we could not determine the population-based prevalence. viruses associated with low prevalence and mild symptoms may not be fully described, either. however, comparable numbers of the archived samples were selected in each study year; thus, we believe that the data obtained in the present study may represent the overall activities of viruses responsible for acute lrtis and their epidemiologic characteristics. during the 5-year observation period, hmpv infection appeared to be rare in the 1-year period of 2003-2004. this finding suggests that hmpv-associated lrtis are subject to annual variations, which contrasts with the relatively stable annual incidence of rsv infections [35, 36] . these results may be the consequence of spurious observations affected by the selection bias of this study. however, annual variability has also been demonstrated in other studies of periods of only 2 or 3 years and of archived samples during a longer period [26, 37, 38] . additional prospective studies are needed to characterize the epidemiologic features of hmpv infection. hbov, a potential causative agent of lrti, demonstrated seasonal periodicity during each study year, with a peak prevalence occurring from may to july. the number of hbov infections peaked slightly later than the number of piv-3 infections, and during its peak months, the total prevalence of the other 10 viral agents excluding hbov was 34.7% (50 cases among 144 nasopharyngeal aspirates), which is lower than the for all comparisons between respiratory syncytial virus (rsv) and overall detection rate. the detection rate of 11 viruses during the same period increased from 34.7% to 59%, similar to that of the total detection rate of 60.6%. these findings suggest that hbov is a major viral agent of respiratory episodes during late spring to early summer. however, the issue of a causal relationship remains unresolved. it is noteworthy that the detection of hbov dna was associated with a higher rate of coinfection than other viral agents. as demonstrated above, the prevalence of coinfection of hbov was similar to that of adenovirus, which is frequently observed as a copathogen because of its long shedding period. in 2 previous studies, hbov has also been found with other agents with prevalences of 17.5% and 55.6% [11, 19] . thus, hbov infection might occur incidentally to respiratory infections caused by other viral agents. at the present time, despite the periodicity shown by hbov, its role as a true pathogen remains uncertain. additional studies are required to determine its asymptomatic prevalence in the population, pathogenicity, and viral shedding characteristics. the etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice occurrence of groups a and b of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children viral etiology and epidemiology of acute lower respiratory tract infections in korean children lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis association of rhinovirus infection with increased disease severity in acute bronchiolitis the role of rhinovirus in asthma exacerbations rhinovirus viremia in children with respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans cloning of a human parvovirus by molecular screening of respiratory tract samples coronavirus infection in acute lower respiratory tract disease of infants acute lower respiratory tract infections in nonhospitalized children rapid identification of human adenovirus types 3 and 7 from respiratory specimens via multiplex type-specific pcr practical implementation of a multiplex pcr for acute respiratory tract infections in children rhinovirus identification by bgli digestion of picornavirus rt-pcr amplicons type-specific identification of influenza viruses a, b, and c by the polymerase chain reaction evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children evidence of human coronavirus hku1 and human bocavirus in australian children 50% endpoint calculation human metapneumovirus infections in hospitalized children human metapneumovirus in children tested at a tertiary-care hospital new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong, china seasonality and clinical features of human metapneumovirus infection in children in northern alberta human metapneumovirus-associated lower respiratory tract infections in korean infants and young children human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children human metapneumovirus infection in japanese children detection of human coronavirus nl63 in young children with bronchiolitis human coronavirus nl-63 infections in children: a 1-year study detection of human coronavirus-nl63 in children in japan human coronavirus nl63 associated with lower respiratory tract symptoms in early life croup is associated with the novel coronavirus nl63 croup: an 11-year study in a pediatric practice respiratory syncytial virus and parainfluenza virus genetic diversity and molecular epidemiology of the g protein of subgroups a and b of respiratory syncytial viruses isolated over 9 consecutive epidemics in korea the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience evidence of human metapneumovirus in children in argentina financial support. korean ministry of health and welfare (grant 0412-bm01-716-0001).potential conflicts of interest. all authors: no conflicts. key: cord-260779-riw5xs3j authors: van griensven, johan; de weiggheleire, anja; delamou, alexandre; smith, peter g.; edwards, tansy; vandekerckhove, philippe; bah, elhadj ibrahima; colebunders, robert; herve, isola; lazaygues, catherine; haba, nyankoye; lynen, lutgarde title: the use of ebola convalescent plasma to treat ebola virus disease in resource-constrained settings: a perspective from the field date: 2016-01-01 journal: clin infect dis doi: 10.1093/cid/civ680 sha: doc_id: 260779 cord_uid: riw5xs3j the clinical evaluation of convalescent plasma (cp) for the treatment of ebola virus disease (evd) in the current outbreak, predominantly affecting guinea, sierra leone, and liberia, was prioritized by the world health organization in september 2014. in each of these countries, nonrandomized comparative clinical trials were initiated. the ebola-tx trial in conakry, guinea, enrolled 102 patients by 7 july 2015; no severe adverse reactions were noted. the ebola-cp trial in sierra leone and the evd001 trial in liberia have included few patients. although no efficacy data are available yet, current field experience supports the safety, acceptability, and feasibility of cp as evd treatment. longer-term follow-up as well as data from nontrial settings and evidence on the scalability of the intervention are required. cp sourced from within the outbreak is the most readily available source of anti-evd antibodies. until the advent of effective antivirals or monoclonal antibodies, cp merits further evaluation. convalescent blood or plasma transfusion has been used in clinical settings for >100 years [1] . until the advent of antibiotic therapy, it was widely used for a range of bacterial and viral diseases. a recent meta-analysis suggests that it could have contributed to an absolute reduction in mortality of around 20% during the 1918 influenza epidemic [2] . at present, hyperimmune globulin, manufactured from convalescent donors' plasma, is still employed as prophylaxis or treatment for certain infectious diseases (eg, measles, diphtheria, polio, hepatitis a and b) [3] . passive antibody therapy is not a new intervention but a widely tested, safe, and proven prophylactic and therapeutic intervention. over the last 10 years, convalescent plasma (cp) has been explored for the treatment of viral severe acute respiratory infections such as severe acute respiratory syndrome and (avian) influenza. a recent meta-analysis identified 32 studies that indicated, overall, a 75% reduced risk in the odds of mortality, particularly if cp was administered early after symptom onset [4, 5] . cp is currently being considered as treatment for middle east respiratory syndrome [6] . for hemorrhagic fevers, well-documented, relatively recent experience stems from a randomized controlled trial comparing cp (1 single unit of 500 ml) with normal plasma against argentine hemorrhagic fever [7] . mortality was 1.1% among the 91 patients who received cp compared to 16 .5% in the 97 patients treated with normal plasma. of interest, a delayed neurological condition was observed in some patients after cp therapy, occurring several weeks after apparent cure. cp has also been explored against the hemorrhagic disease lassa fever, with conflicting results [8] [9] [10] [11] . the world health organization (who) guidelines recommend both convalescent whole blood (cwb) and cp for use against ebola virus disease (evd) [12] . during evd outbreaks, both are local and readily available sources of anti-evd antibodies. blood transfusion is routinely done in all 3 high-transmission countries involved in the current evd outbreak; however, plasma has numerous advantages in these settings. using apheresis, a single donor can give substantially more cp, up to 10 ml/kg every 2 weeks, compared with 1 unit of 450 ml whole blood (approximately 250 ml plasma) every 3-4 months for cwb; hence, more patients can be treated. cp can be given intravenously over a much shorter time period (30 minutes vs 3-4 hours) and hence is less demanding for healthcare teams in ebola treatment centers. the pathogen inactivation step during cp production increases the safety of the intervention. cp transfusion is also safer with fewer transfusion reactions; cp needs to be abo compatible, but there is no need for bedside cross-matching [13] . additionally, cp can be stored for much longer periods. hyperimmune globulin has advantages compared with cwb and cp, as it is a concentrated and purified product [14] . however, this product is presently not available against evd, and hence it has not been evaluated in the current ebola outbreak. nevertheless, it represents an interesting longer-term potential therapy, especially if the ongoing studies suggest efficacy of cp. recombinant monoclonal antibodies are currently being evaluated in sierra leone and guinea [15] , but will not be discussed in detail given this article's focus on convalescent blood products. the successful use of zmapp (an antibody cocktail comprising 3 ebola virus-specific recombinant monoclonal antibodies) for treatment of evd in non-human primate models, even when administration is delayed until 5 days after a supralethal viral challenge, provides proof of concept of antibody-based therapy for evd [16] . similarly, the use of immunoglobulin fractions from convalescent animals is effective for treating evd in nonhuman primates [17] . earlier treatment attempts with monoclonal antibodies or immunoglobulins might have failed because antibody concentrations were too low, antibodies had too narrow a spectrum (eg, 1 monoclonal only), or because treatment was not given repeatedly (zmapp is given 3 times [days 0, 3, 6]). early cwb studies with nonhuman primates failed to demonstrate efficacy when rhesus macaques were transfused immediately after challenge [18] . there are no animal studies with convalescent plasma against evd. key questions include whether antibody titers are high enough in cp and cwb to be effective, and if 1 single transfusion, as per current who guidelines, is sufficient. the exact kinetics of neutralizing antibodies in convalescent donors and variation between individuals are currently not well described. clinical data are limited. the most important study on cwb in evd reported on 8 individuals treated during the kikwit outbreak in 1995; 7 survived [19] . however, this was an uncontrolled small study, with patients enrolled relatively late after onset of symptoms. moreover, several factors beyond an antibody-related effect could have contributed to the improved survival, such as improved hydration or the administration of other blood components such as clotting factors. moreover, a subsequent analysis after adjustment for age, sex, and number of days since onset of symptoms showed no survival benefit of cwb [20] . one well-documented case occurred in 1976 when a laboratory technician survived evd after receiving 2 plasma transfusions combined with interferon injections [21] . several evd patients (mainly expatriates) have been treated with cwb, cp, or recombinant monoclonal antibodies during the current outbreak [22] [23] [24] [25] [26] . however, most of these patients received multiple anti-evd interventions and high-quality supportive care, so it is not possible to assess the efficacy, if any, of passive antibody transfer. in conclusion, whereas the data on zmapp provide proof of concept, clinical data on and experience with convalescent blood products for the treatment of evd are very limited. consequently, who prioritized the need to evaluate cp in clinical trials, with the aim to determine its efficacy, safety, and feasibility. notably, these interventions can be organized by harnessing or increasing existing national capacities quickly within the affected countries, without the complications or limitations associated with shipping commercial products, which are often in limited supply. an overview of clinical trials is provided in table 1 . the ebola-tx trial, in conakry, guinea, is designed to assess the feasibility, safety, and efficacy of cp against evd [27] . survival at 14 days after transfusion of patients treated with cp plus supportive care will be compared to that of patients receiving supportive care alone, in an open-label phase 2/3, nonrandomized comparative study. all eligible and consenting patients of any age with confirmed evd (including pregnant women) are enrolled; exclusion criteria are limited to contraindications for cp or patients arriving in a close to terminal condition. available abo compatible plasma is given, within 48 hours after diagnosis, on a firstcome, first-served basis. patients for whom there is no available compatible plasma are enrolled as concurrent controls, complemented with historical controls. the first plasma collection started on 9 february 2015, followed by the first cp administration on 19 february 2015. as of 7 july 2015, 102 patients have been recruited. the main analysis is planned when a cohort of 130 cptreated patients have reached day 14 posttransfusion. due to the fluctuating number of new patients and the decline of the outbreak in guinea, it is difficult to estimate when this will happen. the ebola-cp consortium in sierra leone, which emerged out of the ebola-tx initiative, is conducting a parallel study in freetown using a similar protocol, crf, and data management. the first patient was recruited 19 march 2015 as a control (no cp given); due to the declining outbreak in sierra leone, as of 23 july 2015, 3 patients have received cp. the evd001 trial is a phase 1/2 pilot study with viral load changes as the primary outcome [28] . children and pregnant women are excluded. the study started in monrovia in november 2014 but subsequently closed due to the decline in caseload. a total of 4 cp-treated patients and 2 controls were included. the ebola medical treatment team in the 34th regiment hospital freetown, sierra leone, has been administering cwb, as per who guidance, for compassionate use from december 2014 to march 2015 (http://awoko.org/2014/12/09/ sierra-leone-news-convalescent-blood-treatment-started/). a total of 52 patients opted for cwb transfusion with 24 included as controls. none of the ongoing clinical studies have reported findings. designing trials, and recruiting patients, to evaluate cwb and cp has been challenging. only ebola-tx has achieved a relatively high sample size (>80 cp-treated patients). the design that would provide the best evaluation would be a randomized trial in which one or other intervention would be compared with control patients not receiving the intervention. however, such randomized designs have proved unacceptable in the volatile settings of the current ebola outbreak, and researchers have at the early stages resorted to nonrandomized study designs [29] . all cp trials were designed to include concurrent control patients. for example, in the ebola-tx trial, controls were to be patients presenting when no abo-compatible plasma was available. however, when the trial started, the supply of cp increased significantly and the number of patients decreased, resulting in sufficient supply of cp to treat nearly all patients. consequently, it will compare the mortality of treated patients with that of historical controls, with the inherent possibility of biased comparisons. key concerns in all trials regarded potential confounding factors including variations in patient characteristics, ebola virulence on presentation, and differences in standards of supportive care over time. for example, the systematic placement of intravenous access lines in patients for cp treatment could encourage more aggressive intravenous hydration during the trial period. how easy it will be to interpret the results of the trials will depend, to a large extent, on the size of the mortality reduction, if any, associated with the intervention. plasma transfusion is considered a relatively safe procedure, particularly if pathogen reduction is done. however, for use as a treatment of evd, there are additional considerations. safe production, storage, and distribution need to be organized in the affected countries. plasmapheresis technology needs to be available or introduced, including quality-assured testing for transfusion-transmissible infections and an effective cold chain, all of which have been major challenges in the currently affected west african countries. although standards for blood group typing are clearly defined in the national guidelines, errors can result from field realities, such as lack of resources, supervision, and poorly incentivized staff. additionally, weak systems of documentation can lead to poor or mistaken identification. the infection control environment of ebola treatment centers brings with it difficult operational challenges, both for care of patients and, in particular, for the implementation of clinical research. short and intermittent patient contact by staff in protective clothing, with potentially confused patients, is the norm. therefore, mistakes are more likely to occur, such as in patient identification, labeling of blood tubes, and request forms during sample collection and packaging. moreover, introducing the procedure for blood group typing into already overloaded evd diagnostic laboratories might engender additional errors. this can potentially increase the risks of adverse reactions due to preanalytical, analytical, or postanalytical errors. these errors could potentially lead to severe reactions if abo-incompatible plasma with high titers of hemolysins were administered. the ability to monitor patients for and to react to severe, acute adverse reactions is clearly limited by the environment of many ebola treatment centers. some (severe) transfusion reactions might erroneously be attributed to evd [29, 30] . for instance, respiratory difficulties, often seen during evd, might be due to transfusion-related acute lung injury. patient management options for adverse events may also be limited. it has been suggested for a number of viruses, including ebola, that the transfer of antibodies might enhance pathogenicity, possibly by antibody-enhanced cell entry [31] . although more recent studies have suggested that this is unlikely to have clinical significance, it still requires special attention and careful evaluation when employing cp, regardless of whether it is for clinical trials or compassionate use [32] . no severe adverse reactions or safety risks for healthcare staff have been noted in the ebola-tx trial by 7 july 2015. while this suggests that cp can be employed safely for evd treatment, the use of such treatment, if found efficacious, should be evaluated in noncontrolled settings before introducing it as a standard of care in public health facilities. the quality of blood transfusion services in many west african countries is generally poor, and in many countries, plasma production is not routinely performed [33] . during the evd out-break, effective plasmapheresis teams were assembled relatively quickly, mainly in mobile plasma units or "plasma mobiles" (http://www.gatesfoundation.org/media-center/press-releases/ 2014/11/support-to-ebola-affected-countries-announcement), and national blood transfusion teams trained. this was made possible by the substantial funding made available through international research consortia. as the situation dictated an emergency response, the plasmapheresis material introduced for use in the present trials is unlikely to be an optimal choice for the concerned national transfusion centers. a transition to simpler, nonautomated systems might be indicated. furthermore, pathogen reduction is an expensive procedure and unlikely to be a priority for resource-constrained settings, where the prerogative is the provision of safe basic blood banking services. the experience of the present trials, which has identified the need to further improve the overall quality of blood banking centers in all 3 countries, should result in longer-term capacitybuilding projects once the outbreak is contained. the use of convalescent blood products is further complicated by the requirement to carefully and properly manage the engagement of ebola survivors, who are often stigmatized in their communities. in many west african countries there is reticence toward blood collection, donation, and transfusion, based on superstitions and beliefs in the community [34, 35] . experience during the 2014-2015 outbreak suggests that if there are appropriate community consultations and discussions, there is a reasonable acceptability among plasma donors, surviving patients, risk with abo incompatibility acceptability in evd context and their family members, at least in the short term. however, a better understanding of plasma donor motivation is required to ascertain whether possible degrees of coercion have occurred. in all 3 high-transmission countries, evd survivors have organized themselves into survivor associations. although this has probably substantially eased donor mobilization, more indepth assessments of their potential role are merited. other key concerns include confidentiality and privacy, especially if members of the survivors association are involved in the recruitment process. additionally, confronting individuals recently recovered from evd with positive serological tests for other infections can have negative consequences. lack of free access to care for infections such as hepatitis b and c further compounds the situation [36] . hence, longer follow-up and more in-depth anthropological assessments among plasma donors and patients are required to further document the acceptability and feasibility of cp as evd treatment. it has been suggested that involving survivors as donors in a potentially life-saving treatment could support their social reintegration and reduce stigma, but evidence for this is currently lacking. although hopefully there will not be another ebola outbreak on the scale of the present one, it would be possible to stockpile plasma for emergency use. there are currently >15 000 ebola survivors in the 3 high-transmission countries. a single donor, donating 600 ml every 2 weeks, could provide sufficient plasma to treat around 35 patients. with 500 regular donors (approximately 3% of the potential total), a total of 17 500 treatments would potentially be available. prequalification of donors with high titers of neutralizing antibodies would make the process most efficient. such scaling up of cp would require either decentralized plasma production or safe transport and storage of cp from a central location to more remote areas. blood transfusion regulations recommend that people who received a blood transfusion should not be permitted to donate blood for 12 months after the date of transfusion, mainly related to the risk of pathogen transmission. applying this to evd survivors following cp treatment would seriously reduce the donor pool. however, if cp is shown to have a substantial effect, the benefits of using such patients as donors would probably outweigh potential risks. it should also be confirmed that cp-treated patients develop sufficient level of neutralizing antibodies against the virus, particularly if treated early in the disease course. the logical next step is to produce hyperimmune globulins from the donated plasma, and several initiatives are focusing on this. as purified and concentrated products, hyperimmune globulins are generally considered to be safer and with higher, less variable antibody titers than cp (table 2) . they can also be stored for prolonged periods. animal production of hyperimmune globulins is also under exploration (http://www.faben techcom/technologie/technology/). pending the building up of such expertise within africa, production could be outsourced. current field experience supports the use of cp against evd as acceptable, feasible, and safe. efficacy data are pending. in consideration of the study design limitations described in this article, these trials may not yield definitive data on the extent to which such treatment reduces case fatality rates. however, it is expected that they will, at minimum, provide some indication of the utility of cp and the challenges in delivering such treatment, in future trials. additional research on cp, hyperimmune globulins, and monoclonal antibodies in animal and clinical studies is required to identify the optimal treatment regimen and better understand the mechanism of action. long-term studies are also required to document better the feasibility and acceptability of cp donation outside of a research setting, to assess the willingness of survivors to become cp donors and to identify any negative immunological, medical, or psychosocial effects of repeated cp donations. longer followup of cp treated patients is also indicated to detect late adverse events. although hyperimmune globulins and recombinant monoclonal antibodies have several advantages, the use of cp sourced from within an outbreak is arguably the most readily available source of anti-evd antibodies and will always have the advantage that it is likely to be active against the circulating strain. in each new outbreak, available hyperimmune globulins or monoclonal antibodies will first have to be evaluated against the causative strain and possibly followed by the production of a new product, all of which takes time. until the advent of potent, safe, affordable, and effective antivirals, and the development of effective vaccines, the use of convalescent blood products should remain part of the potential response to evd. financial support. the ebola-tx project is funded by the european union's horizon 2020 research and innovation program (grant agreement number 666 094). additional funding is provided by the department of economy, science and innovation of the flemish government. potential conflicts of interest. all authors are involved in the ebola-tx trial. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. passive antibody therapy for infectious diseases meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? polyclonal immunoglobulins and hyperimmune globulins in prevention and management of infectious diseases the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis hark back: passive immunotherapy for influenza and other serious infections passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome passive antibody therapy of lassa fever in cynomolgus monkeys: importance of neutralizing antibody and lassa virus strain enhanced treatment of lassa fever by immune plasma combined with ribavirin in cynomolgus monkeys lassa fever. effective therapy with ribavirin the use of lassa fever convalescent plasma in nigeria use of convalescent whole blood or plasma collected from patients recovered from ebola virus disease for transfusion, as an empirical treatment during outbreaks. interim guidance for national health authorities and blood transfusion services world health organization. the clinical use of blood handbook clinical uses of intravenous immunoglobulin putative investigational therapeutics in the treatment of patients with known ebola infection reversion of advanced ebola virus disease in nonhuman primates with zmapp passive immunization of ebola virusinfected cynomolgus monkeys with immunoglobulin from hyperimmune horses ebola hemorrhagic fever: evaluation of passive immunotherapy in nonhuman primates treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee ebola hemorrhagic fever, democratic republic of the congo, 1995: determinants of survival a case of ebola virus infection clinical care of two patients with ebola virus disease in the united states the use of tkm-100802 and convalescent plasma in 2 patients with ebola virus disease in the united states world health organization. compassionate use of experimental treatments for ebola virus disease: outcomes in 14 patients admitted from ebola virus convalescent blood products: where we are now and where we may need to go minipool caprylic acid fractionation of plasma using disposable equipment: a practical method to enhance immunoglobulin supply in developing countries emergency evaluation of convalescent plasma for ebola viral disease (evd) in guinea clinical trial to evaluate the efficacy and safety of convalescent plasma for ebola treatment (evd001). nct02333578 randomised controlled trials for ebola: practical and ethical issues acute respiratory distress syndrome after convalescent plasma use: treatment of a patient with ebola virus disease antibody-dependent enhancement of ebola virus infection evidence against an important role for infectivity-enhancing antibodies in ebola virus infections ebola: a call for blood transfusion strategy in sub-saharan africa blood as medicine: social meanings of blood and the success of ebola trials community perception and beliefs about blood draw for clinical research in ghana large-scale convalescent blood and plasma transfusion therapy for ebola virus disease key: cord-002514-pp06m5xk authors: venkatesan, sudhir; myles, puja r.; leonardi-bee, jo; muthuri, stella g.; al masri, malak; andrews, nick; bantar, carlos; dubnov-raz, gal; gérardin, patrick; koay, evelyn s. c.; loh, tze ping; memish, ziad; miller, elizabeth; oliva, maria e.; rath, barbara a.; schweiger, brunhilde; tang, julian w.; tran, dat; vidmar, tjasa; waight, pauline a.; nguyen-van-tam, jonathan s. title: impact of outpatient neuraminidase inhibitor treatment in patients infected with influenza a(h1n1)pdm09 at high risk of hospitalization: an individual participant data metaanalysis date: 2017-05-15 journal: clin infect dis doi: 10.1093/cid/cix127 sha: doc_id: 2514 cord_uid: pp06m5xk background. while evidence exists to support the effectiveness of neuraminidase inhibitors (nais) in reducing mortality when given to hospitalized patients with a(h1n1)pdm09 virus infection, the impact of outpatient treatment on hospitalization has not been clearly established. we investigated the impact of outpatient nai treatment on subsequent hospitalization in patients with a(h1n1)pdm09 virus infection. methods. we assembled general community and outpatient data from 9 clinical centers in different countries collected between january 2009 and december 2010. we standardized data from each study center to create a pooled dataset and then used mixed-effects logistic regression modeling to determine the effect of nai treatment on hospitalization. we adjusted for nai treatment propensity and preadmission antibiotic use, including “study center” as a random intercept to account for differences in baseline hospitalization rate between centers. results. we included 3376 patients with influenza a(h1n1)pdm09, of whom 3085 (91.4%) had laboratory-confirmed infection. eight hundred seventy-three patients (25.8%) received outpatient or community-based nai treatment, 928 of 2395 (38.8%) with available data had dyspnea or respiratory distress, and hospitalizations occurred in 1705 (50.5%). after adjustment for preadmission antibiotics and nai treatment propensity, preadmission nai treatment was associated with decreased odds of hospital admission compared to no nai treatment (adjusted odds ratio, 0.24; 95% confidence interval, 0.20–0.30). conclusions. in a population with confirmed or suspected a(h1n1)pdm09 and at high risk of hospitalization, outpatient or community-based nai treatment significantly reduced the likelihood of requiring hospital admission. these data suggest that community patients with severe influenza should receive nai treatment. to 2009 also suggests that nais given to high-risk community patients with influenza may reduce subsequent hospitalization [6] . to the best of our knowledge, similar data on hospitalization during the 2009-2010 pandemic period are absent. we therefore performed a global individual participant data (ipd) metaanalysis to address this question. research centers participating in the post-pandemic review of anti-influenza drug effectiveness (pride) study were identified while conducting a metaanalysis of published studies on the effectiveness of nai treatment in hospitalized patients [4] . a detailed description of the pride study has been published previously [5] . in total, the pride consortium obtained data on 170 858 potentially eligible patients from 81 research centers in 38 countries across 6 world health organization regions. a subset of these centers provided community or outpatient data, which were then used for the current analysis. no data were provided or funded for collection by pharmaceutical companies. the protocol for this study was registered with the prospero register of systematic reviews (crd42011001273) [7] . data were available on community patients or those attending outpatient clinics with laboratory-confirmed or clinically diagnosed influenza a(h1n1)pdm09 from 9 centers (argentina, canada, france, germany, israel, saudi arabia, singapore, slovenia, and the united kingdom). these were standardized using a data dictionary (supplementary table 1 ) before pooling for analysis. the primary outcome variable was influenza-related hospital admission as determined by case records linking admission to the influenza illness episode. the primary exposure variable was treatment with an nai initiated in any community or outpatient setting as compared to no nai treatment in the community or outpatient setting. if data were available, we further distinguished early treatment (nai started ≤2 days after symptom onset) vs later treatment (>2 days after symptom onset). we excluded from all our analyses those patients who received nai treatment in the community on the day of hospital admission, on the grounds that treatment would not have had sufficient opportunity to work in these patients. this exclusion also accounts for any physician decisions to prescribe nais taken after a decision to admit the patient to the hospital, amounting to confounding by indication. covariates adjusted for in the final multivariable model were outpatient or community-based antibiotic treatment (yes/no) and propensity score (by quintile) for receiving nai treatment in the community. we computed propensity scores for the likelihood of community-based nai treatment for individual patients within each contributing dataset using the method described by hirano and imbens [8] . multivariable logistic regression models developed to calculate propensity scores included the following covariates: age, sex, presence of a comorbid condition (yes/no), and an indicator of disease severity (in order of preference: documented severe respiratory distress or shortness of breath). the resulting propensity scores were then categorized into quintiles for use in subsequent analyses. in the primary analysis, we used a mixed-effects logistic regression model to investigate the association between community-based nai treatment and subsequent hospital admission using the xtmelogit command in stata (version 14). to account for differences in baseline outcome between individual datasets, we included individual study centers as a random intercept. we ran both unadjusted and adjusted models, the latter containing covariates for community-based antibiotic treatment and propensity scores. to allow for comparisons between the unadjusted and adjusted models, we included missing data as a dummy variable category. the c-statistic (area under the receiver operating characteristic curve) was used to assess model fit. where data were available, we explored the potential impact of timing of nai administration (early nai treatment vs later nai treatment) on hospitalization. we also performed stratified analysis in patients with laboratory-confirmed a(h1n1)pdm09 influenza and adults (aged ≥16 years) and children. furthermore, we carried out an additional analysis restricted to patients with high-risk conditions. patients were classified as having a high-risk condition if they had at least 1 chronic illness recorded that would trigger seasonal influenza vaccination [9] or were aged ≥65 years. results from our mixed-effects logistic regression models are presented as odds ratios (ors) with 95% confidence intervals (95% cis). we received outpatient data on 130 077 patients with clinically or laboratory-confirmed influenza from 25 centers. however, 16 centers (n = 125 049 patients) offered surveillance data that did not contain clinical data on either nai use or subsequent hospitalization status. therefore, the final study population comprised 3376 patients from 9 study centers ( figure 1 ). data from 7 of the 9 included study centers (n = 1183 patients) came from outpatient (ambulatory care) clinics attached to hospitals. of the remaining 2 study centers, 1 provided community surveillance data collected by a local ministry of health (n = 1762) and the other provided data from primary care (n = 431). three of the 9 included centers (canada, germany, and israel; total n = 535) were from pediatric outpatient clinics and comprised entirely of patients aged <18 years. patients from the german study center were particularly young with a median age of 1.4 years (supplementary table 3 ). of 3376 patients in our pooled dataset, 3085 (91.4%) had laboratory-confirmed a(h1n1)pdm09 infection; 1747 (51.8%) were children (<16 years) and 67 (2.0%) were aged >64 years. overall, 1705 (50.1%) were admitted to the hospital and 928 (928/2395; 38.7%) had clinically observed shortness of breath or respiratory distress as a marker of severity. where data were available (n = 473), the median interval between date of symptom onset and date of nai treatment initiation was 1 day (interquartile range [iqr], 0-3) for the whole study population, 1 day (iqr, 0-2) for nonhospitalized patients, and 1 day (iqr, 0-4) for hospitalized patients. of the hospitalized patients, where calculable (n = 1363), the median interval between date of symptom onset and date of hospital admission was 2 days (iqr, 1-3). general characteristics of the study population are presented in table 1 . about one half (50.1%) of the cohort was eventually admitted to the hospital, and 38.8% had 1 or more indications of severe respiratory illness as denoted by observed shortness of breath or respiratory distress. in addition, fewer than 2% of the cohort were elderly and about 72% had no recorded comorbid conditions, suggesting that most patients were young and previously healthy. indeed, 51.8% were children and 37.1% of women aged 15-44 years were pregnant, reflecting the inclusion of 1 obstetric clinic (n = 81) within the data. of the 1705 patients who were hospitalized, we had data on the subsequent course in 1433 patients. of these hospitalized patients, 1155 (80.6%) were treated with nais in-hospital, 147 patients ( of subsequent hospital stay in a smaller subgroup of 522 patients where the median length of stay was 3 days (iqr, 2-5; median, 3 days [iqr, [2] [3] [4] [5] in treated and median, 2 days [iqr, 1.5-5] untreated). of the 1647 patients who were excluded from the analyses because they were hospitalized on the same day as nai treatment initiation, 116 (7%) were admitted to critical care facilities and 23 (1.4%) died. in 1595 of these patients on whom we had pneumonia data, 154 (9.7%) were found to have had clinical signs of pneumonia; in 31 (1.9%) of these patients the pneumonia was radiologically confirmed. the median length of hospital stay in an even smaller subgroup (n = 186 where data were available) was found to be 3 days (iqr, 2-5). in patients with laboratory-confirmed or clinically diagnosed a(h1n1)pdm09 influenza, after adjustment for community-based antibiotic treatment and propensity score, the likelihood of hospital admission in patients with outpatient or community-based nai treatment was 0.24 (95% ci, 0.20-0.30) when compared to no nai treatment in the community (table 2) . a c-statistic of 0.813 (95% ci, 0.799-0.827) suggested that the predictive performance of our model was acceptable. when restricted to laboratory-confirmed a(h1n1)pdm09 patients, the estimate was very similar to the estimate for the overall study population ( table 2) . nai treatment, when in the pooled dataset, 1019 patients (30.1%) were recorded to have at least 1 high-risk condition. in this subpopulation of higher-risk patients, we also observed a reduction in the odds of hospital admission (or, 0.27; 95% ci, 0.19-0.38) in those treated with nais in the community compared with no nai treatment. full results of the sensitivity and stratified analyses are summarized in table 2 . the hospital admission rate varied widely among each of the 9 included study centers, ranging from 3.94% to 69.3%. to separate any effects that hospital admission rates among centers may have had on the association between nais and hospitalization, we did a post hoc stratified analysis by median hospitalization rate (50.6%). after adjusting for community-based antibiotic treatment and propensity score, the pooled or for the association between nai treatment and subsequent hospitalization in study centers with a hospital admission rate <50.6% (n = 991) was 1.00 (95% ci, 0.61-1.64), whereas in centers where the admission rate was >50.6% (n = 2385), the or was 0.17 (95% ci, 0.14-0.22). in this study, we assembled data from a large cohort of community-based patients who had pandemic influenza in 2009-2010, of whom 91% had laboratory confirmation of influenza a(h1n1)pdm09 infection. the demographic and clinical findings (table 1) reveal that most patients were young and previously healthy, yet with relatively severe influenza (indicated by the presence of either documented severe respiratory distress or shortness of breath at presentation). as such, we recognize that our results are not generalizable to a wider range of community-based patients with mild pandemic influenza and may not be generalizable to the elderly. our main findings ( table 2) suggest that nai treatment in the community for patients with severe pandemic influenza substantially reduced the likelihood of hospital admission due to influenza a(h1n1)pdm09. in a pandemic context, individuals generally have little or no preexisting cross-reactive immunity to the infecting virus; therefore, effect size might be lower for seasonal (interpandemic) influenza, and our findings should be interpreted with more caution in that context. in a sensitivity analysis restricted to patients with laboratory-confirmed a(h1n1)pdm09, this finding was unaltered; and in patients with underlying at-risk conditions, the risk reduction was greater. a limitation of our analysis is that we did not have data on body mass index and therefore could not include obesity as a high-risk condition. we also explored potential differences in effect size between adults and children and found that the effect of nais in reducing the likelihood of hospital admission was maintained and broadly consistent in both age groups. these findings contrast with our previous analysis of mortality data, in which we failed to demonstrate significantly reduced mortality in hospitalized children treated with nais [5] . this discrepancy is potentially explained by the relatively high attack [10] and hospitalization rates in children with a(h1n1)pdm09 [11] compared with a relatively low case fatality rate [12] but could also relate to statistical underpowering in the mortality study [5] . a question of considerable clinical relevance relates to the timing of antiviral treatment in relation to the magnitude of benefit obtained, especially since data already exist to suggest that symptom alleviation and mortality reduction are both diminished by delayed treatment [5, 13] . we were able to perform a sensitivity analysis on 473 nai-treated patients in whom we had specific data on the timing of symptom onset and antiviral treatment. this revealed that earlier treatment (within 48 hours of symptom onset) was significantly more beneficial than later treatment. because the dataset contained so few elderly patients, perhaps reflecting the low incidence of a(h1n1)pdm09 infection in the elderly [14, 15] , we were unable to cast any further light on the effectiveness of nais in this particular subgroup of patients. although smaller than our previous ipd analysis that focused on mortality reduction in hospitalized patients [5] , one strength of this study is the relatively large number of patients included from 9 geographically diverse clinical centers. although we were unable to adjust specifically for disease severity in our multilevel models because of the heterogeneity of severity measures used across individual datasets, we nevertheless included physician-recorded breathlessness and severe respiratory distress when deriving propensity scores. however, we acknowledge that confounding by indication [16] may still be present. if it is, we surmise that physicians may have been more likely to treat severe cases than milder ones or to treat putative at-risk groups such as pregnant women with nais. therefore, the treated group would have a higher underlying likelihood of being admitted to the hospital; this in turn would produce a bias in the analysis, tending toward underestimation of any treatment effect. likewise, we recognize that some nai treatment may have been given immediately prior to hospital admission when there was no practical window in which an antiviral drug could have had time to work or perhaps even when the physician had already decided that the patient needed to be admitted. therefore, we think there is sound clinical rationale for excluding patients in whom nai treatment was initiated on the day of hospital admission. another potential limitation of our propensity scoring approach is that we lacked data on vaccination, albeit knowledge of vaccination status might be associated with physicians' decisions to prescribe nais and to hospitalize patients. to explore this further, we determined that patients whose illness onset was on or before 15 october 2009 could not have been vaccinated due to nonavailability prior to this date. even if they had been vaccinated, they would not have had time to seroconvert. we subsequently performed a stratified analysis around this date by dividing the overall pooled dataset into "early pandemic" and "later pandemic. " we had onset dates in 2175 patients of whom 903 were on or before 15 october 2009. the adjusted ors (95% cis) in both groups were very similar (early pandemic group, 0.12 (0.06-0.24) and late pandemic group, 0.11 (0.07-0.18)]. on this basis, we believe that vaccination is unlikely to have been a major confounder in our study. other residual confounding is nevertheless possible as these are observational data. two obvious drawbacks in our data are the overall high rate of hospitalization in the cohort studied, which limits generalizability to patients at high risk of hospitalization, and the substantial variability between hospitalization rates across individual centers, ranging from 4% to 70%. the dataset with the lowest hospitalization rate was from a surveillance system in the united kingdom where a national policy was in place for 2009-2010 to offer nai treatment to all patients with clinically apparent influenza, irrespective of severity. stratifying the analysis around median hospitalization rate revealed no effect of nais in centers below the median but revealed a strong positive effect in centers above the median. we surmise that these data confirm the beneficial effect of nais (beyond symptom relief) in patients who are severely unwell and at high risk of hospitalization vs those with milder illness. to our knowledge, this is the first individual participant data metaanalysis to investigate the association between preadmission nai antiviral use and hospitalization relating to the 2009-2010 influenza pandemic. as such, these data have potential importance for future pandemic stockpiling and treatment policies and may be of relevance to seasonal epidemics, especially for community patients with relatively severe influenza and those with underlying comorbidities. we note that our point estimates of treatment effectiveness are somewhat higher than the 25% reduction in hospitalization for the treatment of seasonal influenza previously reported by hsu and colleagues [6] . however, the disparity in effect size might be explained by the fact that the 4 studies [17] metaanalyzed by hsu et al included patients with generally milder influenza than in the present study. in addition, 3 of these 4 studies were based on diagnoses of influenza-like illness (ili) without laboratory confirmation and therefore were highly vulnerable to misclassification bias. an earlier pooled analysis of clinical trial data from patients with laboratory-confirmed seasonal influenza also showed a 59% reduction in hospitalization [18] . this was confirmed in recent ipd analysis of seasonal influenza patients that reported a risk reduction of 63% in treated patients (intention-to-treat infected population) [19] , which is somewhat similar to our own data. another study not included in hsu's metaanalysis also suggested a 29% reduction in hospitalization associated with oseltamivir but was again based on diagnoses of ili without laboratory confirmation [20] . a recent study from british columbia, based on clinically diagnosed cases of pandemic influenza a(h1n1)pdm09, also noted 16% effectiveness of nais in reducing hospitalization [21] ; however, the hospitalization rate in this cohort was 0.6%, suggesting cases were comparatively very mild. we therefore recognize that our findings reflect the experience of nai use in a cohort of community patients at high risk of hospitalization. in addition, we noted higher effectiveness in patients with 1 or more comorbidities that would have placed them in a target group for annual seasonal influenza vaccination. placed in the context of the limited previous work on this subject, our findings suggest that the greatest benefit from community use of nais is likely to be achieved by targeting individuals for treatment who have clinically suspected or proven influenza and who are also in a recognized at-risk group or clinically assessed to have severe influenza (irrespective of comorbid status). in these 2 groups of patients, substantial reductions in the likelihood of hospitalization can be achieved, especially if treatment is commenced within 48 hours of symptom onset. our data support current advice on nai treatment given by major public health agencies [22, 23] and the findings of a recent independent report from the uk academy of medical sciences and the wellcome trust, which recommend against use of nais in the community for the treatment of mild influenza, but advise that patients with severe influenza should be treated as soon as possible [24] . supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. financial support. the pride study is funded via an unrestricted educational grant from f. hoffmann-la roche, switzerland (the manufacturers of oseltamivir [tamiflu]). the funder had no role in protocol design, no opportunity to comment on it, and no opportunity to see it other than via the prospero website; no access to any data (and no rights to future access); no role in analysis or interpretation; no opportunity to preview results/findings before entry into the public domain; and no opportunity to contribute to, preview, or comment on manuscripts and presentations arising from this work. the research contract between the university of nottingham and the funder is freely available for inspection (commercial details redacted) at: http://www.nottingham.ac.uk/research/groups/healthprotection/projects/pride.aspx potential conflicts of interest. b. a. r. reports grants from f. hoffmann-la roche to her institution (charité universitätsmedizin berlin) outside the submitted work. d. t. reports grants from the canadian institutes of health research/sickkids foundation new investigator (xg08-049r), the canadian institutes of health research catalyst (cat86860), and the university of toronto dean's fund pilot study grant during the conduct of the study. j. s. n.-v.-t. reports that a grant to the university of nottingham from f. hoffmann-la roche funded the current study; he also reports grants to the university of nottingham from glaxosmithkline for research in the area of influenza; and nonfinancial support from the european scientific working group on influenza to lecture on influenza outside the submitted work. all other authors: no potential conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. antivirals for influenza: where now for clinical practice and pandemic preparedness? evidence and policy for influenza control neuraminidase inhibitors for preventing and treating influenza in adults and children impact of neuraminidase inhibitor treatment on 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influenza-related complications, hospitalization and healthcare expenditure in healthy adults and children effectiveness of neuraminidase inhibitors in preventing hospitalization during the h1n1 influenza pandemic in british columbia influenza antiviral medications: summary for clinicians phe guidance on use of antiviral agents for the treatment and prophylaxis of seasonal influenza (2015−16) version 6.0 use of neuraminidase inhibitors in influenza key: cord-010599-nwp2if8d authors: hayden, frederick g.; herrington, darrell t.; coats, teresa l.; kim, kenneth; cooper, ellen c.; villano, stephen a.; liu, siyu; hudson, spencer; pevear, daniel c.; collett, marc; mckinlay, mark title: efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials date: 2003-06-15 journal: clin infect dis doi: 10.1086/375069 sha: doc_id: 10599 cord_uid: nwp2if8d the novel capsid-binding antiviral pleconaril inhibits in vitro replication of most rhinoviruses and enteroviruses. oral pleconaril treatment was studied in 2 parallel randomized, double-blind, placebo-controlled trials. among 1363 picornavirus-infected participants (65%) in the studies combined, the median time to alleviation of illness was 1 day shorter for pleconaril recipients than for placebo recipients (p > .001). cold symptom scores and frequency of picornavirus cultured from nasal mucus specimens were lower among pleconaril recipients by day 2 of treatment. no treatment effects were seen in those without picornavirus infection. pleconaril was associated with a higher incidence of nausea (6% vs. 4%) and diarrhea (9% vs. 7%) and with small increases in mean serum cholesterol levels and platelet counts, compared with baseline measurements. a subsequent 6-week prophylaxis study found that pleconaril induces cytochrome p-450 3a enzymes, which metabolize a variety of drugs, including ethinyl estradiol. early pleconaril treatment was well tolerated and significantly reduced the duration and severity of colds due to picornaviruses in adults. gether cause ∼50% of colds annually [1] . the incidence of colds due to picornaviruses increases to 60%-80% during peak months in the fall and spring in the northern hemisphere [1, 2] . no antiviral therapy of proven value for colds due to picornaviruses is currently available, and prior studies of investigational antivirals did not show therapeutic benefit for established colds [3, 4] . the pathogenesis of cold symptoms is not fully understood [5] , and the importance of ongoing viral replication to symptom causation remains uncertain. pleconaril is a novel, orally absorbed viral capsidfunction inhibitor that specifically inhibits the replication of ∼90% of rhinoviruses and 199% of enteroviruses [6] [7] [8] . in experimentally induced human coxsackievirus a21 infections, oral pleconaril significantly reduced viral shedding and illness measures [9] . more recently, retrospective analysis of 2 phase ii randomized, double-blind, placebo-controlled studies found that pleconaril treatment provided clinical benefit for colds due to picornaviruses in previously healthy adults [10] . consequently, 2 large, randomized, double-blind, placebo-controlled, multicenter trials were conducted to evaluate the efficacy and safety of oral pleconaril for treatment of naturally occurring colds presumptively due to picornaviruses in adults. participants in both studies were otherwise healthy adults (age, р18 years) with self-diagnosed colds who were enrolled within 24 h after symptom onset. participants had moderate to severe rhinorrhea and у1 other respiratory symptom (nasal congestion, cough, or sore throat) that was rated moderate or greater in severity. subjects were excluded if they had fever (oral temperature, 137.8њc), if they had allergic rhinitis that had been treated within the previous 2 weeks, if they had received asthma treatment within the previous 2 months, or if they had chronic cough, any known immunodeficiency, or an underlying medical condition that would confound the study results. pregnant or nursing women were excluded, and urine pregnancy tests were done at entry. smokers were allowed. the institutional review board of each participating site approved the protocol, and written informed consent was obtained from each participant at the time of enrollment into the study. participants were compensated for participation. study design and drug administration. two prospective, multicenter, randomized, double-blind trials of identical design were conducted from august through november 2000; each enrolled participants from geographically diverse areas of the united states (150 sites) and canada (47 sites). participants were randomized in a 1:1 ratio to receive either pleconaril at 400 mg (two 200-mg tablets; picovir; viropharma) or matching placebo tablets 3 times per day for 5 days. to enhance oral absorption, participants were instructed to take the study medication within 15 min after a meal or snack. randomization was stratified by the subject's preenrollment smoking status and preenrollment use of cold symptom-relief medication to ensure that these subjects were balanced between the treatment arms. acetaminophen and dextromethorphan were provided for disabling symptoms, because these agents were unlikely to affect the prominent nasopharyngeal symptoms of colds. the concomitant use of prescription and other over-the-counter cold symptom-relief medications was not permitted. clinical monitoring. participants were evaluated at enrollment and again on study days 3, 6 (end of treatment), and 18 (end of the study) for clinical assessment and obtainment of samples for laboratory testing. study personnel contacted participants every other day by telephone until their cold had resolved or through day 18. participants recorded the severity of 6 individual cold symptoms (rhinorrhea, nasal congestion, sore throat, cough, malaise, and myalgia) in study diaries twice daily, grading each as "not present," "mild" (noticeable but not bothersome), "moderate" (bothersome), or "severe" (limiting usual activities), which were scored as 0, 1, 2, and 3, respectively, for data analysis. once per day, subjects also recorded data on the number of facial tissues used, sleep disturbance, impairment of daily activity as a result of cold symptoms, and use of cold symptom-relief medications or other medications for any reason. safety laboratory studies (hematological study, clinical chemistry, and urinalysis) and physical assessments were done at enrollment and on study day 6. virology assessments. nasal mucus samples were obtained for virological studies at baseline and on study days 3 and 6. subjects were asked to blow nasal mucus directly onto plastic wrap; mucus was induced, if necessary [11] . the sample was transferred into a tube containing viral transport medium (starswab multitrans collection and transport system; starplex scientific) and shipped for storage at ϫ80њc until assayed. the presence of picornavirus rna in nasal mucus samples was identified using a real-time, quantitative rt-pcr assay (taqman; applied biosystems). the pcr primers and probe used in the taqman assay were derived from conserved sequences within the 5 nontranslated region of sequenced human rhinovirus (hrv) genomes. the forward primer sequence was 5 -gtgaagagcc(g/c)c(a/g)tgtgct-3 , corresponding to nucleotides 414-432 of hrv89. the reverse primer sequence was 5 -gct(g/c)cagggttaaggttagcc-3 , corresponding to the reverse complement of nucleotides 461-481 of hrv89. the double-labeled fluorescent probe sequence was 5 -(fam)-tgagtcctccggcccctgaatg-(tamra)-3 , corresponding to nucleotides 438-459 of hrv89. in this assay, the lower limit of detection for the virus (hrv1b) used to generate the standard curve was 10 pfu/ml, or 10,550 genome equivalents/ml (211 genome equivalents per reaction). if all 3 samples obtained from a patient had negative or indeterminate results for this assay, the baseline sample was retested by a modification of an enzyme-linked oligosorbent rt-pcr assay, which detects all prototype rhinoviruses and culturable enteroviruses [12] [13] [14] [15] . a patient was considered to be positive for picornavirus infection if nasal mucus specimens tested positive with either rt-pcr assay on any sampling day. for subjects who had positive rt-pcr results, an aliquot of the baseline mucus sample (200 ml) was submitted for viral culture on monolayers of hela-i cells by a previously described technique [16] . if culture of the baseline sample yielded positive results, aliquots of samples obtained on days 3 and 6 were also cultured. taqman assays were performed at viromed biosafety laboratories (st. paul, minnesota), enzyme-linked oligosorbent (3) 17 (3) 13 (2) 19 (4) lost to follow-up 11 (2) (7) 47 (9) 31 (6) 34 (7) rt-pcr assays were performed at viropharma incorporated (exton, pennsylvania), and viral cultures were performed at the university of virginia (charlottesville) or the university of rochester medical center (rochester, new york). study personnel at each laboratory were blinded to treatment and sample collection day. efficacy end points. the primary efficacy population included any randomized participant with у1 nasal mucus sample that tested positive for picornavirus rna on any sampling day by either quantitative or qualitative rt-pcr methods. the secondary efficacy population included all randomized participants. these participants are referred to as the intent-to-treat infected (itt-i) and intent-to-treat (itt) populations, respectively. the primary end point was the time from initiation of therapy to alleviation of illness, defined as the number of days until complete resolution of rhinorrhea and the other 5 cold symptoms self-assessed as absent or mild for у48 h without use of cold symptom-relief medication. prospectively defined secondary end points were the time to subject-assessed "no cold," times to complete resolution of individual symptoms, total cold symptom severity scores, tissue counts, proportion of nights with disturbed sleep, duration of cold symptom-relief medication use, and frequency of viral shedding in nasal mucus. other end points were the time to у50% reduction in symptom score and changes in viral rna levels over time. data analysis. the distribution of time to resolution of symptom scores was estimated by the kaplan-meier method [17] , and the wilcoxon-gehan statistic [18] was used to test the difference in median resolution times between treatment groups. these analyses included stratification for smoking status and preenrollment use of cold symptom-relief medication. combined analyses of both studies also included stratification by study. in these time-to-event analyses, subjects who discontinued the study were included up to the point of the last recorded observation. the distribution of time to у50% reduction from baseline in total cold symptom severity score was analyzed in the same manner. the treatment effect for change from baseline in daily total symptom severity score and total symptom severity score over the 18-day study was analyzed by analysis of covariance, with effects for treatment, study, smoking status, preenrollment use of cold symptom-relief medication, and baseline total symptom severity score. the last observation carried forward was used to impute missing individual symptom severity scores. treatment effect for presence of picornavirus by culture and percentage of subjects using cold medications was evaluated using fisher's exact test. analysis of variance was used to compare the treatment groups for reduction in virus levels (measured by pcr) from baseline to days 3 and 6, proportion of nights with sleep disturbance, and tissue use. all study participants who received у1 dose of study medication were included in the safety analysis. adverse events that began or worsened at any time after receipt of the first dose of study drug through 5 days after the last dose were summarized. all analyses were done using sas statistical software, version 6.12 (sas institute) [19] , and a 2-sided test at the 5% level was used for all comparisons. sample size. calculations indicated that enrollment of 1000 subjects in each study was required to detect a 25% relative difference between treatment groups in the proportion of picornavirus-infected subjects reaching the primary end point (or an estimated 2-day difference in median time) with 90% power (2-sided test at the 5% level of significance [20] ). the 2 studies randomized 2096 participants (1046 in the pleconaril group and 1050 in the placebo group); 190% of subjects completed treatment (table 1). the (30) 107 (32) 119 (33) 120 (35) a current smokers or those who stopped smoking р3 months before the start of study. most common reason that subjects did not complete treatment was an adverse event (3% of subjects each in the pleconaril and placebo groups). overall, 65% of participants were infected with picornavirus, with a narrow range (62%-68%) across treatment groups in each study. the pleconaril and placebo groups were similar at baseline with regard to relevant demographic and illness characteristics (table 2). the mean age of the itt-i population was 36 years, 69% were female, and 28% were smokers. the median time from symptom onset to receipt of the first dose of study drug was 20 h. illness resolution. among the picornavirus-infected (itt-i) population, the time to reach the primary end point of illness alleviation was significantly shorter among pleconaril-treated subjects than among placebo recipients in each study (figure 1). overall, pleconaril-treated patients experienced a 1-day reduction in the median duration of illness, compared with placebo-treated subjects ( ; table 3 ). the treatment effect p ! .001 was similar for subjects who had positive results of viral cultures at baseline (combined median time to illness alleviation, 7.9 days for the placebo group and 6.8 days for the pleconaril group; ) and for those with negative results of viral p p .002 culture at baseline (7.0 days for the placebo group and 6.0 days for the pleconaril group; ). no treatment effect was p p .048 observed among participants without detectable picornavirus infection (table 3 ). in the itt population, the magnitude of the treatment benefit was smaller than that observed among itt-i subjects but favored pleconaril in both studies. among itt-i subjects, the self-assessed times to "no cold" and to resolution of each individual cold symptom were reduced in pleconaril recipients (table 4). the total cold symptom alleviation of illness is defined as the absence of rhinorrhea and presence of no or mild other cold symptoms for у48 h without use of cold symptom-relief medication. itt, intent-to-treat; itt-i, intent-to-treat infected. severity score was reduced by 19% over the duration of the study for pleconaril recipients (table 4) , who also experienced significant reductions from baseline in symptom severity scores by day 2 of treatment, compared with placebo recipients (figure 2). a reduction in total symptom severity score of у50% occurred earlier among pleconaril recipients than among placebo recipients (combined medians of 2.9 and 3.9 days, respectively; ). pleconaril treatment was associated with fewer tissues p ! .001 used for nose blowing (24% reduction), fewer nights of sleep disturbance (1 night reduction), and fewer days of cold symptom-relief medication use (table 4) . analyses of the combined study results were conducted with figure 2 . change from baseline value in total cold symptom severity scores among picornavirus-infected subjects, days 1-6. pleconaril recipients experienced significant ( ) reductions from baseline by day 2 p ! .05 of treatment in each study. "most common" is defined as incidence of у3% in either treatment group. a total no. of subjects who experienced у1 adverse event, regardless of its relationship to use of the study drug. a cox regression model to assess 2-way interaction effects between treatment and the variables age, sex, race, smoking status, and preenrollment cold medication use. the only interaction with significant impact on treatment effect was smoking status ( ). the time to reach the primary end point was p p .013 shorter for pleconaril recipients than for placebo recipients among nonsmokers (6.0 vs. 7.3 days; ), but it was not p ! .001 different among smokers (8.3 vs. 7.4 days; ). however, p p .692 additional analysis regarding effects of pleconaril for smokers is limited by the fact that smokers constituted only 28% of the study population. virologic analysis. among subjects with detectable picornavirus rna in baseline nasal mucus samples, 827 (65%) of 1263 subjects had positive results of viral cultures. acid stability testing of 69 randomly selected isolates determined that 68 (99%) were acid-labile and presumed rhinoviruses, whereas 1 was acid-stable and a presumed enterovirus. among those who had positive results of culture at baseline, fewer pleconaril recipients had positive culture results on day 3 (range, day 2-4) than did placebo recipients (53% vs. 72%; ; figure 3 ). additional analysis of the subset of subjects p ! .001 who had samples obtained for culture on day 2 revealed that significantly fewer pleconaril recipients than placebo recipients had positive viral culture results (27 [60%] of 45 subjects vs. 49 [84%] of 58 subjects; ). p p .007 nasal mucus viral rna levels decreased rapidly in both pleconaril and placebo treatment groups. subjects in the pleconaril group showed a larger median percentage reduction from baseline in virus levels on study day 3, compared with subjects in the placebo group (97.7% vs. 90.3%; ). by day 6 (range, p ! .001 day [5] [6] [7] [8] [9] , the median percentage reduction in virus levels was 199% in both treatment groups. safety. pleconaril was generally well tolerated. the most commonly reported adverse events were headache, diarrhea, and nausea (table 5); у95% of adverse events were mild or moderate in severity. four subjects receiving pleconaril and 2 receiving placebo reported a serious adverse event, none of which was considered by the investigators to be related to study drug except for 1 case of inadvertent overdose of pleconaril (with no adverse sequelae). no differences were noted in vital signs or physical examination findings in either treatment group, and there were no clinically significant laboratory abnormalities (data not shown). the only laboratory findings associated with pleconaril use were small median increases from baseline values in the pleconaril group for nonfasting serum cholesterol levels (an increase of 5 mg/dl [or 3%], compared with a decrease of 4 mg/dl [or 2%] in placebo recipients; ) and for platelet counts (an in-p ! .001 crease of platelets/mm 3 [or 6%], compared with an 3 15 ϫ 10 increase of platelets/mm 3 [or 3%] in placebo recipients; 3 7 ϫ 10 ). p ! .001 these prospective, double-blind, placebo-controlled studies found that early pleconaril treatment significantly reduces the duration and severity of colds due to picornaviruses in adults; these findings establish pleconaril as the first antiviral to have proven therapeutic value for such illnesses. these results confirm and extend those of an earlier retrospective analysis of adults with colds due to picornaviruses, which found a 1.5-day reduction in the time to alleviation of illness in pleconaril recipients, compared with placebo recipients [10] . pleconaril use was associated with significant reductions in symptom severity scores and the frequency of recovery of picornaviruses within 1 day after initiation of therapy. in addition, pleconaril therapy resulted in a significant reduction in the duration of each individual cold symptom monitored in the study, a finding consistent with the hypothesis that ongoing viral replication is an important contributor to the pathogenesis of cold symptoms. the rapid decrease in viral rna levels in both pleconaril and placebo groups illustrates the importance of early initiation of antiviral therapy. we also observed that substantial proportions of both placebo recipients and, less often, pleconaril recipients continued to have positive culture results on study day 6 or later, although at very low levels of viral rna. this prolonged recovery of virus is consistent with earlier data from natural and experimentally induced rhinovirus infections [21, 22] , but it raises the issue of emergence of drug-resistant variants. in the current studies, viruses with reduced susceptibility to pleconaril (у10-fold change from baseline value) were recovered during or after therapy from ∼10% of patients who received pleconaril. however, subgroup analyses indicate that clinical benefit for these participants was as good as or better than that for pleconaril recipients with no reduction in virus susceptibility to the drug (unpublished data). further phenotypic and genetic characterization of viruses from these and other pleconaril trials is ongoing, to determine relationships between in vitro susceptibility and clinical outcomes. pleconaril was shown to be safe and generally well tolerated. compared with placebo, there were only small (2%) excess frequencies of headaches, nausea, and diarrhea in patients receiving pleconaril. the small increases from baseline in cholesterol levels and platelet counts are not clinically significant, an observation that was confirmed in a subsequent 6-week prophylaxis study (unpublished observations). in that study, an excess of mild or moderate menstrual disorders (most commonly breakthrough bleeding or spotting) was reported from women taking oral contraceptives and pleconaril. subsequent investigations revealed that pleconaril induces hepatic cytochrome p-450 3a enzymes. pleconaril reduced the area under the curve of plasma levels of ethinyl estradiol by 34% following single-dose administration (g. rhodes, personal communication). retrospective review of all randomized, placebo-controlled trials in which pleconaril was administered for 5-7 days revealed that menstrual irregularities were reported by 3.5% of 310 pleconaril-treated women who were using oral contraceptives and by none of 291 placebo-treated women. none of the menstrual irregularities led to discontinuation of treatment. additional studies are ongoing to better characterize the magnitude and duration of cytochrome p-450 3a induction and to determine the clinical significance for coadministration of pleconaril with other drugs metabolized by cytochrome p-450 3a. one limitation of the current studies is that most participants were generally healthy young adults. other studies have established that rhinoviruses can cause both upper and lower respiratory tract complications, including asthma exacerbations in both adults and children [23] [24] [25] , acute exacerbations of chronic obstructive pulmonary disease [26] , acute otitis media in children [27] , and sinusitis in adults [13] . others at risk for lower respiratory complications due to rhinovirus infection include patients with cystic fibrosis [28] , elderly individuals [29] , and immunocompromised persons [30] . the positive findings in the current trials indicate that studies of pleconaril should be extended to children, smokers, and those with underlying airway disease. in summary, early pleconaril treatment of colds due to picornaviruses reduces the duration and severity of illness in adults. pleconaril at this dosage was well tolerated, although additional data are needed to better characterize its potential for drug interactions. tucson: a. adamczyk; brandywine clinical research, downingtown, pa: l. alwine; credit valley hospital armstrong tn: i. biaggioni; primary physicians research brankston; alpine clinical research center central austin internists herridge community health clinic w. gaman; allergy and asthma practice miami valley clinical trial resources children's & adult's clinical research foundation, chapel hill, nc: f. henderson; new hanover medical research, wilmington, nc: c. herring; west texas medical associates, san angelo: d. herrington; nsdea welstar health system, marietta medical group at marple commons d. orchard; nfld drive family practice pierone; clinical research ar: k. roberts; university of missouri columbia, columbia yeoman; northern california research, fair oaks: d. young; the male health centres viruses and bacteria in the etiology of the common cold frequency and natural history of rhinovirus infections in adults during autumn intranasal recombinant alfa-2b interferon treatment of naturally occurring common colds intranasal pirodavir (r77,975) treatment of rhinovirus colds clinical virology analysis of three structurally related antiviral compounds in complex with human rhinovirus 16 treatment of the picornavirus common cold by inhibitors of viral uncoating and attachment activity of pleconaril against enteroviruses clinical activity of pleconaril in an experimentally induced coxsackievirus a21 respiratory infection oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: efficacy and tolerability in phase ii clinical trials improved method for collection of nasal mucus detection of human rhinovirus rna in nasal washings by pcr detection of rhinovirus in sinus brushings of subjects with acute community-acquired sinusitis by reverse transcription-pcr rapid detection of human rhinoviruses in nasopharyngeal aspirates by a microwell reverse transcription-pcr-hybridization assay detection of herpes simplex virus dna in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization comparative susceptibility of human embryonic fibroblasts and hela cells for isolation of human rhinoviruses nonparametric estimation from incomplete observations cancer therapy: prognostic factors and criteria sas/stat user's guide. version 6 tables of the number of patients required in clinical trials using the log rank test the seattle virus watch. v. epidemiologic observations of rhinovirus infections, 1965-1969, in families with young children sites of rhinovirus recovery after point inoculation of the upper airway respiratory viruses and exacerbations of asthma in adults the relationship between upper respiratory infections and hospital admissions for asthma: a time trend analysis association of rhinovirus infections with asthma respiratory viral infections in adults with and without chronic obstructive pulmonary disease detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction severe viral respiratory infections in infants with cystic fibrosis risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study community respiratory virus infections in immunocompromised patients with cancer we thank marissa seligman and sandra norris for editorial assistance in manuscript preparation. key: cord-001800-644lf8vn authors: biggerstaff, matthew; reed, carrie; swerdlow, david l.; gambhir, manoj; graitcer, samuel; finelli, lyn; borse, rebekah h.; rasmussen, sonja a.; meltzer, martin i.; bridges, carolyn b. title: estimating the potential effects of a vaccine program against an emerging influenza pandemic—united states date: 2015-05-01 journal: clin infect dis doi: 10.1093/cid/ciu1175 sha: doc_id: 1800 cord_uid: 644lf8vn background. human illness from influenza a(h7n9) was identified in march 2013, and candidate vaccine viruses were soon developed. to understand factors that may impact influenza vaccination programs, we developed a model to evaluate hospitalizations and deaths averted considering various scenarios. methods. we utilized a model incorporating epidemic curves with clinical attack rates of 20% or 30% in a single wave of illness, case hospitalization ratios of 0.5% or 4.2%, and case fatality ratios of 0.08% or 0.53%. we considered scenarios that achieved 80% vaccination coverage, various starts of vaccination programs (16 or 8 weeks before, the same week of, or 8 or 16 weeks after start of pandemic), an administration rate of 10 or 30 million doses per week (the latter rate is an untested assumption), and 2 levels of vaccine effectiveness (2 doses of vaccine required; either 62% or 80% effective for persons aged <60 years, and either 43% or 60% effective for persons aged ≥60 years). results. the start date of vaccination campaigns most influenced impact; 141 000–2 200 000 hospitalizations and 11 000–281 000 deaths were averted when campaigns started before a pandemic, and <100–1 300 000 hospitalizations and 0–165 000 deaths were averted for programs beginning the same time as or after the introduction of the pandemic virus. the rate of vaccine administration and vaccine effectiveness did not influence campaign impact as much as timing of the start of campaign. conclusions. our findings suggest that efforts to improve the timeliness of vaccine production will provide the greatest impacts for future pandemic vaccination programs. factors, including the size, speed, and number of waves of the pandemic outbreak, the number of doses administered, the timing of the vaccination program relative to the spread of the novel influenza virus, and the vaccine effectiveness (ve) [10] . to help public health officials and policy makers evaluate the impact of a hypothetical vaccination program against a future influenza pandemic, we developed a spreadsheet-based model that allowed quick exploration of the number of hospitalizations and deaths averted in the united states under various vaccination scenarios. we adapted a spreadsheet model (excel, microsoft corporation, redmond, washington) that was originally created to estimate the effects of a vaccine program against influenza a(h1n1) pdm09 [10] . the model user enters an epidemic curve (the number of persons becoming ill by time) and other variables that define the impact of both the pandemic and the vaccination campaign. these variables include the timing of the vaccination program relative to the introduction of no. of initial cases 100 [11] cumulative attack rate, % 20 or 30 [11] case hospitalization ratio, % [11, 12] all cases into the united states, the number of doses administered per week and the allocation by age group, the clinical attack rate, and the ratios of health outcomes to the number of cases (eg, the case hospitalization and case fatality ratios) ( table 1) . we adjusted calculations to account for individuals who were naturally immunized through infection but who may still be vaccinated. to estimate the number of infections prevented by the vaccination program, we took [the number of persons fully vaccinated 2 weeks prior to the current week in the model] × [the probability of not having been previously infected with influenza before being fully vaccinated and having developed immunity] × [ probability of becoming infected with influenza after being fully vaccinated and having developed immunity] × [ve] [10] . we utilized standardized epidemic curves, using 20% and 30% clinical attack rates in 1 wave of illnesses and different levels of clinical severity and assumed that the pandemic began with 100 persons initially infected [11] (table 1) . for our model, we assumed that 2 doses of vaccine administered 3 weeks apart would be needed to be fully effective, based on data indicating that previous h5 and h7 influenza vaccines have low immunogenicity [15] [16] [17] . we further assumed that, during a pandemic with moderate or high mortality, demand for vaccine would be such that 80% of the us population would receive 2 doses of vaccine. we prioritized persons returning for their second dose of vaccine over persons who were receiving their first dose. we also assumed that vaccine was allocated in a pattern similar to the doses administered among 4 age groups (6 months-9 years, 10-19 years, 20-59 years, and ≥60 years) during the 2012-2013 influenza season [18] (table 1) . once 80% of an age group was fully vaccinated, we assumed vaccination would end in that age group. remaining vaccine would then be allocated to other age groups until they reached 80% coverage. we allowed for a 2-week delay in protection against the virus after administration of the second dose of the vaccine [13] . we ran multiple scenarios to explore the effects of the quantity of the doses administered, the timing of the vaccine program, and the ve of the first and second doses. we first assumed that the program would administer either 10 million doses per week (approximating the maximum number of doses administered per week during seasonal influenza programs [14] ) or 30 million doses per week; the latter has yet to be achieved during seasonal influenza vaccination programs. to explore the effects of timing of the vaccine program, we modeled programs starting at 5 different time points, separated by 8-week intervals: 16 weeks before, 8 weeks before, the same week as, 8 weeks after, and 16 weeks after the first cases of the novel influenza virus were introduced into the united states. we also assumed that 1 dose of vaccine was 0% effective for figure 1 . the estimated epidemic curve without vaccination and the cumulative number of persons protected by an influenza vaccination program with the following assumptions: an overall clinical attack rate of the influenza pandemic of 20% or 30%; administered 10 million (left) or 30 million (right) vaccine doses; vaccination programs that begin 8 or 16 weeks before, the same week, or 8 or 16 weeks after the first cases of a novel influenza virus occur in the united states (us); and the vaccine effectiveness (ve) equivalent to the h1n1pmd09 monovalent vaccine. 2009 h1n1-like ve: 2 doses of vaccine administered 3 weeks apart required to be fully effective (62% for persons aged <60 years and 43% for persons ≥60 years) in protecting against subclinical and clinical cases, hospitalizations, and deaths. we assumed 1 dose of vaccine to be 0% effective for all age groups. all age groups and 2 doses of vaccine were 62% effective in protecting against subclinical and clinical cases, hospitalizations, and deaths for persons aged <60 years and 43% for persons aged ≥60 years (table 1) . these values were based on the ve of the monovalent, inactivated, nonadjuvanted influenza a(h1n1)pdm09 vaccine [10] . for the second scenario, we clinical attack rate of the influenza pandemic is 20% and the overall case fatality ratio is 0.53% (high-severity scenario); 10 million doses (left) or 30 million doses (right) of vaccine are administered each week; the vaccination program begins 16 weeks after, 8 weeks after, the same week as, 8 weeks before, and 16 weeks before the first cases of a novel influenza virus occur in the united states; and the efficacy is "h1n1pmd09 monovalent vaccine-like." 2009 h1n1-like vaccine effectiveness: 2 doses of vaccine administered 3 weeks apart required to be fully effective (62% for persons aged <60 years and 43% for persons ≥60 years) in protecting against subclinical and clinical cases, hospitalizations, and deaths. we assumed 1 dose of vaccine to be 0% effective for all age groups. assumed a high ve due to the use of higher concentrations of hemagglutinin antigen [19] or the addition of an adjuvant to the vaccine [20] . in the high ve scenario, we assumed some ve with 1 dose (40% ve for persons aged <60 years and 30% for persons ≥60 years) and higher ve with 2 doses (80% effective for persons aged <60 years and 60% for persons ≥60 years) ( table 1) . clinical attack rate of the influenza pandemic is 20% and the overall case fatality ratio is 0.084% (low-severity scenario); 10 million doses (left) or 30 million doses (right) of vaccine are administered each week; the vaccination program begins 16 weeks after, 8 weeks after, the same week as, 8 weeks before, and 16 weeks before the first cases of a novel influenza virus occur in the united states; and the efficacy is "h1n1pmd09 monovalent vaccine-like." 2009 h1n1-like vaccine effectiveness: 2 doses of vaccine administered 3 weeks apart required to be fully effective (62% for persons aged <60 years and 43% for persons ≥60 years) in protecting against subclinical and clinical cases, hospitalizations, and deaths. we assumed 1 dose of vaccine to be 0% effective for all age groups. to calculate the number of vaccine-associated averted outcomes (hospitalizations and deaths), we assumed that 50% of infected cases were symptomatic and either 1.05% of symptomatic cases were hospitalized and 0.084% of symptomatic cases would die (low-severity scenario) or 4.2% of symptomatic cases were hospitalized and 0.53% of symptomatic cases would die (high-severity scenario) ( table 1) . we adjusted the risk of hospitalization and death by age group ( table 1) . the values for hospitalizations and deaths were based on estimates predicted for a pandemic with high clinical severity, and the adjustments for age were based on historic pandemics [11, 12] . for the scenario with a cumulative clinical attack rate of 20%, without any other intensive interventions, the simulated pandemic peaked in the united states 20 weeks after the introduction of the first 100 cases and resulted in 127 000 000 infections, 63 500 000 clinical cases, and 669 000 hospitalizations and 54 000 deaths in the low-severity scenario or 2 700 000 hospitalizations and 336 000 deaths in the high-severity scenario (figures 1-3) . for the scenario with a cumulative clinical attack rate of 30%, the simulated epidemic peaked 12 weeks after the start and resulted in 188 000 000 infections, 94 000 000 clinical cases, and 1 070 000 hospitalizations and 86 000 deaths (low-severity scenario) or 4 300 000 hospitalizations and 538 000 deaths (high-severity scenario) (figure 1 ). vaccination programs distributing 10 million doses per week would take 54 weeks to achieve 80% coverage of a 2-dose vaccine series among all age groups, whereas programs distributing 30 million doses per week would take 21 weeks (figure 1 ). for an influenza pandemic with a 20% overall cumulative attack rate and high clinical severity, we estimated that a vaccination program beginning 8 weeks before the pandemic started in the united states and that administered 10 million doses of vaccine with the moderate ve per week could avert 568 000 hospitalizations and 71 000 deaths (21% reduction relative to no vaccine). starting the vaccination program 16 weeks before the pandemic started would avert 777 000 hospitalizations and 97 000 deaths (29% reduction) (tables 2 and 3; figures 2 and 3) . a vaccine program administering 30 million doses per week that started 8 or 16 weeks before the pandemic would avert 1 000 000 hospitalizations and 130 000 deaths (39% reduction) (tables 2 and 3 ; figures 2 and 3) . assuming that the ve would be 80% and 60% for persons aged <60 years and ≥60 years, respectively (compared with the base assumption of 62% and 43% for persons aged <60 years and ≥60 years, respectively) would further reduce hospitalizations and deaths by at least an additional 10% relative to no vaccine for both the 10 and 30 million administration scenarios (tables 2 and 3) . for an influenza pandemic with a 30% overall cumulative attack rate and high clinical severity, we estimated that a vaccination program beginning 8 weeks before the pandemic started in the united states that administered 10 million doses of vaccine with the moderate ve per week could avert 570 000 hospitalizations and 71 000 deaths (13% reduction relative to no vaccine). starting the vaccination program 16 weeks before the pandemic started would avert 924 000 hospitalizations and 116 000 deaths (21% reduction) (tables 2 and 3 ; figures 2 and 3) . a vaccine program administering 30 million doses per week that started 8 weeks before the pandemic would avert more than 1 400 000 hospitalizations and 180 000 deaths (33% reduction), whereas one that started 16 weeks before the pandemic would avert 1 700 000 hospitalizations and 207 000 deaths (38% reduction) (tables 2 and 3 ; figures 2 and 3 ). using the high ve would further reduce hospitalizations and deaths relative to no vaccine for both the 10 and 30 million administration scenarios by at least an additional 7% (tables 2 and 3) . for an influenza pandemic with a 20% overall cumulative attack rate and high clinical severity, we estimated that a vaccination program beginning the same week as the pandemic started in the united states that administered 10 million doses of vaccine with the moderate ve per week could avert 375 000 hospitalizations and 47 000 deaths (14% reduction). a vaccine program administering 30 million doses per week that started the same week as the pandemic would avert 916 000 hospitalizations and 114 000 deaths (34% reduction) (tables 2 and 3 ; figures 2 and 3) . using the high ve would further reduce hospitalizations and deaths by at least an additional 7% relative to no vaccine for both the 10 million and 30 million administration scenarios (tables 2 and 3) . for an influenza pandemic with a 30% overall cumulative attack rate and high-severity scenario, we estimated that a vaccination program beginning the same week as the pandemic started in the united states that administered 10 million doses of vaccine with the moderate ve per week could avert 260 000 hospitalizations and 32 000 deaths (6% reduction) (tables 2 and 3 ; figures 2 and 3) . a vaccine program administering 30 million doses per week would avert 777 000 hospitalizations and 97 000 deaths (18% reduction) (tables 2 and 3 ; figures 2 and 3) . using the high ve would reduce hospitalizations and deaths by at least an additional 4% relative to no vaccine for both the 10 million and 30 million administration scenarios (tables 2 and 3 ). for an influenza pandemic with a 20% overall cumulative attack rate and high clinical severity, we estimated that a vaccination program beginning 8 weeks after the pandemic started in the united states that administered 10 million doses of vaccine with the moderate ve per week could avert 183 000 hospitalizations and 23 000 deaths (6.8% reduction relative to no vaccine). beginning the vaccination program 16 weeks after the pandemic started would avert 17 000 hospitalizations and 2000 deaths (0.6% reduction) (tables 2 and 3; figures 2 and 3) . a vaccine program administering 30 million doses per week that started 8 weeks after the pandemic would avert more than 538 000 hospitalizations and 67 000 deaths (20% reduction), whereas one that started 16 weeks after the pandemic would avert 50 000 hospitalizations and 6000 deaths (1.9% reduction) (tables 2 and 3 ; figures 2 and 3) . using the high ve would reduce hospitalizations and deaths by at least an additional 1% relative to no vaccine for both the 10 million and 30 million administration scenarios (tables 2 and 3) . for an influenza pandemic with a 30% overall cumulative attack rate and high clinical severity, we estimated that no vaccination program that began 8 or 16 weeks after the pandemic started in the united states would avert more than 36 000 hospitalizations and 4600 deaths (<1% reduction), regardless of whether 10 million or 30 million doses of vaccine per week with the moderate ve were administered (tables 2 and 3 ; figures 2 and 3) . using the high ve would reduce hospitalizations and deaths by no more than 5% relative to no vaccine for the vaccination programs beginning 8 weeks after the pandemic started in the united states. for the high ve scenarios starting 16 weeks after the pandemic started in the united states, no additional reductions in hospitalizations or deaths were observed (tables 2 and 3) . in our analysis, the clinical attack rate and case hospitalization and case fatality ratios had the greatest impact on the number of severe outcomes averted in the united states, whereas the vaccination program factor with the greatest impact was the timing of the start of vaccination relative to the start of a pandemic. for example, under the 30% clinical attack rate and 30 million doses per week scenario, a vaccination program starting 16 weeks before the start of the pandemic in the united states results in a 38% reduction in hospitalizations and deaths. delaying the start of vaccination to the same week as the pandemic starts in the united states drops the reductions to 18%. the number of vaccine doses administered each week is also very important. decreasing the doses administered to 10 million per week causes the impact of vaccination in the above 2 scenarios to decline to 21% and 6% reductions, respectively. the assumptions related to ve of the first and second doses were relatively less important. this study highlights several key components to pandemic influenza preparedness, especially for a severe pandemic, including the importance of ensuring readiness to initiate large-scale vaccination programs as early as possible and ideally before the introduction of a novel influenza virus into the united states [18, 21] . factors that may impact vaccine dose availability include how soon we develop an appropriate vaccine virus candidate, growth characteristics of vaccine virus candidates, influenza vaccine production capacity, efficiency of vaccine allocation and distribution, and vaccine administration capacity. increased investment and research in vaccine production technologies, including the use of cell-derived recombinant proteins [22] , virus-like particles [23] , or adjuvants (by conserving the use of hemagglutinin antigen), have the potential to increase the speed with which the number of vaccine doses can be produced. also important is the need to identify ways to invest in improvements that will notably increase the capacity to administer large number of doses of pandemic influenza vaccine. currently, the peak administration rate for seasonal influenza in the united states is between 5 and 12 million doses per week. the cdc is working with state and local health officials and vaccine providers to identify means to enhance vaccination administration capabilities. in addition to exercising large-scale b two doses of vaccine administered 3 weeks apart required to be fully effective (62% for persons aged <60 years and 43% for persons ≥60 years) in protecting against subclinical and clinical cases, hospitalizations, and deaths. we assumed 1 dose of vaccine to be 0% effective for all age groups. c two doses of vaccine administered 3 weeks apart required to be to fully effective (80% for persons aged <60 years and 60% for persons ≥60 years) in protecting against subclinical and clinical cases, hospitalizations, and deaths. we assumed 1 dose of vaccine is 40% effective for persons aged <60 years and 30% for persons ≥60 years. mass vaccination clinics, this includes increasing partnerships with nontraditional vaccine providers, such as pharmacies, supermarket chains, and other community vaccine providers including diverse health, faith, and community based-organizations that reach vulnerable, at-risk, hard-to-reach, and minority populations [24, 25] . we evaluated a wide range of vaccine program initiation times relative to disease introduction in the united states in this article. although this information cannot be known in advance, beginning vaccination 16 weeks prior to the introduction of disease in the united states might be possible if the pandemic virus was identified, a stockpiled influenza vaccine were available and appropriate for use, officials were prepared to administer vaccine, and the decision to vaccinate was made at least 28 weeks before the establishment of the virus in the united states (based on current estimates of 12 weeks to fill and finish and begin distribution of stockpiled pandemic vaccine) [26] . this timeline is dependent on robust novel influenza virus surveillance that can identify influenza viruses with pandemic potential before widespread transmission has occurred. during the 2009 h1n1 pandemic, however, the first cases were identified in the united states after widespread transmission had already occurred, and no stockpiled vaccine or vaccine candidate seed viruses were available. in this example, the first doses of vaccine became available 26 weeks after identification of the first case in the united states and 8 weeks after the start of the main wave of pandemic illness in the fall of 2009 [27] . this situation is demonstrated by the scenarios beginning 8 or 16 weeks after the introduction of the virus into the united states [28] . this study has several limitations. most important, because infections with influenza a(h7n9) so far have been rare [5] , the modeled number of pandemic-related hospitalizations and deaths, and the numbers of such that would be prevented by a vaccination program, can only be considered as illustrative and are not based on the current epidemiology of h7n9 or other novel influenza virus illnesses. these results, therefore, should not be interpreted as a prediction of the impact of a widespread outbreak of h7n9 or any other novel influenza a virus with pandemic potential. additionally, we did not account for the effects of other interventions (eg, nonpharmaceutical interventions such as canceling mass gatherings or closing schools), the seasonality of when a novel virus might be introduced into the united states, or "waves of illness," which are thought to have occurred in 3 modern pandemics [1, 2] . these factors could slow the course of the pandemic and thereby increase the amount of time to initiate and complete a vaccine program, increasing the number of hospitalizations and deaths averted. for ease of estimation, we also did not account for any adverse events associated with vaccination or for the indirect effects of vaccination (eg, herd immunity). theoretically, accounting for indirect effects would likely increase the number of hospitalizations and deaths averted for those vaccination programs assumed to start before the pandemic; this effect would likely be lower for those programs assumed to start the same time as or after the pandemic. another important assumption is that 80% of the population would want to be vaccinated. this is distinctly different from recent seasonal influenza coverage estimates of approximately 45% [14] . we do not know the precise correlation between severity of an influenza pandemic and public demand for vaccination, but 80% coverage may be an overestimate. another potential limitation is that no data are available on the ve of a possible h7n9 vaccine. thus, we based our estimates of ve of either an nonadjuvanted influenza vaccine, using data from the 2009 h1n1 pandemic, or a hypothetical vaccine with high ve based on data from adjuvanted 2009 h1n1 vaccine ve estimates. limited data indicate that h7 vaccines have lower immunogenicity than seasonal influenza vaccines, which may result in lower ve [29, 30] . the population coverage or the effectiveness of a h7n9 or other future pandemic vaccine may be lower than what is assumed here, leading to a smaller number of averted outcomes. historically, influenza pandemics have been largely unpredictable events, and it is likely that the set of assumptions used in this study will vary from the actual events seen in the next pandemic, even if influenza a(h7n9) is the virus involved. however, the finding that variations in the timing of vaccination administration yield the greatest effect on the reduction in hospitalizations and deaths than do variations in rate of vaccine administration or effectiveness would likely remain consistent. continued research and investment in work that improves the timeliness of vaccine production and administration will have the greatest benefits in the event of another influenza pandemic. global epidemiology of influenza: past and present epidemiology of 2009 pandemic influenza a (h1n1) in the united states human infection with a novel avian-origin influenza a (h7n9) virus clinical findings in 111 cases of influenza a (h7n9) virus infection human infection with avian influenza a (h7n9) virus-update receptor binding by an h7n9 influenza virus from humans biological features of novel avian influenza a (h7n9) virus population-level antibody estimates to novel influenza a/h7n9 emergence of avian influenza a(h7n9) virus causing severe human illness-china effects of vaccine program against pandemic influenza a(h1n1) virus, united states standardizing scenarios to assess the need to respond to an influenza pandemic novel framework for assessing epidemiologic effects of influenza epidemics and pandemics sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic h1n1 virus in u.s. populations fluvaxview influenza vaccination coverage h5n1 avian influenza: preventive and therapeutic strategies against a pandemic a randomized clinical trial of an inactivated avian influenza a (h7n7) vaccine a phase i clinical trial of a per.c6 cell grown influenza h7 virus vaccine influenza vaccination coverage: how well did we do in 2012-13 randomized, double-blind controlled phase 3 trial comparing the immunogenicity of high-dose and standard-dose influenza vaccine in adults 65 years of age and older and pandemic vaccines, to prevent influenza hospitalizations during the autumn 2009 influenza pandemic wave in castellon, spain. a test-negative, hospital-based, case-control study final estimates for 2009-10 seasonal influenza and influenza a (h1n1) 2009 monovalent vaccination coverage-united states protective efficacy of a trivalent recombinant hemagglutinin protein vaccine (flublok(r)) against influenza in healthy adults: a randomized, placebo-controlled trial virus-like particle (vlp)-based vaccines for pandemic influenza: performance of a vlp vaccine during the 2009 influenza pandemic vaccinations administered during off-clinic hours at a national community pharmacy: implications for increasing patient access and convenience public health and faith community partnerships: model practices to increase influenza prevention among hard to reach populations news release: hhs boosts national capacity to produce pandemic flu vaccine surveillance for influenza during the 2009 influenza a (h1n1) pandemic-united states report to the president on reengineering the influenza vaccine production enterprise to meet the challenges of pandemic influenza low immunogenicity predicted for emerging avian-origin h7n9: implication for influenza vaccine design a recombinant viruslike particle influenza a (h7n9) vaccine disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention (cdc).supplement sponsorship. this article appears as part of the supplement titled "cdc modeling efforts in response to a potential public health emergency: influenza a(h7n9) as an example," sponsored by the cdc.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-257553-479x7av6 authors: kortepeter, mark g.; seaworth, barbara j.; tasker, sybil a.; burgess, timothy h.; coldren, rodney l.; aronson, naomi e. title: health care workers and researchers traveling to developing-world clinical settings: disease transmission risk and mitigation date: 2010-12-01 journal: clin infect dis doi: 10.1086/657115 sha: doc_id: 257553 cord_uid: 479x7av6 with the recent emphasis on funding and training opportunities for global health and humanitarian aid and the increased interest in the field, many health care workers and medical researchers are traveling from resource-replete to resource-limited settings. this type of travel brings unique disease risks not routinely considered for the business or vacationing traveler. this review provides practical advice for this special population of travelers, targeted to specific health care-related risks (needlestick, hemorrhagic fever viruses, severe viral respiratory disease, and tuberculosis), with suggestions for risk mitigation. retrovirals and antibiotics) on hand must all be considered (table 1) . pretravel advice for managing needlestick injuries should start with a discussion of prevention. needlesticks may be more likely to occur in crowded, rushed, and unfamiliar work settings [1] . advise hcws to set up a "sharps container" if one is not available. potential substitutes include an empty container, such as a soda can or plastic laundry detergent bottle. awareness of the disease epidemiology of the region and among potential patients will help in risk assessment should a needlestick injury occur. in addition to human immunodeficiency virus (hiv) and hepatitis viruses, other viruses can be transmitted by needlestick, such as dengue virus and other hemorrhagic fever viruses. other potential disease risks include syphilis, mycobacterial and rickettsial infections, trypanosomiasis, visceral leishmaniasis, and malaria [2] . malaria is particularly notable, because the risk of disease from needlestick may be higher than that of hepatitis viruses or hiv and because fatal and near-fatal cases have occurred as a result of diagnostic delays [3] . knowing the prevalence of drug resistance for needlestick-transmissible endemic diseases is also useful. if a needlestick injury occurs, hcws should note the source patient's region of origin, presenting complaints, history, and physical findings and perform a rapid hiv test or obtain a blood sample for later testing. wash the injury site thoroughly with warm water and soap. caustic antiseptics (eg, bleach and povidone iodide) are not recommended, because they may actually increase the risk of infection as a result of local tissue injury and recruitment of inflammatory cells [4] . a clear plan for follow-up assessments and points of contact is essential. given that diagnostic testing for hepatitis viruses and hiv may be limited at overseas destinations, hcws should consider completing baseline tests for them before travel. hepatitis b virus (hbv) is one of the most transmissible viruses related to needlestick and is generally more common in resource-limited settings than in the united states. the risk to unvaccinated persons is 37%-62% if the source is positive for hepatitis b e antigen (hbeag) and is 23%-37% if the source is negative for hbeag and positive for hepatitis b surface antigen (hbsag). transmission from environmental surfaces can occur even without a cutaneous injury [4] . fortunately, hbv vaccination is highly effective and eliminates this risk for the 90% who respond to the primary series. for those who do not respond, repeat vaccination may protect 30%-50%. before repeating the series, check antibody to hepatitis b core antigen and hbsag to ensure that the patient is not a chronic hbv carrier. deep intramuscular injection with a longer needle may be needed for larger recipients [4] . if serostatus is negative after 6 immunizations, there is no benefit to additional vaccine. seronegative hcws remain at high risk for hepatitis b if exposed, and hepatitis b immunoglobulin (0.06 ml/kg intramuscularly) should be offered after injury and repeated in 1 month [4] . hepatitis b immunoglobulin started within 1 week of exposure provides ∼75% protection [4] . tenofovir and lamivudine have activity against hbv, but there is no guidance for prophylactic use in this setting. consider administration of hepatitis b immunoglobulin before departure for seronegative hcws when concern exists for hbv exposure in a remote setting. passive antibody half-life is 3 weeks, and levels are measurable for ∼2 months [5] . although no prophylaxis for hepatitis c virus (hcv) exposure exists, the needlestick transmission risk is lower (1.8%), and up to 20% of transmitted infections resolve spontaneously. the incubation period is 4-12 weeks. exposed individuals should be monitored with hcv rna testing, if available. immediate treatment is not recommended in light of the significant toxicity and duration of therapy and because some infections will resolve spontaneously, but treatment should be considered if the hcv infection remains established after 12 weeks [6] . most feared is hiv infection after needlestick, but it has the lowest risk of transmission (0.3%). time to initiation of postexposure prophylaxis (pep) is critical. pep is most effective if started immediately and definitely should be started within 3 days of exposure; pep must be continued for 4 weeks [4, 7, 8] . unlike hiv treatment, for which the use of triple-drug combinations is an essential success factor, 3 drugs are unlikely to be significantly better than 2 for pep and paradoxically may be less effective, given that adherence to 3-drug regimens is poorer because of toxicity of the multidrug regimen. zidovudine monotherapy is 79% effective in preventing infection [7] . the world health organization (who) recommends using 2 nucleoside reverse-transcriptase inhibitors from the country's first-line regimen and 3 drugs if the source patient is known to be infected with drug-resistant hiv or if there is 115% resistance in the community [8] . the centers for disease control and prevention (cdc) recommends a 3-drug pep regimen if the source patient is known to be infected with hiv and the source device is a hollow-bore needle or has visible blood contamination. the cdc recommends zidovudine, stavudine, or tenofovir plus emtricitabine or lamivudine. when a third drug is included, both the cdc and who recommend a ritonavirboosted protease inhibitor, not nonnucleoside reverse-transcriptase inhibitors [8, 9] . hcws are strongly encouraged to bring postexposure medications to their destination. follow up for needlestick injury should include a high level of suspicion for any febrile illness in the weeks after the incident; serological tests for hiv, hepatitis, and syphilis at ∼3 months; and adherence support if antiretrovirals are given. hiv rna should be checked if there is an acute febrile illness after an hiv-positive needlestick exposure. if hcv rna measurements are available, they can be monitored at 2, 6, 12, and 24 weeks after a high-risk exposure. viral hemorrhagic fever (vhf) is an acute febrile syndrome, accompanied with a bleeding diathesis, caused by 4 families of single-stranded rna viruses (filoviruses, arenaviruses, bunyaviruses, and flaviviruses) [10, 11] . nosocomial spread and infection of hcws are potential risks but can be mitigated by early recognition and appropriate precautions. the first step in protection is to know which viruses are endemic to the local area, as shown in table 2 [12, 14] . humans are infected via the bite of an arthropod vector, inhalation or ingestion of rodent excretions, or mucosal or percutaneous contact with infected blood or secretions of patients [10] . in underresourced settings, spread of these viruses in the hospital environment is often facilitated by lack of recognition, reuse of equipment (such as contaminated needles), and inconsistent enforcement of basic infection-control practices, frequently because of inadequate supplies. clinical diagnosis of vhf requires an index of suspicion and is challenging, especially in underdeveloped regions, where the broad differential diagnosis of acute febrile illness includes malaria, typhoid, rickettsial illness, and leptospirosis. common presenting symptoms include acute onset of fever, malaise, headache, myalgias, and prostration, frequently accompanied by gastrointestinal complaints. early signs may include pharyngitis, conjunctivitis, cutaneous flushing, or a maculopapular rash. hemorrhagic manifestations may include oozing at intravenous puncture sites, petechiae, purpura, large ecchymoses, or frank hemorrhage; however, manifestations, frequency, and severity of bleeding varies depending on the agent [11, 12] . case fatality rates can range from !1% for rift valley fever to nearly 90% with ebola and marburg fever [15] . specific diagnosis can be made on the basis of detection of virus or viral antigens in serum, plasma, or whole blood by antigen-capture or antibody-detection enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. viral isolation should be attempted only in a biosafety level 4 containment laboratory, such as those at the cdc or the us army medical research institute of infectious diseases [11] . all hemorrhagic fever viruses except dengue virus are known to infect via artificially produced aerosols in a laboratory environment. potential spread between laboratory animals has raised concern about potential person-to-person airborne transmission [10, 16] . however, the epidemiology of vhf outbreaks indicates that if person-to-person airborne transmission does occur, it is rare [15] . in the 1995 zaire ebola epidemic, infections of hcws declined dramatically after the institution of isolation precautions [17] . however, achieving universal adherence to strict isolation precautions in hot, humid conditions in such environments is a challenge, as demonstrated in a 2000 ebola outbreak in uganda [18] . specific guidance for infection control is available [19] . standard, contact, and droplet precautions should be used in a private room for both inpatient and outpatient settings. although airborne precautions are not required in earlier stages of illness, they should be considered early (1) to eliminate the need for later transfer to an airborne isolation room, (2) for severe pulmonary involvement, and (3) for certain activities that might stimulate coughing or generate aerosols (eg, bronchoscopy and endotracheal intubation) [19, 20] . the who and cdc have developed vhf infection-control recommendations for african health care settings that are applicable to other austere environments [21] . their handbook recommends practical measures for establishing standard precautions for all patients, routine hand washing, and safe handling and disposal of used needles and syringes. upgrading to vhf precautions includes isolating the patient and wearing protective clothing (scrub suit, gown, apron, gloves, mask, headcover, eyewear, and rubber boots). hcw training should occur in advance, and access to patient care areas should be limited to those with a definite need. the cdc identifies 3 levels of risk (casual, close, and high-risk contacts) for potential exposure, along with follow-up recommendations [22] . the only vhf vaccine currently licensed in the united states is for yellow fever. investigational vaccines exist for several other vhfs; however, they are undergoing animal or clinical testing, are only for laboratory workers, or are not generally available [10] . medical countermeasures are limited. in a postexposure situation, expert consultation for treatment or prophylaxis measures is recommended. recently, emergent severe respiratory viral diseases have prompted intensified research and public health concern. a coronavirus causing severe acute respiratory syndrome (sars-cov) spread rapidly in 2003, resulting in a pandemic with frequent nosocomial spread [23] . the reemergence in the human population of avian influenza a(h5n1) in 2003 raised international alarm over a potential novel influenza pandemic. widespread infection in poultry and wild birds continues to cause occasional human cases in southeast asia and the middle east. although human-to-human transmission is limited, the case fatality rate exceeds 60% [24] . most recently, influenza a(h1n1), a reassortment zoonotic influenza virus, emerged in 2009 in north america and spread throughout the world, resulting in a pandemic declaration by june 2009. as of april 2010, 1300,000 confirmed cases (producing at least 17,700 deaths) have been reported from 1200 countries, and the pandemic virus has effectively replaced previously circulating seasonal influenza strains [25] . researchers and hcws face obvious risks for occupational acquisition of respiratory diseases. limited data to quantify the actual risk exist, although compelling anecdotes document the potential danger [26] . protective measures to mitigate risk for those with exposure include contact and respiratory precautions. optimal strategies depend on the nature of the pathogen, its known or suspected transmission routes, and exposure circumstances. transmission of sars-cov appears to occur predominantly through close interactions with infected persons. during the pandemic, transmission to hcws occurred after close, unprotected contact with symptomatic persons and was significantly mitigated once infection-control precautions were implemented; the degree of risk was related to the type and intensity of exposure (endotracheal intubation was significantly associated with contracting sars) [27, 28] . airborne transmission of sars-cov is clearly suggested by modeling studies, retrospective evaluation of nosocomial outbreaks, and anecdotal reports of transmission aboard commercial aircraft [29] [30] [31] . transmission of human influenza viruses occurs via large droplets, direct and indirect contact, and droplet nuclei. the relative contribution of each route is not known and likely varies with the setting and circumstances. there are conflicting opinions regarding the importance of airborne transmission of influenza viruses in humans [32] [33] [34] . direct contact may be more important than inhalation in conditions of high humidity and temperature [35] [36] [37] . frequent and diligent hand hygiene is likely the most important intervention for the interruption of transmission for many respiratory viruses, particularly influenza virus. when clean water is not available in field settings, alcohol-based hand gels should be used. these gels and/or washing with soap and water have demonstrated effectiveness [38] . personnel should be trained in and use protective clothing, gloves, shoe covers, goggles, and masks. the principal question with respect to respiratory protection is whether the mask should be a fitted, high-efficiency, particlefiltering n95 respirator. the answer depends on the relative contribution of aerosols versus large droplets to disease trans-mission. the who cites evidence from observational studies in hospitals suggesting that droplet transmission is responsible for the majority of nosocomially acquired cases to recommend standard plus droplet precautions for persons with seasonal influenza [39] . recent results suggest that surgical masks are effective for preventing seasonal influenza, although some controversy exists [40] . during the 2009 h1n1 pandemic, an institute of medicine panel recommended that hcws use n95 masks, given the uncertainty regarding risks of transmission [41] . in resource-limited settings, priority should be given to risk-reduction interventions focusing on hand hygiene, contact precautions, expedient respiratory protection, and the use of ppe that is effective for aerosol respiratory protection, particularly during exposures likely to generate aerosols (eg, intubation). although heterotypic protection against novel strains and pathogens cannot be expected, influenza vaccination is recommended for individuals involved in respiratory disease research and for hcws caring for such patients, unless contraindicated. vaccination is recommended to decrease the risk of illness due to seasonal influenza virus, reducing the frequency of diagnostic and management dilemmas for those occupationally involved in close contact with individuals with severe respiratory disease, such as that caused by influenza a(h5n1) or sars. prevention of influenza by means of chemoprophylaxis with neuraminidase inhibitors is a potential strategy and may be indicated in certain settings, such as direct contact with sources of avian influenza [39] . in general, tuberculosis is a potential threat to hcws and researchers who travel to work in high-burden countries [42] . approximately 500,000 new cases of multidrug-resistant (mdr) tuberculosis, defined as tuberculosis resistant to isoniazid and rifampin, were reported in 2008, and 1% were treated according to who standards [43] . extensively drug-resistant (xdr) tuberculosis, defined as mdr tuberculosis plus resistance to fluoroquinolones and a second-line injectable (kanamycin, amikacin, or capreomycin), has been reported in 58 countries [44] . mdr and xdr tuberculosis pose special risks. recognition of infectious individuals is difficult because of the lack of laboratory infrastructure for culture and susceptibility testing. treatment of mdr and xdr tuberculosis is prolonged, with significant toxicity and poor outcomes. treatment completion or cure is reported for 46%-87% of mdr cases and for 29%ϫ71% of xdr cases [45] [46] [47] [48] [49] . the epidemiology of drug-resistant tuberculosis is described in the who fourth global report [50] . information is incomplete for some regions, especially africa, but medical literature identifies a serious problem with mdr and xdr tuberculosis there [51, 52] . china represents 25% of the global burden, and india represents 120%. countries of the former soviet union have the highest proportion of drug resistance; 20% of all cases are mdr tuberculosis. other countries with a high proportion of mdr tuberculosis (rates of 13%) are vietnam, thailand, korea, the philippines, peru, and guatemala [50] . because of good infection control, at-risk screening, and treatment of latent tuberculosis, nosocomial transmission is rare in the united states. exposure overseas may occur in locations with uncontrolled transmission and inadequate infection-control practices. exposure is likely not only in hospitals but also in clinics, hospices, prisons, factories, mines, orphanages, and offices [51] . most hcws are not counseled regarding tuberculosis risks before departure, nor are they evaluated on their return. many are unaware of the risk, and if symptoms develop the diagnosis is often delayed, allowing potential transmission to vulnerable populations in us medical facilities. before departure, the risk of tuberculosis at the destination should be assessed. hcws should be screened for latent tuberculosis by an interferon g release assay (igra) or the tuberculin skin test (tst). igras are more specific and are preferred for those with a history of bacille calmette-guérin (bcg) vaccination. if the results of tst and igra are negative, vaccination with bcg may be considered 2-6 months before departure (b.j.s., personal communication). two months after returning, rescreen with an igra. fit testing with either a disposable filtering facepiece respirator or a reusable elastomeric respirator is advised. for those with facial hair, an adequate seal is not possible and a powered air purifying respira-tor is needed. respiratory protection should be brought to the destination. who infection-control guidelines for resource-limited countries suggest measures to limit transmission, including patient cohorting, individual respiratory protection, and natural ventilation [53] . engineering controls (such as negative air pressure) are usually not available; therefore, hcws should minimize the time spent in high-risk areas with poor ventilation when possible. personal respiratory protection is the main option for limiting exposure [54] . treatment of latent tuberculosis is strongly recommended for us hcws. if latent tuberculosis is detected before departure, it is often best to delay treatment until return. the cdc recommends treatment of latent tuberculosis with 9 months of isoniazid or 4 months of rifampin [55] . when latent tuberculosis is diagnosed after travel to high-risk areas, infection with mdr or xdr tuberculosis must be considered. if exposure to mdr or xdr tuberculosis is not reported or is unlikely, standard treatment is indicated. when the risk is significant or definite exposure occurs, treatment based on the suspected or known susceptibility of mdr and xdr tuberculosis exposure can be considered. this approach is often taken in the united states but is based on expert opinion and is controversial. treatment may include 6-12 months of either ethambutol or pyrazinamide with levofloxacin or moxifloxacin [56] . the cdc recommends clinical and radiographic follow-up for 24 months for those with latent tuberculosis likely due to mdr or xdr pathogens, regardless of whether treatment of latent tuberculosis occurs. if tuberculosis develops, rapid identification of resistance is critical. molecular detection of drug resistance is available at the cdc and at some state health departments. consultation with an expert is strongly advised. assistance is available from health departments or the cdc's regional training and medical consultation centers [57] . medical researchers and health care workers who travel to resource-limited settings to conduct patient care or research compose an understudied travel population. the nature of their work, restricted provision of supplies, and potentially limited use of infection-control practices puts them at unique risk for acquiring specific infections. this review provides practical guidance to mitigate the risk of potential occupational infectious diseases transmission in such settings and aspires to raise 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aerosol transmission of influenza a virus: a review of new studies virus survival as a seasonal factor in influenza and poliomyelitis transmission of influenza virus in temperate zones is predominantly by aerosol, in the tropics by contact: a hypothesis high temperature (30њc) blocks aerosol but not contact transmission of influenza virus influenza virus transmission is dependent on relative humidity and temperature efficacy of soap and water and alcohol-based hand rub preparations against live h1n1 influenza virus on the hands of human volunteers avian influenza guidelines, recommendations, descriptions surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a: a letter report tuberculosis in health care workers in kwazulu-natal, south africa update: tuberculosis facts world health organization. countries that had reported at least one xdr-tb case by time to sputum culture conversion in multidrug-resistant tuberculosis: predictors and relationship to treatment outcome treatment outcome of multidrug-resistant tuberculosis among vietnamese immigrants extensively drug-resistant tuberculosis in california treatment outcomes and long-term survival in patients with extensively drug-resistant tuberculosis multidrug-and extensively drug-resistant tuberculosis anti-tuberculosis drug resistance in the world emergence of increased resistance and extensively drug-resistant tuberculosis despite treatment adherence extensively drug resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and hiv in a rural area of south africa who policy on tb infection control in health-care facilities, congregate settings and households tuberculosis infection control in resource-limited settings in the era of expanding hiv care and treatment targeted tuberculin testing and treatment of latent tuberculosis infection management of persons exposed to multidrug-resistant tuberculosis potential conflicts of interest. all authors: no conflicts. key: cord-280763-4bnv2t3f authors: piñana, josé luis; pérez, ariadna; montoro, juan; giménez, estela; gómez, maría dolores; lorenzo, ignacio; madrid, silvia; gonzález, eva maría; vinuesa, víctor; hernández-boluda, juan carlos; salavert, miguel; sanz, guillermo; solano, carlos; sanz, jaime; navarro, david title: clinical effectiveness of influenza vaccination after allogeneic hematopoietic stem cell transplantation: a cross-sectional, prospective, observational study date: 2019-06-01 journal: clin infect dis doi: 10.1093/cid/ciy792 sha: doc_id: 280763 cord_uid: 4bnv2t3f background: vaccination is the primary method for preventing influenza respiratory virus infection (rvi). although the influenza vaccine is able to achieve serological responses in some allogeneic hematopoietic stem cell transplantation (allo-hsct) recipients, its clinical benefits are still uncertain. methods: in this prospective, cross-sectional study, we retrospectively analyzed the effect of inactivated trivalent influenza vaccination on the prevalence of influenza rvi in a consecutive cohort of 136 allo-hsct adult recipients who developed 161 rvi over 5 flu seasons (from 2013 to 2018). respiratory viruses in upper– and/or lower–respiratory tract specimens were tested using multiplex polymerase chain reaction panel assays. results: overall, we diagnosed 74 episodes (46%) of influenza rvi in 70 allo-hsct recipients. influenza rvi occurred in 51% of the non-vaccinated compared to 36% of the vaccinated recipients (p = .036). a multivariate analysis showed that influenza vaccination was associated with a lower prevalence of influenza rvi (odds ratio [or] 0.39, p = .01). a multivariate risk factor analysis of lower–respiratory tract disease (lrtd) identified 2 conditions associated with the probability of influenza rvi progression: influenza vaccination (or 0.12, 95% confidence interval [ci] 0.014–1, p = .05) and a high-risk immunodeficiency score (or 36, 95% ci 2.26–575, p = .011). influenza vaccination was also associated with a lower likelihood of an influenza-related hospital admission (14% vs 2%, p = .04). conclusions: this study shows that influenza vaccination may have a clinical benefit in allo-hsct recipients with virologically-confirmed rvi, in terms of a lower influenza rvi prevalence, slower lrtd progression, and lower likelihood of hospital admission. the influenza virus has a significant impact on morbidity and mortality in allogeneic hematopoietic stem cell transplantation patients (allo-hsct), leading to complications ranging from self-limited upper-respiratory tract infections to life-threatening or fatal pneumonias [1] [2] [3] [4] . the particular threat influenza poses for allo-hsct recipients was well documented during the 2009 influenza a/h1n1 pandemic, as well as during consecutive seasonal influenza epidemics. it showed increased risks of subsequent bacterial pneumonia, hospital admissions, mechanical ventilation requirements, and mortality [5, 6] . vaccination is the primary method for preventing influenza infections, but it is less effective in immunocompromised patients than in healthy individuals [7] [8] [9] [10] . based on studies analyzing serological responses, the inactivated influenza vaccine is strongly recommended annually for patients beyond 6 months post-transplant [11] [12] [13] [14] ; beyond 3 months after allo-hsct if without graft-versus-host disease (gvhd) or immunosuppression [10] ; or during community outbreaks [13, 15] . however, allo-hsct recipients respond poorly to vaccinations: only onethird of allo-hsct recipients will achieve protective influenza antibody titers [7, 9, 10] , which obviously limits the efficacy of such a vaccine, challenging reports on its clinical benefits. in fact, clinical evidence of influenza-inactivated vaccine effectiveness after allo-hsct is very limited [16] . strategies based on an influenza vaccine with a higher antigen dose [17] , multiple vaccine doses [18] , or adjuvanted vaccines [19] might improve immune responses, although no controlled trials have been performed. we conducted a prospective, cross-sectional, observational epidemiological study of community-acquired respiratory virus (carv) respiratory tract disease (rtd) in allo-hsct recipients who developed upper rtd (urtd) and/or lower rtd (lrtd) symptoms after transplant. patients' influenza vaccination status was prospectively recorded at the time of their respiratory virus screening. in this study, we report the prevalence of influenza rtd according to the vaccination status over 5 consecutive influenza seasons in a consecutive series of allo-hsct recipients with virologically-documented respiratory virus infections (rvis). this was a prospective, cross-sectional study of rvis in adult (>18 years) allo-hsct recipients that was conducted at 2 spanish transplant centers. the whole cohort comprised consecutive allo-hsct recipients with respiratory symptoms who were prospectively screened for respiratory viruses at the hospital clinic universitari of valencia (hcuv) between december 2013 and may 2016 and at the hospital universitari i politècnic la fe in valencia (hlf) from june 2016 to may 2018. in december 2013 at the hcuv and in may 2016 at the hlf, we implemented the medical information/education for recipients and caregivers, explaining in detail the risks of having rvis in the context of immunosuppression. the specific information provided included a description of the respiratory symptoms that merit urgent notification to the transplant team; recommendations concerning screening for respiratory viruses; details of available therapies; and infectious prevention control measures for patients and caregivers. a telephone number (on-call 24 h) for emergent conditions was also provided [20] . the local ethics committee approved the study protocol. we prospectively recorded participants' clinical and biological characteristics at the time of carv polymerase chain reaction (pcr) test screening, including the month and year of vaccination and the influenza vaccination status at each flu season. variables included the immunodeficiency scoring index (isi) [21] and basel immunodeficiency grading [22, 23] results; hospital admissions; the use of immunosuppressant drugs; the presence of signs or symptoms of acute or chronic gvhd; and transplant characteristics. with the aim of retrospectively comparing the influenza rtd prevalence between vaccinated and non-vaccinated participants, we included 1 recipient/rv episode/vaccination status in each flu season, following the selection algorithm described in figure 1 . we designed the case selection as follows: the first inclusion criterion was to retrospectively select all rvi episodes (irrespective of the carvs detected) with known vaccination statuses. the next step was to divide the carv episodes into 2 groups according to the vaccination status at each rvi episode (vaccinated or unvaccinated, in each corresponding flu season). following recommendations from the world health organization's guidelines [24] , we excluded the carv episodes taking place outside the flu seasons (between june and november) from both groups. we considered december to may as recruitment periods, according to our local and national epidemiological data, since influenza viruses were circulating in our community. the third step was to exclude episodes that occurred before day 90 after a stem cell infusion from the unvaccinated group, since the vaccine was not given to any recipients during that period and the first recorded influenza vaccine was given at day 91 after transplant. for both cohorts, we also excluded rvi episodes when baseline disease relapses occurred before the carv episode. for recipients with more than 1 rvi episode in the same flu season, we applied the following criteria: recipients with more than 1 episode of carv rvi other than influenza during the same flu season were computed only once and classed as not having an influenza infection in that season. recipients with more than 1 episode of rvi and whose vaccination status changed between 2 consecutive carv rvi episodes in the same season were computed twice: once in the non-vaccinated group and once (after vaccination) in the vaccinated group. if a recipient developed 2 rvi episodes caused by 2 different influenza viruses (ie, influenza a h1/n1 and b) at different time intervals during the same flu season, both episodes were computed in the corresponding group. when a recipient developed 2 influenza virus rvi episodes in the same season and the same influenza virus was detected, only the first episode was computed. finally, when a recipient developed 2 rvi episodes, 1 of them caused by the influenza virus, we only computed the influenza episode in that season. annual influenza vaccination was recommended to all patients at both transplant centers after the third month following allo-hsct. recipients received a seasonal inactivate trivalent influenza vaccine according to the national health vaccination program that ran from november until february in each flu season. for patients with moderate to severe gvhd at the time of the vaccination program who had received gammaglobulin, anti-thymocytic globuline, or rituximab within the 3 months before the flu vaccine period, vaccine administration remained at the physician's discretion. urtd was defined as a combination of upper-respiratory tract symptoms (rhinorrhea, sinusitis, otitis, or pharyngitis), as well as the positive identification of a carv by a pcr test and the absence of lower-respiratory tract infection symptoms and/ or any indication of pulmonary infiltrates in chest x-ray or computed tomography (ct) scan radiology results. we classified lrtd as possible, probable, or confirmed, as previously described [25] . there were no probable episodes, because we did not perform bronchoscopies in patients without the radiological proof of pulmonary involvement. we defined episodes as an urtd or lrtd according to european conference on infections in leukaemia-4 recommendations [26] . acute gvhd was diagnosed and graded according to the standard criteria [27] . patients with urtd symptoms underwent nasopharyngeal aspiration, nasopharyngeal swabs, or an induced sputum test, while bronchoalveolar lavage (bal) was performed in patients with a lrtd whenever possible. carv testing in bal samples were performed with 2 real-time pcr multiplex platforms, as described elsewhere in detail [28] . at the hcuv, samples were tested by real-time pcr using the luminex xtag rvp fast v1 assay (luminex molecular diagnostics, toronto, canada), while at hlf the clart pneumovir dna array assay (genomica, coslada, spain) was performed and interpreted following the manufacturer's recommendations. the clart pneumovir dna array assay differs from the luminex xtag rvp fast assay in that it detects influenza c virus, but not alphacoronavirus nl63 virus or betacoronaviruses hku1 and oc43. the clart pneumovir identifies the new influenza a/h1n1v. the primary objective of the study was to compare the prevalence of influenza urtds and/or lrtds and clinical characteristics among vaccinated and unvaccinated allo-hsct recipients. secondary endpoints included identifying the risk factors for both influenza rtd and progression of the influenza virus from an urtd to a lrtd. frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. differences between medians were compared using the mann-whitney u test. univariate and multivariate analyses of the association of clinical and biological risk factors with the progression of influenza lrtd were calculated using logistic regression models. for multivariate analyses, only variables with parameter estimates showing a p value ≤ .10 in the univariate analysis were finally included. we reported 2-sided exact p values, and p values ≤ .05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. overall, 263 allo-hsct recipients developed 601 episodes of upper-and/or lower-respiratory tract symptoms and were screened for rvis. in total, 231 allo-hsct recipients (87%) developed at least 1 episode of a virologically-documented rvi, accounting for 458 episodes (76%) of proven rvis. according to the algorithm selection (figure 1 ), we finally included 136 allo-hsct recipients with 161 virologically-documented rvi episodes over 5 flu seasons. out of 136 recipients, 8 were computed twice since they changed their vaccination status during the course of the study: 2 during the same season and 6 within 2 consecutive seasons. thus, we finally included the characteristics of 144 recipient cases according to the subjects' vaccination statuses (table 1 ). there were 101 seasonally-non-vaccinated allo-hsct recipients with 115 rvi episodes and 43 seasonally-vaccinated recipients with 46 rvi episodes who accomplished the selection criteria for comparison purposes. of the 136 participants, 21 (15%) had computable rvi episodes in multiple flu seasons: specifically, 19 had 1 computable rvi episode in each of 2 consecutive seasons, whereas 2 recipients had 1 computable episode in each of 3 consecutive seasons. patients' clinical and biological characteristics at the time of their rvi episodes are summarized in table 2 according to their vaccination statuses. as expected, the non-vaccinated group had significantly higher rates of conditions related to poor serological influenza vaccine responses. active gvhd, steroid therapy, ongoing immunosuppression therapy, and hypogammaglobulinemia were significantly over-represented in the non-vaccinated group as compared to the vaccinated group (60% vs 37%, 41% vs 22%, 90% vs 54%, and 30% vs 13%, respectively; p < .05 for all comparisons). of note, the rate of low total lymphocyte counts at the time of rvis at different cut-offs (<500, <200, and <100) were not statistically different among groups. we accounted for 74 proven influenza rvi episodes over 5 flu seasons in 70 recipients, with a median time of onset of 511 days after stem cell infusion (range 98-4752). we observed a higher number of influenza rvi episodes in 2 seasons-2014-2015 (n = 19) and 2017-2018 (n = 30)-as shown in table 2 and figure 2 . together, these 2 seasons represent 64% of all influenza episodes over the 5 flu seasons. there were 55 episodes limited to urtd, whereas 19 (26%) involved the lower respiratory tract (9 possible and 10 proven lrtds). the rate of hospitalization directly attributable to the influenza virus was 23% (17 out of 74 episodes). the most common influenza virus subtype was a, in 44 episodes (59%; 15 h1n1, 10 h3n2, and the remainder not subtypable), and b, in 34 episodes (46%). in 4 cases, influenza a and b were detected in the same episode/sample and were classed as co-infections. the observed 5-season prevalence of influenza rvis was significantly higher in the non-vaccinated (51%) compared to the vaccinated group (36%; p = .036). this statistical difference was even higher regarding the influenza lrtd prevalence (16% in non-vaccinated vs 2% in the vaccinated group, p = .01). the progression rates of urtds to lrtds in recipients with influenza rvis were 30% (18 out of 59) in the non-vaccinated group compared to 7% in the vaccinated group (1 out of 15; p < 0.01). influenza-related hospital admissions were more common in the non-vaccinated group compared to the vaccinated group (14% vs 2%, p < .05). logistic regression univariate and multivariate analyses of risk factors for influenza virus respiratory infections and progressions to lrtds are shown in table 3 . in order to identify the conditions associated with influenza virus infections, we studied the 161 evaluable recipient/episode pairs. a multivariate analysis identified the flu vaccine as the main factor associated with a reduced risk of influenza virus infection (odds ratio [or] 0.39, 95% confidence interval [ci] 0.18-0.8, p = .01). to analyze the risk factors for lrtd progression of an influenza virus infection, we focused the analysis on a multivariate analysis identified 2 independent conditions associated with lrtd progression. again, the flu vaccine was associated with a lower probability of lrtd progression (or 0.12, 95% ci 0.014-1, p = .05). in contrast, a high-risk isi score predicted a higher probability of the influenza virus lrtd progression (or 36, 95% ci 2.26-575, p = .011). this study shows that, irrespective of the flu season, a trivalent influenza inactivated vaccine given after allo-hsct was the most important factor associated with the lower prevalence of influenza rvis among recipients with proven carv rvis. in allo-hsct recipients with influenza rvis, a multivariate analysis showed that influenza vaccination was associated with a lower probability of the influenza virus progressing to lrtd. we also observed that a high-risk isi score was highly predictive of influenza lrtd progression in our cohort of patients. although this study was not designed to assess the vaccination rate in our population, only 28% of the recipients with proven carv rvis received the influenza vaccine over 5 flu seasons. this agrees with prior epidemiological studies and enquiries, where the vaccination rate has ranged from 20-60% after allo-hsct [29] [30] [31] . however, some factors merit consideration when interpreting the reported vaccination rate. we did not detect all vaccinated recipients in our prospective respiratory virus survey, since those who were vaccinated may have had lower incidences of carv rvis and a lower propensity to seek medical attention or testing. however, the lack of consensus across current guidelines [12] [13] [14] [15] regarding the timing and conditions in which the influenza vaccine should be administered, in particular when immunosuppressant therapy is required to treat active/uncontrolled moderate-to-severe gvhd, is likely the most important contributor to this apparently low vaccination rate. in these scenarios, physicians may decide to defer vaccination. thus, based on our findings, we currently recommend flu vaccination at 3 months post-transplant to all of our recipients, irrespective of their immunosuppression status. in our selected cohort, half (51%) of the unvaccinated recipients developed influenza rvis, with moderate to severe clinical consequences, as reflected in higher hospital admission and lrtd progression rates. to address the clinical efficacy of influenza vaccination in recipients with gvhd, corticosteroid therapy, hypogammaglobulinemia, or with ongoing immunosuppressant use, we compared the influenza rvi prevalence in vaccinated recipients according to the presence or absence of such conditions. although the number of cases was limited, we did not find statistical differences regarding vaccinated recipients with or without gvhd (36 % vs 31%, respectively, p = .9), corticosteroids (40% vs 31%, p = .7), immunosuppressants (36% vs 29%, p = .7), or hypogammaglubulinemia (50% vs 30%, p = .1). in fact, our multivariate analysis revealed that vaccination status was the main condition associated with a lower influenza rvi prevalence in recipients with at least 1 episode of a carv rvi. this is an important finding, since we provide clinical evidence that seasonal influenza vaccines could be clinically beneficial in allo-hsct recipients, even when a significant number of vaccinated recipients had gvhd (19%), used immunosuppressants (54%), or used corticosteroids (22%). although the serological vaccination response in such recipients is poor, we can speculate that vaccination may also exert a cellular-mediated response. influenza vaccination was able to mediate peripheral blood t-cell responses, characterized by production of the th1 cytokine ifn-gamma by cd4+ cells, both in patients vaccinated more than 6 months after transplantation and those vaccinated earlier, after stem cell transpantation [10] . these data suggest that, even in cases where the expected serological response could be suboptimal (allo-hsct <6 mo, gvhd, immunosuppressant use, or corticosteroid use), the influenza vaccine could elicit a clinical benefit through a cellularly-mediated effect, reducing the influenza infection prevalence and/or its severity. although the influenza rvi prevalence in vaccinated recipients was still substantial (36%), it should be noted that the risk factors for influenza lrtd progression identified vaccination as a condition associated with a reduced rate of lrtd progression. furthermore, vaccination was also associated with a lower hospital admission rate. given these findings, we speculate that vaccination could mitigate the severity of influenza rvis, even in the presence of conditions related to decreased serological responses. another relevant finding was the usefulness of the isi score in stratifying the lrtd progression risk of influenza rvis, even when we analyzed episodes occurring after day 90 after stem cell infusion. isi was developed by investigators from the md anderson cancer center to predict outcomes in allo-hsct recipients with respiratory syncytial viruses [21] . the same investigators demonstrated its value in predicting lrtd progression in the setting of influenza rvis [32] . therefore, the use of the isi could be applied to assess the need for prophylactic/therapeutic intervention with several doses of the influenza vaccine and/or with high doses of antiviral drugs in prospective studies. abbreviations: alc, absolute lymphocyte count; allo-hsct, allogeneic hematopoietic stem cell transplantation; anc, absolute neutrophil count; atg, anti-thymocytic globuline; basel ig, basel immunodefciency grading; ci, confidence interval; gvhd, graft-versus-host disease; hla, human leucocyte antigen; igg, immunoglobuline g; is, immunosuppressants; isi, immunodeficiency score index; log. regr., logistical regression model; lrtd, lower-respiratory tract disease; ns, not significant; nt, not tested; or, odds ratio; rtd, respiratory tract disease; rvi, respiratory virus infection. a these variables were not included in the final multivariate models, since they were included in the isi score. regarding influenza virus epidemiological data from the current study, most influenza rvi cases occurred in the 2014-2015 and 2017-2018 flu seasons. these data are in accordance with spanish epidemiological data, where the influenza prevalence was significantly higher in 2014-2015 (350 cases/100 000 hab) and 2017-2018 (450 cases/100 000 hab) as compared to the 2013-2014, 2015-2016, and 2016-2017 flu seasons (270, 190 , and 220 cases/100 000 hab, respectively) [33, 34] . these observations confirm that influenza rvi prevalence in our allo-hsct recipients mimicked influenza virus prevalence in the general population. finally, we acknowledge that this study has some important limitations, such as the retrospective nature of the analyses, the small sample size, the somewhat heterogeneous vaccination policy, and the cohort selection method. in addition, the use of 2 different pcr methods, which differed (minimally) in their analytical performance, can be viewed as a limitation. in spite of this, our study has strengths that merit consideration. we used molecular testing. our prospective carv survey mirrored national epidemiological data in influenza rvis in each flu season. we provided data encompassing 5 complete flu seasons, limiting the distortion likely introduced by the vaccination coverage variability between seasons. in conclusion, we provide clinical evidence that influenza vaccination after allo-hsct is associated with a lower prevalence of influenza rvi and a lower severity of the disease. respiratory virus infections after stem cell transplantation: a prospective study from the 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invasive pneumococcal disease in adult hematopoietic stem cell transplant recipients: a decade of prospective population-based surveillance vaccination adherence in hematopoietic stem cell transplant patients: a pilot study on the impact of vaccination cards and reminder telephone calls vaccination guidelines after hematopoietic stem cell transplantation: practitioners' knowledge, attitudes, and gap between guidelines and clinical practice outcomes of influenza infections in hematopoietic cell transplant recipients: application of an immunodeficiency scoring index informe semanal de vigilancia de la gripe en españa key: cord-256583-z3pd339v authors: yen, muh-yong; schwartz, jonathan; hsueh, po-ren; chiu, allen wen-hsian; armstrong, donald title: traffic control bundling is essential for protecting healthcare workers and controlling the 2014 ebola epidemic date: 2015-03-01 journal: clin infect dis doi: 10.1093/cid/ciu978 sha: doc_id: 256583 cord_uid: z3pd339v nan to the editor-a global health crisis, the 2014 ebola outbreak has now struck healthcare workers (hcws) at unprecedented levels. whereas historically, ebola epidemics spread via person-to-person transmission, the current outbreak in west africa has seen unexpectedly extensive spread of nosocomial disease, despite hcws' reliance on previously effective infection control procedures such as patient isolation, barrier nursing procedures, and required personal protective equipment (ppe) [1] . indeed, infection struck even among hcws caring for patients with ebola virus disease (evd) in western hospitals equipped with modern facilities and procedures. this has sparked growing concerns regarding how to protect hcws [2] , even those working outside the illprepared and overwhelmed regions of west africa now grappling with ebola [1] . in our view, the most concerning examples include dr khan [3] , a sierra leonean virologist who contracted ebola despite his extensive experience and careful adherence to procedures; dr spencer [4] , a médecins sans frontières physician who became symptomatic upon returning to new york despite working in well-designed isolation units built specifically to protect hcws from evd infection; and dr sacra, an obstetrician who contracted ebola without having knowingly cared for any evd patients [5] . based on these developments and the knowledge that ebola may remain viable to a certain degree on dry solid surfaces with fomites for approximately 1 day [6, 7] , we hypothesize that fomite transmis-sion of ebola may best explain some of these unanticipated cases. fomite transmission is facilitated by the practice of situating patients with acute symptoms and potentially extremely high viral loads outside isolation rooms in environments where adherence to routine disinfection practices is rare [7] . taiwan's experience with severe acute respiratory syndrome (sars) in 2003 is instructive. we contend that during the height of the sars epidemic, hcws in institutions that failed to identify designated zones of risk simply assumed they were secure from risk as long as they were not in proximity to patients with highly contagious pathogens. however, their confidence in existing barrier precautions and ppe as providing sufficient protection when away from heavily contaminated areas proved unwarranted [8] . as it turned out, consistent use of ppe and negative pressure isolation rooms was insufficient because the main cause of nosocomial sars transmission was casual contact with fomites in contaminated environments either outside of isolation zones or during removal of ppe [8, 9] . unlike when dealing with contamination by nuclear or chemical spills, there exist no distinct boundaries delineating contaminated and clean zones when managing biological disasters. lacking clearly designated zones of risk, fomites far from patient rooms were still found to be positive for sars coronavirus rna [10] . in the mistaken belief that they were well away from contaminated areas and because they encountered no visible contamination, hcws occasionally came into contact with these fomites after removing their ppe. in our view, this same scenario likely explains the infection with ebola suffered by drs khan, spencer, and sacra. realizing the threat of nosocomial infection, the taiwan centers for disease control responded by implementing traffic control bundling (tcb), which included triage and diversion of patients before they enter the hospital; clear delineation of zones of risk between contaminated and clean zones; and gloves-on hand disinfection at checkpoints between zones of risk ( figure 1 ) [11] . tcb proved critical (p < .05) for protecting hcws [9] . indeed, infection rates among hcws caring for sars patients dropped to zero following its implementation, ultimately contributing to nationwide sars control [7] . a key aspect of successful tcb is installation of alcohol dispensers in all zones to encourage hand disinfection. the dispensers not only demonstrate to hcws the significance of zones of risk, but also strengthen adherence to, and frequency of, hand disinfection. during the taiwan response to sars, alcohol dispensers were situated along the path from the contaminated zones through the intermediate zones and into the clean zones ( figure 1 ). as a result, compliance by hcws with hand disinfection rose to 100% [9, 11] . having already disinfected their hands, even when hcws touched their surroundings after leaving contaminated zones and while removing ppe, they were already decontaminated and thus did not make contact with nor left fomites. in conclusion, we suggest that tcb is a powerful, convenient, and economical tool for protecting hcws against highly contagious diseases. we recommend that it be implemented as part of ongoing efforts to contain and control the current ebola outbreak. figure 1 . conceptual scheme of traffic control bundling. following triage outside the hospital entrance, all the way until being hospitalized in the isolation room, the patients remained contained inside a zone of risk (red arrow), which is distinguished from "clean zones" through the "intermediate zone." dispensers with 75% alcohol for gloves-on hand sanitation are installed at checkpoints positioned in between zones of risk. healthcare workers (hcws) in a clean zone are required to don personal protective equipment (ppe) before entering the zones of risk. when departing a zone of risk, but prior to entering a clean zone, hcws are required to undergo decontamination and remove ppe in an intermediate zone. here hcws disinfect their hands, gloved or not, between every single step of the decontamination process and removal of ppe to avoid casual contact of skin/mucosa with the virus. ebola and compliance with infection prevention measures in nigeria guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in u.s. hospitals, including procedures for putting on (donning) and removing (doffing) sierra leone's top ebola doctor gets virus nyc doctor craig spencer followed proper protocol after returning from ebolastricken west africa us doctor with ebola lands in nebraska for treatment persistence in darkness of virulent alphaviruses, ebola virus, and lassa virus deposited on solid surfaces assessment of the risk of ebola virus transmission from bodily fluids and fomites using an integrated infection control strategy during outbreak control to minimize nosocomial infection of severe acute respiratory syndrome among healthcare workers taiwan's traffic control bundle and the elimination of nosocomial severe acute respiratory syndrome among health care workers from sars in 2003 to h1n1 in 2009: lessons learned from taiwan in preparation for the next pandemic key: cord-273839-oasgagpc authors: bisno, alan l.; gerber, michael a.; jack m., gwaltney; kaplan, edward l.; schwartz, richard h. title: diagnosis and management of group a streptococcal pharyngitis: a practice guideline date: 1997-09-17 journal: clin infect dis doi: 10.1086/513768 sha: doc_id: 273839 cord_uid: oasgagpc this is the second in a series of practice guidelines commissioned by the infectious diseases society of america through its practice guidelines committee. the purpose of these guidelines is to provide assistance to clinicians when making decisions on treating the conditions specified in each guideline. the targeted providers are pediatricians, family practitioners, and internists. the targeted patients and setting for the acute pharyngitis guideline are pediatric, adolescent, and adult outpatients with a complaint of sore throat. funding was provided by the idsa. panel members represented experts in adult and pediatric infectious diseases. the guidelines are evidence-based. a standard ranking system was used for the strength of the recommendations and the quality of the evidence cited in the literature reviewed. the document has been subjected to external review by peer reviewers as well as by the practice guidelines committee and was approved by the idsa council. an executive summary, algorithms, and tables highlight the major recommendations. indicators of quality will assist in guideline implementation. the guideline will be listed on the idsa home page at http://www.idsociety.org. outcomes 10-day course to achieve maximal pharyngeal eradication of group the desired outcomes are: (1) prevention of acute rheumatic a streptococci, but the use of certain newer agents has been reported fever; (2) prevention of suppurative complications (e.g., peritonsillar to achieve comparable bacteriologic and clinical cure rates among abscess, cervical lymphadenitis, or mastoiditis); (3) abatement of patients with streptococcal pharyngitis when these agents are given clinical symptoms and signs; (4) a rapid decrease in infectivity so for £5 days. however, definitive results from comprehensive studas to reduce transmission of group a b-hemolytic streptococci to ies are not available; thus, final evaluation of these proposed shorter family members, classmates, and other close contacts and to allow courses of oral antibiotic therapy is not possible, and they cannot the rapid resumption of usual activities; (5) minimization of potenbe recommended at this time. moreover, these antibiotics have tial adverse effects of inappropriate antimicrobial therapy. much broader spectrums than penicillin, and most, even when administered for short courses, are more expensive. except under special circumstances, neither repeated bacteri-evidence ologic testing (culture or radt) of patients who have success-we reviewed a large number of clinical trials of diagnostic fully completed a course of antimicrobial therapy nor routine and treatment strategies for group a streptococcal pharyngitis. testing of asymptomatic household contacts of patients with the reports were examined for indicators of quality. for examgroup a streptococcal pharyngitis is recommended. ple, studies of treatment were evaluated for randomization, a small percentage of patients will have recurrences of acute blinding, use of streptococcal typing to differentiate treatment pharyngitis that are associated with throat cultures (or radts) failures from new infections, duration and timing of follow-up positive for group a streptococci within a short period following examinations, and statistical power [1, 2] . completion of a course of antimicrobial therapy. such episodes may be treated with one of the antimicrobial agents appropriate for treatment of the initial illness. if these episodes were previously values treated with oral agents and compliance is in question, retreatment in evaluating diagnostic options, we placed a high value with intramuscular benzathine penicillin g should be considered. on selecting the diagnostic test that was most accurate for when multiple episodes occur over the course of months or years, differentiating acute pharyngitis due to group a b-hemolytic it may be difficult to differentiate viral infections in a streptococcal streptococci from that due to other agents. for evaluation of carrier from true group a streptococcal infections. certain antimitreatment, particularly high values were assigned to proven crobial agents, such as clindamycin and amoxicillin/clavulanate, clinical and bacteriologic efficacy, safety, spectrum of antimimay be beneficial because they have been shown to yield high crobial activity, and relative cost. rates of pharyngeal eradication of streptococci under these particular circumstances. the group a b-hemolytic streptococcus is the most common definition bacterial cause of acute pharyngitis [3] . accurate diagnosis, followed by appropriate antimicrobial therapy, is important for group a streptococcal pharyngitis (pharyngotonsillitis) is an acute infection of the oropharynx and/or nasopharynx with the reasons previously stated (see the section on outcomes). although acute pharyngitis is one of the most frequent illnesses streptococcus pyogenes. for which pediatricians and other primary care physicians are consulted, less than half of patients with this condition are infected by group a streptococci. moreover, the signs and objective symptoms of group a streptococcal and nonstreptococcal pharyngitis overlap so broadly that accurate diagnosis on clinical the objective of this practice guideline is to provide recommendations for the accurate diagnosis and optimal treatment grounds alone is usually impossible [4] . with the exception of very rare infections by certain of the of group a streptococcal pharyngitis. other pharyngeal bacterial pathogens listed in table 1 (e.g., corynebacterium diphtheriae and neisseria gonorrhoeae), antimicrobial therapy is of no proven benefit in the treatment of acute options pharyngitis due to bacteria other than the group a streptococcus. it is therefore extremely important for physicians to be able to physicians caring for patients with acute pharyngitis must formulate differential diagnoses and determine which, if any, exclude the diagnosis of group a streptococcal pharyngitis to prevent inappropriate administration of antimicrobials to large confirmatory tests should be performed. if clinical and laboratory evaluations result in a diagnosis of group a b-hemolytic numbers of patients with pharyngitis. the administration of such therapy unnecessarily exposes patients to the associated expense streptococcal pharyngitis, one of several antimicrobial agents and treatment schedules may be selected. and hazards, and it may also contribute to the emergence of / 9c37$$se43 08-25-97 15:12:42 cidal uc: cid temperate climates, it usually occurs in the winter and early spring. patients with group a b-hemolytic streptococcal pharyngitis commonly present with sore throats (generally of sudantibiotic-resistant bacteria that is being reported with increasing den onset), pain on swallowing, and fever. headache, nausea, frequency in the united states and elsewhere. vomiting, and abdominal pain may also be present, especially if a diagnosis of group a streptococcal pharyngitis is conin children. physical examination reveals tonsillopharyngeal firmed, the clinician must select the most appropriate antimicroerythema with or without exudates and tender enlarged anterior bial agent known to be effective against the group a streptococcervical lymph nodes (lymphadenitis). other findings may incus. the cost of an effective course of antimicrobial therapy clude a beefy red swollen uvula, petechiae on the palate, excorimay vary as much as 20-fold, depending on the drug chosen. ated nares (especially in infants), and a scarlatiniform rash. the regimens recommended herein are judged to be optimal however, none of these findings is specific for group a bin regard to specificity, safety, and cost. hemolytic streptococcal pharyngitis, and they may occur with other upper respiratory infections. conversely, the absence of recommendations fever or the presence of clinical features such as conjunctivitis, cough, hoarseness, coryza, anterior stomatitis, discrete ulcerative lesions, viral exanthem, and diarrhea strongly suggests a viral rather than a streptococcal etiology. differential diagnosis. viruses are the most common nonbacterial causes of acute pharyngitis (table 1) [3] . respiratory viruses such as adenovirus, parainfluenza virus, rhinovirus, and who should be tested for group a b-hemolytic streptococcal respiratory syncytial virus frequently cause acute pharyngitis. other viral agents of acute pharyngitis include coxsackievirus and echo viruses as well as herpes simplex virus. epstein-when attempting to decide whether to perform a laboratory test for a patient who presents with acute pharyngitis, the clini-barr virus is a frequent cause of acute pharyngitis that is often accompanied by the other clinical features of infectious mono-cal and epidemiological findings mentioned above should be considered before the test is performed. a history of close nucleosis (e.g., generalized lymphadenopathy and splenomegaly). systemic infections with measles virus, cytomegalovirus, contact with a well-documented case of streptococcal pharyngi-/ 9c37$$se43 08-25-97 15:12:42 cidal uc: cid tis is helpful, as is an awareness of a high prevalence of group a positive throat cultures that would not otherwise be identified. thus, while initial therapeutic decisions may be made on the b-hemolytic streptococcal infections in the community. testing usually need not be performed for patients with acute pharyngi-basis of the results of an overnight culture, it is advisable to reexamine plates at 48 hours that are negative at 24 hours [20] tis whose clinical and epidemiological features do not suggest a group a streptococcal etiology. selective use of diagnostic (category a, grade ii). the clinical significance of the number of group a b-hemostudies for group a b-hemolytic streptococci will result in an increase in both the proportion of positive test results and the lytic streptococcal colonies present on the throat culture plate is problematic. while cultures are more likely to be strongly percentage of patients with positive tests who are truly infected rather than merely streptococcal carriers (category a, grade ii). positive for patients with true acute group a streptococcal pharyngitis than for patients who are streptococcus carriers, efforts have been made to incorporate clinical and epidemiological features of acute pharyngitis into scoring systems that there is so much overlap that the differentiation cannot be made accurately on the basis of the degree of positivity of the throat attempt to predict the probability that a particular illness is caused by group a b-hemolytic streptococci [10, 11] . how-culture alone [19] (category a, grade ii). the most widely used test for differentiating group a strepto-ever, at best these clinical scoring systems predict positive results of throat cultures or radts only £80% of the time. cocci from other b-hemolytic streptococci in physicians' offices is probably the bacitracin disk test. this test provides a therefore, unless the diagnosis of group a streptococcal pharyngitis can be confidently excluded on clinical and epidemio-presumptive identification based on the observation that ú95% of group a streptococci show a zone of inhibition around a logic grounds (see clinical diagnosis above), bacteriologic studies should be performed (category a, grade ii). disk containing 0.04 units of bacitracin, while 83% -97% of non-group a streptococci do not [21, 22] . an alternative and highly specific method of identifying throat cultures streptococcal serogroups is the detection of the group-specific cell-wall carbohydrate antigen directly on isolated bacterial culture of a throat swab on a sheep blood agar plate remains the standard for the documentation of the presence of group a colonies. commercial kits containing group-specific antisera are available for this purpose. such tests are appropriate for streptococci in the upper respiratory tract and for the confirmation of the clinical diagnosis of acute streptococcal pharyngitis use by microbiology laboratory personnel, but most physicians who perform throat cultures would find it difficult to justify [12] (category a, grade ii). a single throat swab cultured correctly on a blood agar plate has a sensitivity of 90% -95% in the additional expense for the minimal improvement in accuracy that serogrouping with an antigen detection test would detecting the presence of group a b-hemolytic streptococci in the pharynx [13] (category a, grade ii). provide [19] . several variables impact on the accuracy of the throat culture results. for example, the manner in which the swab is obtained has an important impact on the yield of streptococci from the throat culture [14, 15] . throat swab specimens should be ob-a disadvantage of culturing a throat swab on blood agar plates is the delay (overnight or longer) in obtaining the culture tained from the surface of both tonsils (or tonsillar fossae) and the posterior pharyngeal wall. other areas of the oropharynx results. radts have been developed for the identification of group a b-hemolytic streptococci directly from throat swabs. and mouth are not acceptable sites for sampling, and these sites should not be touched before or after the appropriate areas although these rapid tests are more expensive than blood agar cultures, the advantage they offer over the traditional procedure have been sampled. in addition, false-negative results may be obtained if the patient has received antibiotics shortly before is the speed with which they can provide results. rapid identification and treatment of patients with streptococcal pharyngitis or at the time the throat swab specimen is collected. it has also been reported that the use of anaerobic incubation can reduce the risk of the spread of group a b-hemolytic streptococci, allowing these patients to return to school or work and selective culture media may increase the proportion of positive cultures [16, 17] . however, the data on the impact of sooner, and can reduce the acute morbidity associated with this illness [13, 23 -25] (category a, grade ii). the use of radts the incubation atmosphere and the culture media are conflicting, and, in the absence of a definite benefit, the increased cost vs. throat cultures for certain populations (e.g., patients seen in emergency departments) has been shown to significantly and effort associated with anaerobic incubation and selective culture media are difficult to justify, particularly for physicians increase the number of patients appropriately treated for streptococcal pharyngitis [26] . who process throat cultures in their own offices [16, 18, 19] (category a, grade ii). most of the radts that are currently available have an excellent specificity ( §95%) when compared with blood agar another variable that can impact on the yield of the throat culture is the duration of incubation. tomatic pharyngitis after the organism's presence in the throat the first radts were based on latex agglutination methodis confirmed by microbiological or immunologic means (figure ology, were relatively insensitive, and had unclear endpoints. 1). when there is clinical or epidemiological evidence that newer tests based on eia techniques offer more sharply deresults in a high index of suspicion, antimicrobial therapy can fined endpoints as well as increased sensitivity. more recently, be initiated while laboratory confirmation is pending provided radts for which optical immunoassay and chemiluminescent such therapy is discontinued if the diagnosis of streptococcal dna probes are used have become available. data on these pharyngitis is not confirmed. early initiation of antimicrobial newer tests suggest that they may be more sensitive than other therapy results in faster resolution of the signs and symptoms radts and perhaps even as sensitive as standard throat cul[23 -25] (category a, grade i) of the infection, but two facts tures on sheep blood agar plates. however, in view of someshould be recalled. first, group a streptococcal pharyngitis is what conflicting data [28] , additional corroborative information usually a self-limited disease; fever and constitutional sympis needed before these tests can be recommended for routine use toms disappear spontaneously within 3 or 4 days of onset, even without confirmatory throat cultures for negative test results. when antimicrobial therapy is not administered [32] . second, titers of antibodies to streptococci reflect past and not presit has been shown that therapy can be safely postponed £9 days ent immunologic events and are of no value in the diagnosis after the onset of symptoms and still prevent the occurrence of of acute pharyngitis. these titers are valuable for confirming the major nonsuppurative sequel, acute rheumatic fever [33] prior streptococcal infections in patients suspected of having (category a, grade i). acute rheumatic fever or acute glomerulonephritis. they also these facts allow the clinician flexibility in initiating therapy are helpful in prospective epidemiological studies that are conduring the evaluation of an individual patient with presumed ducted to separate patients with acute infection from those who group a streptococcal pharyngitis. the results of the initial are carriers. therapeutic studies were reported nearly 50 years ago; since guideline: the diagnosis of acute group a streptococcal then numerous antimicrobial agents have been examined in pharyngitis should be suspected on clinical and epidemiological grounds and then supported by the results of a laboratory test. either a positive throat culture or radt provides adequate confirmation of the presence of group a b-hemolytic streptococci in the pharynx, but a negative radt result should be confirmed with a throat culture (category a, grade ii). most asymptomatic patients with group a b-hemolytic streptococci present in the upper respiratory tract after a complete course of appropriate therapy are streptococcal carriers [29, 30]. therefore, follow-up throat cultures are not routinely indicated for asymptomatic patients who have received a complete course of therapy for group a streptococcal pharyngitis (category a, grade ii). however, there are special situations when follow-up throat cultures should be performed for asymptomatic patients. throat cid clinical trials and have been shown to be capable of eradicating been shown to be resistant to erythromycin [40] . sulfonamides and tetracyclines are not recommended for treatment of group group a streptococci from the upper respiratory tract. however, it must be recognized that the only antimicrobial actually exam-a streptococcal pharyngitis because of the higher rates of resistance to these agents among group a streptococci and the ined in controlled studies and shown to be capable of preventing initial attacks of rheumatic fever has been intramuscular reposi-frequent failure of these agents to eradicate even susceptible organisms from the pharynx. tory penicillin [34, 35] (category a, grade i). these studies were performed with procaine penicillin g in oil containing antimicrobial therapy. when selecting an antimicrobial for treatment of group a streptococcal pharyngitis, important is-aluminum monostearate [34, 35] , a preparation that has since been supplanted by benzathine penicillin g. (for this reason, sues include efficacy, safety, antimicrobial spectrum (narrow vs. broad), dosing schedules, compliance (or adherence), and no regimens listed in table 2 have been assigned a grade of a1.) there are data, although not definitive, indicating that cost. these factors influence the cost-effectiveness of antimicrobial therapy. benzathine penicillin g is effective in primary prevention of rheumatic fever (prevention of an initial attack of rheumatic a number of antibiotics have been shown to be effective for treating group a streptococcal pharyngitis. these agents in-fever following an episode of group a streptococcal pharyngitis) [36, 37] . benzathine penicillin g has also been shown clude penicillin and its congeners (such as ampicillin and amoxicillin), as well as numerous cephalosporins, macrolides, and to decrease the occurrence of rheumatic fever cases during epidemics of streptococcal pharyngitis in military recruit camps clindamycin. however, penicillin remains the treatment of choice because of its proven efficacy, safety, narrow spectrum, [38] . moreover, benzathine penicillin g has been proven effective for preventing rheumatic fever in patients who have had and low cost [31, 41, 42] . erythromycin is a suitable alternative for patients who are allergic to penicillin. first-or second-a previous attack of the disease (secondary prophylaxis) [39] (category a, grade i). other antimicrobials can effectively erad-generation cephalosporins are also acceptable for penicillinallergic patients who do not manifest immediate hypersensitiv-icate group a streptococci from the upper respiratory tract, and it is assumed that such eradication is an adequate measure of ity to b-lactam antibiotics. most oral antibiotics must be administered for 10 days to effectiveness in the primary prevention of rheumatic fever. antimicrobial resistance has not been a significant issue in achieve maximal pharyngeal eradication of group a streptococci, but certain new agents have been administered in shorter the treatment of group a streptococcal pharyngitis in the united states. there has never been a clinical isolate of group a courses. it has been reported that azithromycin [43 -45] , cefuroxime [46] , cefixime [47] , and cefpodoxine [48] can be used to streptococcus documented to be resistant to penicillin anywhere in the world. although there have been geographic areas where achieve comparable bacteriologic and clinical cure rates among patients with streptococcal pharyngitis when these drugs are isolates have been highly resistant to macrolide antibiotics (specifically erythromycin), this has not been and currently is not given for £5 days. however, definitive results from comprehensive studies are not available, and thus it is not possible to a clinically significant problem in north america. less than 5% of group a streptococci isolated in the united states have endorse these proposed shorter courses of oral antibiotic ther* amoxicillin is often used in place of oral penicillin v in young children; the efficacy of amoxicillin appears to be equal to that of penicillin v, and this choice is primarily related to acceptance of the taste of the suspension. † see text. ‡ for patients weighing less than 60 lbs (27 kg). § two milliliters of c-r bicillin(900/300) contains 900,000 units of benzathine penicillin g and 300,000 units of procaine penicillin g; this preparation thus contains less benzathine penicillin g than is conventionally used in the treatment of adolescents or adults. available data indicate that orally administered first-and second-generation cephalosporins also are effective in eradicating group a streptococci from the upper respiratory tract; these agents should not be used in patients with immediate hypersensitivity to b-lactam antibiotics. first-generation cephalosporin a ii second-generation cephalosporin a ii. # these are total daily doses (maximum daily dose, 1 g per day). / 9c37$$se43 08-25-97 15:12:42 cidal uc: cid apy at this time. moreover, the spectra of these antibiotics are special circumstances are present (see the section on repeated diagnostic testing). because routine retesting is no longer much broader than that of penicillin, and, even when they are administered for short courses, they are more expensive. advised, only those patients whose signs and symptoms of acute pharyngitis return within the succeeding few weeks will antimicrobials for group a streptococcal upper respiratory tract infections may be given either orally or parenterally. , noncompliance with the pre-group a streptococcal pharyngitis. intramuscular benzathine penicillin g is preferred for patients who are unlikely to com-scribed antimicrobial regimen [52], or a new infection with a group a streptococcus acquired from family, classroom, or plete a full 10-day course of oral therapy. guideline: patients with acute streptococcal pharyngitis community contacts. a second episode of pharyngitis with the original infecting group a streptococcal strain (i.e., treatment should receive therapy with an antimicrobial agent in a dosage and for a duration that is likely to eradicate the infecting organ-failure) cannot be ruled out, but this occurs only rarely. streptococcal carriers do not ordinarily require further anti-ism from the pharynx. on the basis of penicillin's narrow spectrum of antimicrobial activity, the infrequency with which microbial therapy. these individuals have group a b-hemolytic streptococci present in their throats but have no evidence of it produces adverse reactions, and its modest cost, it is the drug of choice for nonallergic patients. an immunologic reaction to this organism [53] . during the winter and spring in temperate climates, £20% of asymptom-management of close contacts and pharyngeal carriers. approximately 25% of individuals within the household of an index atic school-aged children may be streptococcus carriers. they may be colonized by group a b-hemolytic streptococci for patient may also harbor group a streptococci in their upper respiratory tracts. however, it is usually not necessary to perform several months, and during that period they may have episodes of intercurrent viral pharyngitis. when tested, these patients throat cultures for these contacts or treat them if they are asymptomatic. in those situations in which repeated testing is indicated are found to have group a b-hemolytic streptococci in their pharynges and appear to have acute streptococcal pharyngitis. (see the section on repeated diagnostic testing), performing cultures for asymptomatic family contacts and treating those who streptococcal carriers are unlikely to spread the organism to their close contacts and are at low risk, if any, for developing are positive are advisable. when a larger group (e.g., schools, day care centers, or domiciliary institutions) is involved in a suppurative complications or nonsuppurative complications (e.g., acute rheumatic fever) [53] . documented outbreak of group a streptococcal upper respiratory infections or scarlet fever, throat cultures should be performed moreover, it is more difficult to eradicate group a streptococci from the upper respiratory tracts of streptococcal carriers for all patients; however, only those with positive throat cultures should be treated with antimicrobials. the administration of intra[29] . this has been shown to be true with penicillin therapy and also may be true with some other antimicrobials. in fact, muscular injections of benzathine penicillin g has been shown to be very effective in terminating such outbreaks. many of the published studies showing relatively high rates of failure to eradicate group a streptococci from the upper strains of group a streptococci that cause invasive infections may spread to close contacts of the index case. secondary cases respiratory tract with penicillin therapy were likely ''contaminated'' with carriers. of severe invasive infection have rarely occurred in family and institutional contacts and in health care workers [49 -51] . data in practice it is difficult to differentiate a carrier with an intercurrent non-group a streptococcal infection from a patient are as yet too limited to assess with precision the risk of secondary illness or to make a firm recommendation regarding the with acute streptococcal pharyngitis. helpful clues include the patient's age, season of the year, local epidemiology (e.g., the advisability of routinely performing cultures and treating close contacts of patients with group a streptococcal infections such presence of influenza or enteroviral illnesses), and the precise nature of the presenting signs and symptoms (see the section as necrotizing fasciitis or the toxic shock -like syndrome. guideline: it is not necessary to perform throat cultures or on clinical diagnosis above). in many instances, however, the clinician may not be able provide treatment for household contacts of patients with group a streptococcal pharyngitis, except in specific situations in confidently to distinguish persistent carriage from acute infection and will elect to administer another course of antimicrobi-which there is increased risk of frequent infections or of nonsuppurative sequelae (category b, grade iii). als. for single episodes of symptomatic, culture-confirmed or radt-confirmed group a streptococcal pharyngitis that occur shortly after completion of a course of appropriate antimicrobial therapy, any of the agents listed in table 2 is appropriate. because patient compliance with oral antimicrobials often is b-hemolytic streptococci an issue, a regimen of intramuscular benzathine penicillin g should be considered. for these single repeated episodes, it is performing routine throat cultures (or rapid antigen testing) for asymptomatic patients after completion of antibiotic therapy not necessary to reculture the throat after the second course of therapy unless the patient remains or becomes symptomatic or for group a streptococcal pharyngitis is not necessary unless / 9c37$$se43 08-25-97 15:12:42 cidal uc: cid unless special circumstances are present (see the section on there have been no definitive controlled studies of therapy for multiple, repeated symptomatic episodes of culture-positive repeated diagnostic testing above). an even more challenging clinical circumstance is a pa-acute pharyngitis in the same patient; however, the regimens listed in table 3 have been reported to result in low bacteriologi-tient -usually a school-aged child or adolescent -who has multiple episodes of acute pharyngitis and cultures or radts cal failure rates [55 -58]. guideline: a small percentage of patients will have recur-positive for group a streptococci within a period of months to years. it is likely that most patients in this category are rences of acute pharyngitis and throat cultures (or radts) positive for group a streptococci within a short period of time streptococcal carriers with nonstreptococcal infections. for patients who have frequent distinct episodes of infection, informa-following completion of a course of antimicrobial therapy. a single such episode may be retreated with the regimens listed tion regarding the clinical response to antibiotic therapy and the presence or absence of group a streptococci in throat cultures in table 2. when multiple episodes occur over the course of months or years, it may be difficult to differentiate viral infec-performed during asymptomatic intervals is helpful in distinguishing persistent carriage from repeated episodes of strepto-tions from true group a streptococcal infections in a streptococcal carrier. use of certain antimicrobial agents has been shown coccal pharyngitis. serotyping of repeated streptococcal isolates from an individual patient may also assist in arriving at to yield high rates of streptococcal eradication in the pharynx under these particular circumstances (category a, grade ii). this determination, but such studies can be done only in specialized research laboratories. suggested regimens with these agents are listed in table 3. when physicians suspect ping-pong spread to be associated with multiple repeated episodes of group a streptococcal infections in one family, performing simultaneous cultures for all d. indicators of quality family contacts and treating those whose cultures are positive may be helpful (category b, grade iii). there is no credible indicators of quality of care for patients with acute pharyngitis include: (1) performance of throat cultures or radts for evidence that family pets are reservoirs for group a streptococci or that they contribute to familial spread. patients suspected of having group a streptococcal pharyngitis; (2) performance of throat cultures for patients with negative continuous antimicrobial prophylaxis for group a streptococcal infection is not recommended because there is insuffi-radts; (3) prescription of one of the antimicrobial regimens recommended in table 2 for patients with acute pharyngitis cient evidence to show that it is effective, except for preventing recurrences of acute rheumatic fever. surgical removal of the and positive tests for group a streptococci; (4) withholding or discontinuing antimicrobial therapy for patients with throat tonsils may be considered for the rare patient whose symptomatic episodes do not diminish in frequency over time and for cultures negative for group a streptococci; (5) omission of routine follow-up cultures for patients who have received an whom no alternative explanation for the recurrent pharyngitis is evident. tonsillectomy may decrease recurrences of symp-adequate course of antimicrobial therapy; (6) avoidance of routine throat cultures for asymptomatic family contacts of patients tomatic pharyngitis in selected patients, but only for a limited period of time [54] (category a, grade i). with group a streptococcal pharyngitis; (7) avoidance of con† although shorter courses of some newer macrolides and cephalosporins have been reported to be effective for treating group a streptococcal upper respiratory tract infections, the evidence is not yet sufficient to recommend these agents for therapy at this time (this is also true for patients with repeated infections or for those in whom the organism is difficult to eradicate). ‡ maximum dose, 750 mg of amoxicillin per day. § benzathine penicillin g is useful for patients whose compliance with previous courses of oral antimicrobials is questionable. limited data suggest that the addition of rifampin ( streptococcal pharyngitis: placebotinuous long-term antimicrobial prophylaxis for preventing recontrolled double-blind evaluation of clinical response to penicillin thercurrent episodes of acute pharyngitis. apy the effect of penicillin therapy on the symptoms and signs of streptococcal pharyngitis clinical evaluation of a latex aggluti-references nation test for streptococcal pharyngitis: performance and impact on treatment rates streptococcal pharyngitis: current therapy and criteria for evalua-27 antigen detection test for streptococcal pharyngitis: evaluation of purpose of quality standards sensitivity with respect to true infections immunoassay for rapid detection of group a b-hemolytic streptococci: 97(6pt2)(suppl):949 -54. should culture be replaced? perplexity and precision in the diagnosis of streptococ-29 do the b-hemolytic respiratory tract gerber ma. treatment failures and carriers: perception or problems? pedi-587 -601. atr association of group c b-hemolytic streptococci with endemic pharyn-1997 red book: report of the committee on infectious diseases. 24th gitis among college students clinical and microbiologi-483 -94 effect of streptococci community-wide outbreak tis and pharyngitis the role of the streptococcus diagnosis of pharyngitis: clinical and epidemiologic in the pathogenesis of rheumatic fever prevention of rheumatic fever. treatment of the preceding strep a simple scorecard for the tentative diagnosis of streptococcal tococcic infection streptococcal pharyngitis: evaluation of clinirheumatic fever by treatment of the preceding streptococcal infection with cal syndromes in diagnosis controlled studies of streptococinfections on clinical grounds comparison of throat cultures and rapid strep tests for diagnorate of rheumatic fever streptococcal pharyngitis: optimal site for throat culof rheumatic fever by treatment of previous streptococcal infections. i. ture cultures of streptococcus 466 -71. pyogenes from the oropharynx protection of a military population effect of atmosphere of incubation from rheumatic fever: routine administration of benzathine penicillin g on the isolation of group a streptococci from throat cultures. j lab clin to healthy individuals effect of atmosphere and duration of fever in children and adolescents. a long-term epidemiologic study of incubation on primary isolation of group a streptococci from throat subsequent prophylaxis, streptococcal infections, and clinical sequelae. cultures comparative effectiveness of three prophylaxis regimens in pre ann intern comparison of throat culture methods for the recovery of group a streptococci in a pediatric office setting in vitro susceptibility of recent north american diagnosis of pharyngitis: methodology of throat cultures suitability of throat culture procedures for detection of group streptococcal pharyngitis and prevention of rheumatic fever: a statement a streptococci and as reference standards for evaluation of streptococcal for health professionals. committee on rheumatic fever, endocarditis, antigen detection kits extraction method with pronase b for grouping beta-hemolytic strepto-42. world health organization. rheumatic fever and rheumatic heart disease. cocci bacitracin differentiazation, 1988. tion of presumptive identification of group a b-hemolytic streptococci: 43. hamill j. multicentre evaluation of azithromycin and penicillin v in the comparison of primary and purified plate testing effect of antibiotic 44. weippl g. multicentre comparison of azithromycin versus erythromycin therapy on the clinical course of streptococcal pharyngitis. j pediatr in the treatment of paediatric pharyngitis or tonsillitis caused by group a streptococci / 9c37$$se43 08 a comparison of azithromycin and penicillin v for the treat-51 penicillin v for group a streptococcal pharyngoton comparative efficacy sillitis: a randomized trial of seven vs ten days' therapy. jama and safety of four-day cefuroxime axetil and ten-day penicillin treatment of group a beta-hemolytic streptococcal pharyngitis in children. pediatr 53. kaplan el. the group a streptococcal upper respiratory tract carrier state efficacy of tonsillectomy pharyngitis and/or tonsillitis: comparison with 10-day penicillin for recurrent throat infection in severely affected children: results of v therapy. cefixime study group course treatment of acute group a b-hemolytic streptococcal tonsil-clindamycin treatment of chronic pharyngeal carriage of group a streplopharyngitis: ten days of penicillin v vs 5 days or 10 days of tococci cefpodoxime therapy in children clusters of invasive group a scand streptococcal infections in family, hospital, and nursing home settings eradication of group a streptococci from the dinsa lin v treatment failure penicillin plus coccus among family members and health care workers. clin infect dis rifampin eradicates pharyngeal carriage of group a streptococci key: cord-275349-b35pt3mo authors: lenz, heinz-josef; richardson, peter; stebbing, justin title: the emergence of baricitinib: a story of tortoises versus hares date: 2020-07-06 journal: clin infect dis doi: 10.1093/cid/ciaa940 sha: doc_id: 275349 cord_uid: b35pt3mo nan keywords. covid-19; sars-cov-2; barictinib; anti-viral; cytokine storm; trial. baricitinib is a once daily orally administered jak1/2 inhibitor, which inhibits the signaling of many cytokines and has been approved for the treatment of moderate to severe rheumatoid arthritis (ra) based on long-term randomized dosing against both placebo and standard of care [1] , notably tumor necrosis factor (tnf)alpha blockade. its use for the treatment of coronavirus disease 2019 (covid-19) was originally suggested after a search of the extensive benevolentai knowledge graph for approved drugs that could be used in this pandemic [2] . an advantage of approved drugs is that they have a known safety profile and can therefore be rapidly approved in fast moving pandemics. the artificial intelligence algorithms predicted that baricitinib would inhibit severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection of cells [2] , (an effect later confirmed in human liver spheroids) [3] , combined with its better-known anti-inflammatory properties. the doses required were predicted to be the same as used for the treatment of ra. at these doses, interleukin 6 (il-6) levels were reduced both in covid19 patients as well as dose dependently in ra patients in a previous phase 2b randomized ra trial, the first time this was shown in humans [4] . what was more intriguing was its inhibition of members of the numb associated kinase family, thought in turn to translate to reduced ap-2 mediated viral propagation early in the infectious cycle, suggesting antiviral activity; this was shown in liver spheroid models, which express detectable albeit low levels of the ace2 receptor. in hospitalized patients it is anticipated that residual virus that propagates itself in airway epithelial tissues maintains and amplifies the exaggerated inflammatory response typical of covid-19. consequently, the combined potential antiviral and anti-inflammatory effects of this dually acting drug could be ideal for halting the progression of the disease in hospitalized patients, when taken for a limited duration. at the university of southern california now, a randomized placebo-controlled phase 2 study with remdesivir with/without baricitinib is ongoing to test the efficacy to prevent mechanical ventilation including significant biomarker research. in this issue of clinical infectious diseases, titanji et al describe the outcome of 15 moderate-to-critically ill patients with covid-19 treated with baricitinib and hydroxychloroquine. this therapy was associated with a reduction in inflammatory markers including fever, c reactive protein (crp), improvement of oxygen requirements, and recovery in 12 of the 15 (80%) patients studied. although it is possible that the antiviral and anti-inflammatory effects of hydroxychloroquine contributed to this positive outcome, this would seem unlikely according to the recently reported national institutes of health (nih) randomized trials) [5] , among many other studies. this new paper in the journal extends the previous published reports of baricitinib treatment in mild-tomoderate covid19 patients and provides further evidence that baricitinib could be a potential treatment for unwell hospitalized patients with this disease, independent of severity. indeed, in mild-to-moderate patients, baricitinib therapy was also associated with a reduction in the requirement for intensive care unit (icu) care when compared with matched case controls [6, 7] . in each series baricitinib treatment has been associated with a reduction in inflammation and recovery from the disease in the vast majority of patients, consistent with the present report, but we do not know if this is simply an association or represents a causative effect of the drug. in this new paper, 3 patients died, 2 of which had very high plasma il-6 levels prior to starting baricitinib therapy consistent with previous data that high il-6 is a predictor of mortality in covd19 infections [8, 9] . these useful results retrospectively justified the testing of the anti-il6/ il6r antibodies kevzara (sarilumab) and actemra (tocilizumab) in severely unwell patients, although randomized studies are yet to show benefits. baricitinib treatment over prolonged periods in ra patients has been associated with increased infections and thromboembolism, but the short duration of treatment in covid-19 patients may mitigate against such side effects. in this report it is difficult to say whether one incident of pulmonary embolism and methicillinresistant staphylococcus aureus (mrsa) infection were due to the complications of covid-19 or baricitinib, but the much larger numbers of patients in the yet to report randomized studies should clarify this. one would be advised to remain vigilant of such signals reflecting thromboembolic or infection risk in randomized controlled trials testing a variety of immunomodulatory therapies in covid-19 patients, either alone or in combination, especially in view of associations between clots and sars-cov-2 infection [10] . specifically, severe endothelial injury associated with intracellular sars-cov-2 virus and disrupted endothelial cell membranes, widespread vascular thrombosis with microangiopathy, and occlusion of alveolar capillaries including significant new vessel growth through a mechanism of intussusceptive angiogenesis has been described [11] . the first report of a significant reduction in covid-19 mortality in any randomized study recently came from the recovery trial in the united kingdom showing that low dose dexamethasone had a significant effect in reducing mortality when given with standard of care (21.6% vs 24.6% of the patients dying within 28 days) [12] ; this effect was greatest in ventilated patients (a 30% reduction in mortality) and slightly less in those provided with noninvasive supplemental oxygen (a 20% reduction) [12] . it is tempting to speculate that a similar or greater reduction in mortality could be expected with baricitinib in randomized trials, especially because baricitinib is now reported to be associated with recovery from covid-19 in different series of patients. it may even be possible to combine the baricitinib with dexamethasone, at least in critically ill patients, although once again it would of course be important to assess the risk of infection when administering these 2 potent anti-inflammatory agents together. as acknowledged by titanji et al, and reiterated by us, randomized trials are required for us to be certain if baricitinib is an effective and safe therapy for covid-19 patients [3] . this report, and others like it, have supported the selection of baricitinib in a number of studies including the actt2 randomized trial, where it is being tested in combination with remdesivir, and a further placebocontrolled phase 3 trial recently commenced. it is anticipated that these trials should provide a robust estimate of the efficacy of baricitinib in covid-19 and would be complementary to each other. these initial case series warrant further confirmation from the ongoing larger, randomized and controlled trials. thus far, these data sets also validate use of artificial intelligence at a time of a biomedical crisis, to help speed drug development. the use of an oral medicine taken once daily for a short duration should also lend itself to low-and middle-income countries. note potential conflicts of interest. p. j. r. is an employee of benevolentai and has received honoraria for giving a lecture to lilly with j. s. and j. s. has sat on sabs for vaccitech, heat biologics, eli lilly, replete, alveo, certis oncology solutions, greenmantle and benevolentai, has consulted with lansdowne partners and vitruvian. he sits on the board of directors for bb biotech healthcare trust. h.-j. l. has no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. baricitinib versus placebo or adalimumab in rheumatoid arthritis baricitinib as potential treatment for 2019-ncov acute respiratory disease mechanism of baricitinib supports artificial intelligencepredicted testing in covid-19 patients efficacy and safety of baricitinib in japanese patients with active rheumatoid arthritis receiving background methotrexate therapy: a 12-week, double-blind, randomized placebo-controlled study rapid systematic review on clinical evidence of chloroquine and hydroxychloroquine in covid-19 critical assessment and recommendation for future clinical trials baricitinib therapy in covid-19: a pilot study on safety and clinical impact retrospective, multicenter study on the impact of baricitinib in covid-19 moderate pneumonia the potential role of il-6 in monitoring coronavirus disease 2019 clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 effect of dexamethasone in hospitalized patients with covid-19: preliminary report key: cord-289247-qc3to2xj authors: yamaoka, yutaro; jeremiah, sundararaj s; miyakawa, kei; saji, ryo; nishii, mototsugu; takeuchi, ichiro; ryo, akihide title: whole nucleocapsid protein of sars-cov-2 may cause false positive results in serological assays date: 2020-05-23 journal: clin infect dis doi: 10.1093/cid/ciaa637 sha: doc_id: 289247 cord_uid: qc3to2xj nan m a n u s c r i p t 2 to the editor the nucleocapsid protein (np) of sars-cov-2 is one of the widely used antigens in serodiagnostics of the novel coronavirus disease . we appreciate guo and colleagues for shedding new light on the humoral response profile of covid-19 [1] . they have generated recombinant whole nucleocapsid protein (rnp) of sars-cov-2 using escherichia coli expression system and used it to develop an enzyme linked immunosorbent assay (elisa) to detect anti-sars-cov-2 antibodies in plasma. the rnp elisa was negative in the plasma of all the 285 non covid-19 individuals tested. the authors have also shown that rnp does not have cross reactivity with antibodies of other common coronaviruses; nl63, 229e, oc43, and hku1. however, we have observed controversial findings and suggest the advantage of n-terminally truncated nucleocapsid protein (δn-np). although the amino acid sequences of the entire nucleocapsid proteins (np) of sars-cov and other common coronaviruses are dissimilar to that of sars-cov-2 [1] , the conserved residues at the nterminal domain of np show a high degree of similarity (figure-1a) . this was pointed out by yu et al in sars-cov, who observed cross reactivity of whole np with other coronaviruses and encountered high rates of false positivity while testing healthy donor sera [2] . they had overcome this problem by using δn-np which was devoid of the homogenous conserved residues at the n-terminal region [2, 3] . since sars-cov-2 np has over 90% homology to sars-cov np [1] and their conserved residues are almost identical (figure-1a) , we expected the possibility of cross reactions with whole/full length np (fl-np). we expressed both fl-np and δn-np of sars-cov-2 in wheat germ cell free protein production system [4] and designed an igg detection elisa to compare their performance. upon after the third week [5] . in the time-course analysis, the δn-np elisa detected the lower level of antibodies that began to increase from second week onwards while fl-np elisa at the same cut-off would have falsely reported these as negative (figure-1c) . detection of anti-viral antibodies can be used both for diagnosis and population surveillance, ideally with the former possessing high sensitivity and the latter high specificity. we aimed to enhance the sensitivity to detect the low level of igg in patients during relatively earlier stages of the disease. with higher sensitivity, fl-np based elisa gives a higher background but this occurs to a much lesser extent with δn-np. we presume this is probably due to the cross reactivity of fl-np with antibodies to other human coronaviruses and this should be studied further. we conclude that δn-np is better suited than fl-np to develop high sensitivity diagnostic assays for covid-19. a c c e p t e d m a n u s c r i p t profiling early humoral response to diagnose novel coronavirus disease (covid-19) evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay recombinant truncated nucleocapsid protein as antigen in a novel immunoglobulin m capture enzyme-linked immunosorbent assay for diagnosis of severe acute respiratory syndrome coronavirus infection development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen antibody detection and dynamic characteristics in patients with covid-19 we thank mayuko nishi and chizu suzuki for their technical assistance. this study was approved by the institutional review board of yokohama city university (irb no. b200200048, b160800009), and the protocols used in the study were approved by the ethics committee. written informed consent was obtained from the patient (or family/guardian) research and development (amed) to ar (19fk0108110h0401). the authors have no conflicts of interest directly relevant to the content of this article. y.y. is a current employee of kanto chemical co., inc. a c c e p t e d m a n u s c r i p t key: cord-266820-exl36jt3 authors: rivera, frida; safdar, nasia; ledeboer, nathan; schaack, grace; chen, derrick j; munoz-price, l silvia title: prevalence of sars-cov-2 asymptomatic infections in two large academic health systems in wisconsin date: 2020-08-19 journal: clin infect dis doi: 10.1093/cid/ciaa1225 sha: doc_id: 266820 cord_uid: exl36jt3 sars-cov-2 asymptomatic infections may play a critical role in disease transmission. we aim to determine the prevalence of asymptomatic sars-cov-2 infection at two hospital systems in two counties in wisconsin. the sars-cov-2 prevalence was 1% or lower at both systems despite the higher incidence of covid-19 in milwaukee county the coronavirus disease 2019 (covid-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is responsible for more than two million covid-19 cases in the united states. [1] as of june 23, 2020, dane and milwaukee counties in wisconsin had reported a covid-19 rate of 215 and 1,085.2 cases per 100,000 people, respectively. [2] some studies suggest that asymptomatic infections could be responsible for amplifying the spread of the disease. [3] however, data regarding the prevalence of asymptomatic infections and their impact on transmission are still scarce. this study aims to determine the prevalence of asymptomatic sars-cov-2 infection at two hospital systems in two counties with markedly different rates of covid-19. this study was performed at froedtert health (879-bed system, milwaukee, wi) and university of a c c e p t e d m a n u s c r i p t 4 combined nasopharyngeal/oropharyngeal swab specimens or nasopharyngeal specimens only were collected at froedtert health and uw health, respectively, according to standard institutional procedures. a nasopharyngeal swab was inserted into the nose until it reached the nasopharynx, rotated for 10 seconds, and removed. the oropharyrngeal swab was collected by swabbing the poster pharynx and tonsillar areas; the oropharyngeal swab was combined with the nasopharyngeal swab into a single tube of transport media and sent to the laboratory. specimens were promptly tested using either the thermofisher taqpath sars-cov-2 assay or the roche cobas 6800 sarsdemographic characteristics were retrospectively assessed from electronic medical records. an asymptomatic infection was defined as the presence of a positive sars-cov-2 test in the absence of any of the following symptoms: fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, or diarrhea. this study was approved by the institutional review boards at both institutions. from april 6, 2020 to june 04, 2020, a total of 11,654 asymptomatic patients were tested for sars-cov-2, and 61 (0.52%) were positive [froedtert health, 38; uw health, 23]. the percentage of positivity did not appear to increase over time. among the total cohort, the median age was 42, iqr 37 and 27 (42%) were men. demographic characteristics by hospital are shown in table 1 patients were followed-up 14 days after testing and seven patients (11.5%) out of 61 originally deemed to be asymptomatic infections developed symptoms consistent with covid-19. during the study period, we observed a low prevalence of asymptomatic sars-cov-2 infections in these two academic health systems in south wisconsin. even though both sites had a prevalence of 1% or less, froedtert had higher prevalence of asymptomatic infections (1% vs 0.3%; p<0.001). this low prevalence of asymptomatic infections has been recently reported in other areas with high covid-19 rates, such as boston and philadelphia [4, 5] ; however, these two studies included pregnant women and children. in contrast, two hospitals in new york city reported a prevalence of sars-cov-2 asymptomatic infections of 14% among women admitted for delivery. [6] our results suggest that the prevalence of asymptomatic infections in individuals undergoing preadmission or preoperative screening may not be predictable based on the incidence of covid-19 in the region. these data could bring into question the need to screen all asymptomatic patients in certain regions; however, in addition to guiding isolation precautions, these screening tests also serve as sentinel data for changes in the epidemiology of the virus. our conclusions are hindered due to possible selection bias of the population surveyed. additionally, rather than asymptomatic infections, these positive results could represent patients who previously experienced covid-19 and are now long-term shedders. further studies are needed to determine the interplay between the prevalence of asymptomatic infections and covid-19 in a region. a c c e p t e d m a n u s c r i p t 6 no funding sources were used for this publication. n.s. reports grants from nih. all no conflicts of interests related to this publication. a c c e p t e d m a n u s c r i p t m a n u s c r i p t 8 table 1 . demographic characteristics of sars-cov-2 asymptomatic infected patients stratified by health system. wisconsin department of health services. covid-19: county data household transmission of sars-cov-2 universal sars-cov-2 testing on admission to the labor and delivery unit: low prevalence among asymptomatic obstetric patients incidence of covid-19 in pediatric surgical patients among 3 us children's hospitals universal screening for sars-cov-2 in women admitted for delivery key: cord-007170-svsfu7fj authors: richt, j. a.; vandewoude, s.; zink, m. c.; clements, j. e.; herzog, s.; stitz, l.; rott, r.; narayan, o. title: infection with borna disease virus: molecular and immunobiological characterization of the agent date: 1992-06-17 journal: clin infect dis doi: 10.1093/clinids/14.6.1240 sha: doc_id: 7170 cord_uid: svsfu7fj borna disease virus (bdv), which seems to be distinct from all other known viruses, exhibits a unique mechanism of pathogenesis. this review highlights several aspects of the biology of infection with this virus and summarizes the preliminary characterization of the agent. studies on bdv may help to illuminate several important areas of neurobiology, including the mechanisms regulating the replication of a new type of rna virus in the nuclei of neural cells, the neuroinvasiveness and neurotropism of such viruses, their t cell-mediated immunopathology, tolerance in newborn animals to persistent viral infection of the central nervous system, and behavioral diseases and eating disorders induced by such agents. mainly in the gray matter of the brain, and pathognomonic intranuclear joest-degen inclusion bodies in neurons and astrocytes of the frontal cortex [8, 9] . mechanisms of transmission of the virus in horses and sheep are not well understood, but experimental data suggest that intranasal infection is the common route [10] . recent seroepidemiological and virological surveys of borna disease in horses have shown that bdv-specific antibodies are present in many horses without clinical signs of the disease. since most of these animals remain clinically healthy for at least 1 year, a long incubation period may be characteristic of the pathogenesis of borna disease [11] . these data suggest further that bdv is more widely disseminated than was previously thought. in fact, bdv-specific antibodies have been found in clinically healthy horses in the united states (authors' unpublished results). bdv has been only partially characterized. although the agent is known to have physical and replicative properties typical of conventional viruses [1, 12, 13] , its nucleic acid has only recently been identified as rna [14] [15] [16] [17] . bdv is highly neurovirulent in most laboratory animals and in cell cultures. it has a tropism for cells derived from the neural crest: neurons, astrocytes, and schwann cells. tissue culture cells of nonneuronal origin exhibit only low-level susceptibility to infection, but this resistance can be overcome by cocultivation with bdv-infected primary brain cultures [18] . the virus replicates noncytopathically in infected cells and is tightly cell associated. infection with bdv is persistent; cultures produce approximately one infectious unit per cell [13] . treatment of infected cells with interferon a/,3 does not influence the establishment or maintenance of persistent infection [19] . persistently infected mdck (madin-darby canine kidney) cells are widely used in indirect immunofluorescence assays for the detection of bdv-specific antibodies in serum and csf of affected animals and humans [18, 20] . the viral antigens in persistently infected cells accumulate in the nuclei (sparing the nucleoli) and in the perinuclear area of the cytoplasm and appear as punctate grains in immunofluorescence reactions. bdv particles have never been visualized in infectious material, but replication of bdv is associated with synthesis of at least three novel polypeptides with respective molecular masses of 14, 24, and 38-39 kd [21] [22] [23] [24] . antibodies to these virus-associated antigens are produced in infected animals and can be recognized readily by radioimmunoprecipitation and western blot analysis [21] [22] [23] [24] . sera from animals previously infected with bdv or immunized with bdv-specific proteins do not neutralize the infectivity of the virus [25] [26] [27] . polyclonal antibodies to all three bdv proteins are found in the serum of infected animals. monoclonal antibodies (mabs) to the 38/39-kd and 24-kd antigens have been obtained after immunization of mice with bdv-specific antigens [22, 24] or after infection of rats [22] . most of these antibodies react in western blot analyses exclusively with the protein used for immunization in lysates of infected cell cultures and infected rat brain. evidence for an antigenic relation between these two proteins has been found by protease digestion and tryptic fingerprint analysis [24] . several identical peptides were revealed after digestion of the 38/39-kd and 24-kd proteins. furthermore, the cross-reactivity of mabs to the purified 38/39-kd and 24-kd proteins indicates that these proteins share antigenic determinants [16, 24] . neither protein is glycosylated, but the 24-kd antigen was found to be phosphorylated at serine residues [24] . bdv has not yet been classified taxonomically because infectious particles have never been visualized, and only recently has this agent been identified as an rna virus [14] [15] [16] [17] 28] . since novel proteins were identified in bdv-infected cells and tissues, it was assumed that bdv-specific mrnas must also be present in infected material. thus the strategy used to isolate bdv-specific molecular clones was to construct subtractive cdna libraries from mrnas isolated from uninfected and bdv-infected cell cultures and brain [15, 16] . no specific nucleic acid probe for bdv was available; the cdna cloning was therefore done in an expression vector, and clones were screened with mabs [16] specific for the bdv-specific 38/39-kd protein [22] or with a radiolabeled subtraction probe enriched for bdv-specific sequences [15] . the bdv-specific clones isolated in our laboratory [16, 17, 28] and in that of another group [14, 15] recognized four bdv-specific rnas of 10.5, 3.6, 2.1, and 0.85 kilobases (kb), respectively, in bdv-infected rat brain; all of these rnas were enriched by polyadenylate selection [16] . lipkin et al. [15] reported that the largest rna species is 8.5 kb in size and is not polyadenylated. in vitro transcription and translation of the bdv-specific cdna clone isolated in our laboratory (clone b8) produced proteins of 24 and 14 kd that were recognized by both polyclonal and monoclonal antibodies to bdv [16, 17, 28] (figure 1). no significant similarities to any known viral or cellular sequences were detected with both the nucleotide and the amino acid sequences of clone b8 [16, 17] . in addition, southern blot hybridization with bdv-specific cdna clones isolated by both laboratories [15, 16] showed that these clones were not of cellular origin and did not hybridize to any dna species in infected material. these data and the identification of four species of rna in infected material clearly demonstrate that bdv is an rna virus [14] [15] [16] [17] 28] . furthermore, since the bdv-specific rnas in infected material are sensitive to digestion with pancreatic rnase, bdv seems to be a single-stranded rna virus [14] . oligonucleotides with negative polarity from different regions of the b8 clone all hybridized to the same four positivestrand rnas identified for the entire b8 clone [16, 17, 28] (figure 2, lane b). this result indicates that all of the nucleotide sequences in the b8 clone are present in all of the larger rnas and thus suggests that the organization of the rnas is a nested set of overlapping rnas, as in coronaviruses [29] [30] [31] . in addition, positive-polarity oligonucleotides from different regions of clone b8 hybridized to three bdv-specific rnas of 10.0, 3.5, and 1.7 kb [17, 28] (figure 2, lane d). these rnas probably represent negative-strand complements of the positive rna species. recently, negativestrand rnas have been identified in coronavirus-infected cells; these rnas are thought to be important in replication of the subgenomic rnas [30, 31] . thus, both the organization of the rnas and the presence of positive-and negativestrand rnas indicate a striking similarity of bdv to the coronavirus family, although it has not yet been resolved whether bdv is a negative-or a positive-strand rna virus. the resolution of this issue will require the isolation of bdv and the identification of the polarity of its genomic rna. bdv is infectious for a wide range of animal species, from chickens to nonhuman primates and possibly humans [1, 3, 20, 32] . the clinical manifestations and outcome of experimental infection vary among animals. rabbits develop an reproduced with permission from [28] . acute, fatal paralytic disease similar to that seen in horses within 4-6 weeks [4, 5] ; tree shrews (tupaia glis) develop an unusual, nonfatal behavioral disease characterized by aberrant social interactions [33] . several strains of rat can be infected with bdv [25] [26] [27] 34] , but only some of these strains develop disease [35] . with the exact manifestation depending on the viral variant, lewis rats can develop a biphasic neurological illness characterized by an initial hyperacute aggressive phase that is followed by a passive somnolent stage, an obesity syndrome with fertility disturbances, or paralysis followed by death [5, 25, 26, 36] . rhesus monkeys develop severe paralytic disease with retinopathy [5, 37, 38] , whereas mice and hamsters show no clinical signs despite replication of bdv in the cns [39, 40] . histologic studies of brains from animals with borna disease show typical meningoencephalomyelitis, with the most severe lesions in the frontal region of the cerebral cortex. no pathological abnormalities have been observed in infected mice and hamsters. tree shrews have been used extensively in studies of social interactions because of their well-defined behavioral pat-terns. sprankel et al. found that these animals, classified at the phylogenetic root of the primates, developed a long-lasting, persistent, productive infection in the cns and unique behavioral abnormalities after is inoculation with bdv [33] . characteristic perivascular infiltration of mononuclear cells and intranuclear joest-degen inclusion bodies in the cns were observed. housing conditions influenced the reaction of these animals to infection. the behavior of all paired animals changed dramatically. these animals showed a need for increased body contact with their partners during the first few weeks after infection. further, infected animals tended to accept their partners much more quickly in grooming social interactions than did uninfected shrews. normally passive females became as aggressive as their male partners and took poor care of their offspring. in contrast, only 25% of the infected animals maintained in solitary cages showed clinical and behavioral changes. these animals exhibited hyperactivity, with drastic shortening of resting time, and developed eating disorders (bulimia) about 4 weeks after infection. the hyperactive phase was followed by a hypoactive phase characterized by retirement into sleeping boxes and decreased interest in self-grooming. only some of these animals devel-retinopathy in humans. thus, in studies of these changes, rhesus monkeys were infected ic with bdv [5, 37, 38] . the animals developed a persistent infection in the cns and produced antibodies to bdv-specific antigens in serum, csf, and aqueous humor. pathological alterations in the cns and retina consisted of infiltrations of mononuclear cells similar to those in naturally infected animals; however, no destruction of the neuronal layers of the retina was observed [37, 38] . clinical signs started with apathy and anorexia 4-8 weeks after infection and progressed to severe neurological manifestations dominated by paralysis of the hind limbs. one animal splenectomized 11 months before infection showed signs of apathy but developed no paralysis and had fewer cellular infiltrates in the cns and the retina than did other animals [37] . these findings suggested that the pathogenesis of borna disease in primates is based on an immunopathological mechanism-an idea confirmed in studies with lewis rats and rabbits. neurotropism of bdv figure 2 . northern blot analysis of rna from rat brain. lanes a and c contain 10µg of total rna from uninfected rat brain. lanes b and d contain 10 tig of total rna from bdv-infected rat brain. lanes a and b were hybridized with a negative-sense 32p-labeled oligonucleotide (247), lanes c and d with a positive-sense 32p-labeled oligonucleotide (305). the blot was stripped and rehybridized with a chicken li-actin probe to ensure that equal amounts of rna were loaded in each lane. reproduced with permission from [28] . oped more severe neurological signs, such as ataxia and partial paralysis of the limbs [33] . the pathological changes in the brain and retina of bdvinfected animals resemble certain types of encephalitis and most studies on the pathogenesis of bdv infection have involved experimentally inoculated lewis rats. as has already been mentioned, the clinical manifestations of the infection in these animals depend on the passage history of the virus used for inoculation. thus a variety of bdv variants have been used, including strains that cause paralysis and death (similar to the disease in horses and sheep), obesity with fertility disturbances, behavioral changes, or inapparent infections. in all cases, ic inoculation of bdv into adult rats resulted in productive viral replication in the nervous system, where viral antigen was confined to astrocytes, schwann cells, and central and ganglionic neurons and their axonal-dendritic processes [10, 41, 42] . infectivity was first detected in brain homogenates 7 days after inoculation with 105-106 id50/ml by day 15 [26] . this infectivity titer in the brain was maintained throughout an observation period of 7 months (figure 3, top). similar data were obtained for black hooded rats, which were found to be resistant to the disease [35] . lower levels of infectivity (10 2-104 id50/ml) were usually found in spinal cord, adrenal glands, and ganglia of the autonomic nervous system. infectivity in the eyes was transient, lasting -4 weeks after virus appeared in the brain. loss of infectivity coincided later with degeneration and loss of retinal neurons. in immunocompetent adult rats no infectivity was found in extraneural tissue at any stage [26, 43] . neither infectious virus nor viral antigens were detectable in lung, spleen, kidney, muscle, peritoneal macrophages, or blood. the same pattern of tropism was seen in cyclophosphamide-treated rats [26, 43] (figure 3, bottom) and in athymic adult rats after ic inoculation of bdv [44] . the lack of susceptibility of cultured macrophages and spleen fibroblasts week-old rats infected with bdv and treated with cyclophosphamide (150 mg/kg) 1 day later. the persistently high levels of infectivity in the brain and eyes and the lack of infectivity in nonneural tissues are shown. rats did not respond immunologically to the virus and did not become ill. reproduced with permission from [26] . to infection with bdv demonstrated the strong tropism of the virus for cells of neural origin [26, 27] . in rats inoculated ic or intranasally at birth, bdv replicated preferentially in the cns and the peripheral nervous system but also spread to nonneural tissues. whereas infectivity in nervous tissue was maintained at a level of 10 5 -106 id50/ml, titers in the nonneural tissues such as heart, lung, liver, spleen, kidney, pancreas, and bladder were approximately two orders of magnitude lower [43] . infectious virus was shed in saliva, lacrimal fluid, and urine [10] . infectivity assays and immunocytochemical assays on sequential tissue samples obtained after inoculation from infected newborn rats showed that the agent first began to replicate in the cns and then disseminated along nerve pathways to nonneural tissues. presumably, infection in the latter tissues was initiated at the point of innervation, with subsequent spread from cell to cell. no viremia was detected in these animals. the similar pattern of dissemination noted in infected adult rats treated for prolonged periods with cyclosporin a [23, 45] may suggest that nonneural cells are innately susceptible to infection but that yet-unknown factors in the host prevent access of the virus to those cells. delineation of the scheme for the dissemination of bdv via neural pathways from and to the cns was based on the initial observation by krey et al. [46] that in rabbits spread of the virus from the brain to the retina could be prevented by ablation of the optic disk. the incubation period of the disease in rats (the period between inoculation of virus at a peripheral site and onset of illness) varied significantly with the route of inoculation [10, 27] . the onset of boma disease could be recognized only after the virus invaded the brain. disease appeared 17 days after ic inoculation, 20-24 days after intranasal inoculation, and 47 days after footpad inoculation [27] . after intranasal infection (probably the natural route), bdv entered the neural receptors in the olfactory epithelium and migrated into the brain (figure 4) [10] . similar results were obtained with inoculation into the hind footpad of adult rats (figure 5). infection and disease were prevented by sectioning of the sciatic nerve before or within 1 day after inoculation of bdv [27] . in contrast, neither iv inoculation of bdv weekly for 3 weeks nor oral inoculation of the virus resulted in infection [27] . thus bdv infection in the host resulted only after obligatory replication in nervous tissues, with subsequent dissemination to nonneural tissues via neural pathways in immunologically impaired animals. once introduced into the cns of the rat, bdv usually caused a persistent infection with continuous productive replication in the brain and spinal cord. all immunocompetent infected animals developed antibodies to bdv-specific antigens, and these antibodies coexisted with infectious virus in the cns. hyperimmune sera or csf from bdv-infected animals did not protect against infection in neutralization experiments in vivo or in vitro [26, 27] . infection of newborn rats. although infection of newborn rats resulted in persistent viral replication in the cns as well as in visceral organs, these animals developed no inflammatory response or signs of borna disease. however, "luxury" functions of the cns seem to be affected, possibly as a result of morphological changes confined to the loss of neurons from the dentate gyms of the hippocampus and of some neurons from the nuclear and photoreceptive layers of the retina [25, 26, 43] . the animals produced normal litters, and no virus was found in brain homogenates of their progeny [26] . (however, when housed in the same cage as their infected newborns, mothers can become infected [10] .) infected newborn animals developed bdv-specific antibodies late after infection [26, 43] . infection of immunocompetent rats. infected immunocompetent rats developed severe disseminated meningoencephalitis in which the most intense inflammatory reaction was centered in the gray matter of the cerebral cortex. the onset of clinical disease coincided with the appearance of meningoencephalomyelitis rather than being a direct effect of viral replication in the brain [26, 27] . the most prominent changes were dense accumulations of mononuclear cells in the perivascular spaces and throughout the neuropilcorresponding to the areas with greatest viral replication [25] [26] [27] 42] . immunocytochemical investigation of the composition of the inflammatory cell population during the course of infection revealed that macrophages and lymphocytes of the cd4+ phenotype were dominant at all stages [42, 47] . cd8 + t cells were less frequent. b lymphocytes and plasma cells became prominent during later stages of the disease, when marked parenchymal deposition of immunoglobulin developed [42] . the severity of the inflammatory reaction reached its max-imum between day 30 and day 40, during the hyperacute phase of the disease. edema and necrosis of cellular elements in the neuropil accompanied the inflammatory changes. this phase was characterized by alertness, loss of fear, and frenzied hyperactivity. infection with an "aggressive" variant led to paralysis and death during this phase. the cell loss was most extensive in the cerebrum between the frontal and temporal cortices, and this effect finally led to hydrocephalus ex vacuo in surviving animals [25, 26] . hydrocephalus progressed slowly, and, despite continuous productive replication of bdv in the brain, the inflammatory response began to decline after day 50, with only minimal inflammatory lesions detectable in the brain by day 200. there was no further loss of brain substance and no further extension of hydrocephalus after the inflammatory cells disappeared. beyond day 75, by which the inflammatory response had become mild, the animals became much calmer, spending most of the time in an inactive state and asleep. no pathological changes were found in the ependymal lining of the ventricles or the choroid plexus throughout the study [25, 26] . despite the dynamic changes in the intensity of inflammation, high levels of viral replication in the cns persisted in these animals ( figure 3, top) . pathological changes in the eyes were similar to those in the brain and developed subsequent to encephalitis. there was a degeneration of rods and cones and a gradual focal disappearance of neurons from the inner and outer nuclear layers [25, 26] . by day 100 most of the animals had become blind since the infection resulted in the complete destruction of the inner and outer nuclear layers of the retina. no inflammatory cells were found in the organs at late stages. viral variants differed in their ability to cause various clinical manifestations in the rat. this observation raises the intriguing possibility that bdv exhibits marked genetic variability and that different strains may cause different pathological manifestations of neurological disease. the obesity syndrome developed gradually in immunocompetent infected rats, starting -5 months after infection ( [48] and s. herzog and r. rott, unpublished results). the obese rats exhibited characteristic behavioral and metabolic alterations, with significant increases in the intake of food and water and the development of high levels of triglycerides, insulin, and glucose in the blood [48] . histologic examination revealed prominent hydrocephalus and meningoencephalitis. in long-term survivors, titers of infectious virus in the cns declined drastically and antibody levels decreased ( [48] and s. herzog and r. rott, unpublished results). prevention of disease in rats. in 1981 gierend and ludwig [49] reported that treatment of bdv-infected rabbits with glucocorticoids and/or cyclophosphamide resulted in a reduction in antibody production, the development of only low-intensity encephalitis, and a delayed onset of clinical disease. similar studies showed that a single ip injection of cyclophosphamide (150 mg/kg), given i day before or after ic inoculation of bdv into 4-week-old rats, prevented the onset of borna disease. these rats did not develop antibodies to bdv, encephalitis, or clinical disease [25, 26] . the level of production of virus in the cns and the neurotropism of the agent were similar in treated and untreated infected animals ( figure 3) . thus a persistent, productive type of viral replication developed in the brain and eyes of cyclophosphamide-treated rats (figure 3, bottom) without the complications of pathological effects or clinical disease. as in infected newborn rats, neurons in the dentate gyms of the hippocampus degenerated and were replaced by glial cells. since other infected neuronal groups did not degenerate, loss of these cells may have been due to down-regulation of trophic factors. in contrast, no necrosis among other cell types in the brain occurred in these animals. similarly, infected newborn, athymic, and cyclosporin a-treated adult rats failed to develop encephalitis or disease [23, 26, 36, [43] [44] [45] . these data suggested strongly that borna disease was caused by the cellular immune response to bdv in immunocompetent animals. reconstitution of bdv-specific inflammation: immunosuppressed animals. the immune basis of borna disease was confirmed in adoptive transfer experiments in which spleen or cervical lymph node cells from infected rats were transferred to infected, 4-week-old, cyclophosphamide-immunosuppressed virus carriers. after adoptive transfer these tolerant virus carriers developed bdv-specific antibodies, encephalitis, and behavioral disease (table 1). the donor note. four-week-old lewis rats were inoculated ic with bdv; some rats were treated with cyclophosphamide (150 mg/kg) 24 hours later. animals rendered tolerant by this treatment were injected iv with various cell suspensions, as indicated, and were maintained for clinical and pathological examination. nmi t cells are described in the text; ppd t cells = purified protein derivative-specific t cells. figure 6 . acetone-fixed astrocytes persistently infected with bdv were stained with tetrarhodamine isothiocyanate-labeled rat antibodies to bdv and fluorescein isothiocyanate-labeled rabbit antibodies to glial fibrillary acidic protein (gfap). arrows point to punctate accumulation of bdv antigens in the nuclei, and arrowheads show diffuse staining pattern of gfap in the cytoplasm. cells had no effect when injected into immunocompetent animals [25] . further, transfer of hyperimmune bdv-specific sera into virus carriers did not cause clinical disease [25] . with the goal of identifying the cell type responsible for inducing disease in virus carriers, a permanent t cell line, nm 1, was developed from the popliteal lymph node cells of lewis rats immunized in their hind feet with affinity-purified, bdv-specific 38/39-kd protein [47, 50] . the nm 1 cells responded specifically to the 38/39-kd bdv antigen in lymphocyte proliferation assays and did not recognize unrelated antigens. cytofluorographic studies with mabs to leukocyte differentiation antigens showed a staining pattern characteristic for cd4 + t cells. inhibition studies with mabs against selective restriction elements of the major histocompatibility complex (mhc) revealed that these cd4 + effector t cells were mhc class ii restricted [47, 50] . the latter cells were functionally characterized in studies with astrocytic cultures used as antigen-presenting cells or targets and pulsed with bdv-specific 38/39-kd protein or infected with bdv. astrocytes are known to become infected during bdv-induced encephalopathy [41, 42] , and primary astrocyte cultures are susceptible to bdv infection when inoculated with infectious rat brain homogenate (figure 6) . the persistent noncytopathic infection of astrocytes with bdv did not result in significant spontaneous expression of mhc class ii antigens, although such expression has been reported for other neurotropic viruses [51, 52] ; however, the expression of this antigen was easily induced by incubation with recombinant interferon-7 (ifn-y). in lymphocyte proliferation experiments, uninfected, ifn-7treated astrocytes were able to induce specific proliferation of nm 1 cells when inoculated exogenously with the 38/39-kd protein [50] . when persistently bdv-infected astrocytes were used as antigen-presenting cells after induction with ifn-y, only slight specific proliferation of the nm 1 cells was found. however, exogenous addition of the bdv-specific 38/39-kd protein to these cultures resulted in antigen-specific proliferation [50] . furthermore, when analyzed in a conventional cytotoxicity assay, bdv-infected astrocytes were found to be susceptible to lysis by nm 1 t cells (j. a. richt and l. stitz, unpublished results). the bdv-specific nm i cells induced acute meningoencephalitis mainly in the gray matter of the brain and produced hyperacute paralytic disease when injected iv into immunosuppressed syngeneic virus carriers [47, 50] (table i ) . the clinical signs became manifest as early as 5 days after transfer, with development of severe apathy and somnolence; most of these animals had to be killed within 24 hours after the onset of disease. inflammatory exudates contained a large number of cd4 + t cells, fewer cd8 + lymphocytes and b cells, and some neutrophils [42, 47, 50] . the animals did not go through the hyperactive phase that typically occurs during regular infection or reconstitution with spleen cells. their disease was hyperacute and rapidly fatal. the fact that disease could be induced after transfer of mhc class ii-restricted, bdv-specific, cytolytic cd4 + t lymphocytes suggested that the lesions in borna disease may be a delayed-type hypersensitivity reaction. the mechanism for the different clinical disease that resulted from reconstitution with nm 1 cells-as opposed to spleen and lymph node cells-is not understood. the nm 1 cells probably caused elaboration of unusually large amounts of acute-phase proteins and cytokines, which in turn could have caused an imbalance in neurotransmitter functions in the brain [53] . the difference in clinical disease caused by the spleen and lymph node cell suspensions may also have been due to the combination of nonselected immune cells used for transfer. carriers. the pathogenesis of borna disease has several features in common with lymphochoriomeningitis (lcm) virus infection in mice. in both cases the virus causes acute cns disease that can be prevented by immunosuppression, and this tolerance can be overcome by reconstitution with specifically sensitized cells. the similarity in the pathogenesis of the two infections applies in newborn animals. newborn mice inoculated with lcm virus become persistently infected, but their growth is severely retarded and they later die from immune complex disease [54] . newborn rats inoculated with bdv also become persistently infected, as has already been described. however, unlike their murine lcm-infected counterparts, persistent bdv-infected rats do not become ill [43] . in contrast to adoptive transfer into cyclophosphamidetreated virus carriers [25] , injection of spleen cell suspensions from diseased syngeneic rats into newborn bdv carriers did not result in inflammation or in clinical boma disease [55] . for evaluation of the role of immunologic tolerance in neonatal virus carriers, these animals were surgically joined to syngeneic normal rats via the peritoneal cavity in a manner that allowed the free flow of humoral and cellular immune elements [55] ; this scheme followed the classical experiment of billingham et al. [56] . the normal-counterpart rats were then inoculated with bdv and developed typical borna disease. examination of the tissues from the persistently infected chimera showed that these animals developed mild lesions in the central and peripheral nervous systems. adoptive transfer of the cd4+ virus-specific t cell line nm 1 into neonatal (6-week-old) infected rats also resulted in mild transient encephalitis [55] ; this development suggested that tolerance to the virus in newborn infected animals can be overcome by transfer of bdv-specific immune cells, although reconstitution was not as efficient as in cyclophosphamide-treated virus carriers. regulatory immune mechanisms present in neonatal infected animals, but not in adult rats rendered tolerant by cyclophosphamide, may be responsible for this unique mechanism of tolerance and resistance to the induction of disease. the behavioral changes in animals infected with bdv are somewhat reminiscent of some types of affective disorders in human beings. indeed, the serum and csf of some patients with psychiatric illnesses (including recurrent unipolar depression, bipolar affective disorders, and residual-type schizophrenia or personality disorders) contain bdv-specific antibodies [32, 57] . antibodies were present in 4%-7% of sera obtained from -5,000 psychiatric patients from germany, the united states, and japan. the percentage of patients seropositive was highest in a region of southern germany where borna disease has been known to be endemic among horses and sheep. approximately 1% of 1,000 randomly collected sera from otherwise hospitalized patients from this area in which borna disease is endemic contained antibodies to bdv [20] . examination of 56 acutely seropositive patients by magnetic resonance imaging revealed that 46% had cerebral lesions in one or both hemispheres, whereas no one in a seronegative control group had such lesions [58] . serological examination by bode et al. of patients infected with human immunodeficiency virus has shown an incidence of bdv antibodies of -8% [59] . the same investigators have reported a high incidence of bdv-specific antibodies among patients with chronic inflammatory neurological disorders such as multiple sclerosis [60] . the bdv specificity of the human antibodies was recently reinforced by the finding that antibodies from three of seven seropositive patients recognized the 24-kd protein expressed by a bdv-specific cdna clone [16, 28] (figure 1). these data suggest either that a bdv-like infection is prevalent in the human population or that some patients with inflammatory neurological diseases have cross-reacting antibodies that recognize antigens expressing epitopes homologous with those of bdv proteins. these observations were substantiated further by attempts to isolate bdv from the csf of three seropositive patients [20] . csf from these patients was either applied to fetal rabbit brain cells or inoculated is into rabbits, which are highly susceptible to borna disease. in cell cultures a small number of immunoreactive cell foci were found with bdv-specific antibodies 10-12 days after inoculation. however, the cells lost their antigen during subcultivation. the inoculated rabbits developed no signs of borna disease; neither lesions in the cns nor bdv-specific antigens were demonstrable in brain sections by immunohistologic techniques. nevertheless, these animals developed bdv-specific antibodies in their sera. that the brain homogenate from one rabbit was infectious for fetal rabbit brain cells was demonstrated by positive cell foci in immunofluorescence assays [20] . however, again, the antigen disappeared during attempts to propagate the agent by subcultivation of the cells. these findings can be interpreted as typical of abortive infection. although these data strongly support the hypothesis that a bdv-like agent causes infection in humans, a defined mental disorder has not been correlated with the presence of bdv-specific antibodies. the development of such a disorder may be related to various factors, such as the genetic properties of the virus; the route of infection; and the age, immune status, and genetic makeup of the infected individual. evidence that these factors play a role in the outcome of borna disease has been found in naturally infected horses and sheep as well as in experimentally infected animals. die bornasche krankheit der pferde und schafe virologisch gesicherter ausbruch der bornaschen krankheit in einer schafherde der schweiz experimentelle untersuchungen fiber die seuchenhafte gehirn-und rackenmarksentztindung der pferde (bornasche krankheit) the cerebrospinal fluid of rabbits infected with borna disease virus handbuch der viruserkrankungen untersuchungen fiber das klinische verhalten der seuchenhaften gehirnrtickenmarksentziindung (bornaschen krankheit) des pferdes nebst angaben fiber diesbeztigliche therapeutische versuche borna disease in a sheep untersuchungen fiber die pathologische histologie, pathogenese und postmortale diagnose der seuchenhaften gehirn-rtickenmarksentziindung (bornasche krankheit) des pferdes die ausbreitung der enzephalitischen reaktion bei der bornaschen krankheit der pferde und deren beziehungen zur encephalitis epidemica, zur heine-medinschen krankheit und zur lyssa des menschen. eine vergleichend-pathologische studie axonal transport of borna disease virus along olfactory pathways in spontaneously and experimentally infected rats seroepidemiologische untersuchungen zur bornaschen krankheit (ansteckende gehirn-riickenmarkentziindung) der pferde borna disease: a persistent virus infection of the central nervous system preliminary studies on the biology of borna disease virus molecular characterization of the borna disease agent isolation and characterisation of borna disease agent cdna clones a borna virus cdna encoding a protein recognized by antibodies in humans with behavioral diseases analysis of borna disease virus-specific rnas in infected cells and tissues replication of borna disease virus in cell cultures influence of interferon on persistent infection caused by borna disease virus in vitro borna disease, a possible hazard for man? isolation and characterization of a 14500 molecular weight protein from brains and tissue cultures persistently infected with borna disease virus purification and properties of an intranuclear virus-specific antigen from tissues infected with borna disease virus atypical dissemination of the highly neurotropic borna disease virus during persistent infection in cyclosporin a-treated, immunosuppressed rats antigenetic relationship and further characterization of two major borna disease virus proteins behavioral disease in rats caused by immunopathological responses to persistent borna virus in the brain pathogenesis of borna disease in rats: immune-mediated viral ophthalmoencephalopathy causing blindness and behavioral abnormalities pathogenesis of borna disease in rats: evidence that intraaxonal spread is the major route for virus dissemination and the determinant for disease incubation molecular and immunopathological studies of borna disease virus infection in rats coronaviruses and their replication coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis minus-strand copies of replicating coronavirus mrnas contain antileaders detection of serum antibodies to borna disease virus in patients with psychiatric disorders behavior abnormalities in tree shrews (tupaia glis, diard, 1920) induced by boma disease virus persistent, tolerant or subacute infection in borna disease virus-infected rats studies on the genetic control of resistance of black hooded rats to borna disease untersuchungen fiber die experimentelle bornavirus-infektion bei der ratte borna disease in rhesus monkeys as a model for uveo-cerebral symptoms virus-induced pigment epithelitis in rhesus monkeys. clinical and histological findings persistent borna virus infection in adult hamsters adaptation of borna disease virus to the mouse astrocytes and schwann cells are virus-host cells in the nervous system of rats with borna disease determination of immune cells and expression of major histocompatibility complex class ii antigen in encephalitic lesions of experimental borna disease replication of borna disease virus in rats: age-dependent differences in tissue distribution effect of borna disease virus infection on athymic rats inhibition of immunemediated meningoencephalitis in persistently borna disease virusinfected rats by cyclosporine a spread of infectious virus along the optic nerve into the retina in borna disease virus-infected rabbits borna disease virus-induced meningoencephalomyelitis caused by a virus-specific cd4+ t cell-mediated immune reaction escape from lethal disease in rats after borna disease virus infection: survival with obesity syndrome influence of immunosuppressive treatment on borna disease in rabbits borna disease, a progressive meningoencephalomyelitis as a model for cd4 + t cell-mediated immunopathology in the brain viral particles induce la antigen expression on astrocytes tumor necrosis factor amplifies measles virus-mediated la induction on astrocytes narayan 0, oldstone mba. neurotransmitter abnormalities in borna disease southern p. virus and immune responses: lymphocytic choriomeningitis virus as a prototype model of viral pathogenesis induction of borna disease and inflammation in rats inoculated with borna disease virus as neonates quantitative studies on tissue transplantation immunity. iii. actively acquired tolerance borna disease virus. a possible etiological factor in human affective disorders? kernspintomographische befunde bei psychiatrischen patienten mit and ohne semm-antikfirper gegen das borna disease virus-specific antibodies in patients with hiv infection and with mental disorders increased incidence of borna disease virus infection in chronically diseased patients key: cord-266696-w9sb038q authors: zhou, yi-hua; chen, zhaochun title: is the immune system impaired in patients with severe acute respiratory syndrome? date: 2004-03-15 journal: clin infect dis doi: 10.1086/382081 sha: doc_id: 266696 cord_uid: w9sb038q nan severe acute respiratory syndrome? sir-cui et al. [1] recently described pronounced lymphopenia and low counts of cd4 + cells, cd8 + cells, and b cells in patients with severe acute respiratory syndrome (sars). on the basis of these low cell counts, cui et al. [1] suggested that sars coronavirus (sars-cov) might damage lymphocytes and concluded that the immune system was impaired during the course of sars. however, cui et al. [1] did not provide direct evidence to support their hypothesis. low counts of both cd4 + and cd8 + cells in the peripheral circulation do not always indicate that the immune system is impaired: redistribution of lymphocytes among peripheral and secondary lymphoid organs and migration of these cells to inflamed tissues caused by infections may also result in lymphopenia. postthymus naive t cells do not reside in any single lymphoid organ but, rather, circulate continuously between blood and lymph through a specialized t cell zone in secondary lymphoid tissues, which forms part of the "recirculating lymphocyte pool." neither splenectomy nor ablation of bone marrow by radioactive isotope therapy reduces the number of lymphocytes, and thymectomy in adult mice causes only a very slow decrease in the size of the recirculating lymphocyte pool. these findings suggest that splenic atrophy and pathological changes in lymph nodes observed in patients with sars [2] [3] [4] were not the causes of lymphopenia. although sars-cov rna was detected in pbmcs obtained from patients with sars [5] , no sars-cov was recovered from splenic, lymphatic, and bone marrow specimens obtained from patients with fatal cases [3] . this finding indicated that the pathological presentations in lymphoid organs were unlikely to have been directly caused by the virus. if sars-cov was directly detrimental to lymphocytes, the damage should start at the beginning of infection and continue through the incubation period, resulting in a decreased lymphocyte count after the onset of sars. however, although most patients had mild to moderate decreased lymphocyte counts during the early phase of illness [6, 7] , many patients had normal lymphocyte counts at the onset of symptoms, and some even had increased cd4 + and cd8 + cell counts during the first week of illness [3] . clinically, there is no evidence that the onset of sars is associated with impairment of the immune system. sars is characterized by respiratory symptoms and signs correlated with pulmonary lesions caused by sars-cov infection. at the time of writing, no report has shown that the initial manifestation of sars is caused by immunosuppression (e.g., aids), and only a small portion of the patients have had secondary bacterial infections-which were easily treatable-during the late course of illness, despite receipt of corticosteroid treatment [8] . the effectiveness of corticosteroid therapy in stopping the progression of pulmonary lesions in patients with sars also suggests that sars is not associated with the impairment of the immune system but is associated with immunopathological damage [9] . cui et al. [1] also found that 76% of patients with sars had a low b cell count. however, the immune function of b cells in patients with sars appeared not to have been impaired, because specific anti-sars-cov was detected as early as day 10 after the onset of illness [8] , 93%-100% of the patients had seroconversion to anti-sars-cov after week 3 [8, 10] , and the level of specific igg remained high for at least 3 months [10] . in conclusion, although lymphopenia does occur in patients with sars, no evidence supports the position that sars-cov damages lymphocytes. humoral immune response to sars-cov is not damaged in patients with sars. before in vitro and in vivo cellular immune responses are investigated, it would be cautious to conclude that the immune system is impaired in patients with sars. [3] demonstrated that, in patients with sars, lymphopenia was observed in various lymphoid organs, there were scanty numbers of lymphocytes in exudates from inflamed lungs, and lymphocytosis was not found in any organs. in another article [4] cited by zhou and chen [1] , hemorrhagic necrosis and attenuation of lymphocytes were also evident in lymph nodes and spleen specimens obtained from patients with sars, which is direct evidence of damaged immune organs and tissues in patients with sars. on the basis of these findings, lang et al. [4] concluded that the severe damage to the pulmonary and immunological systems was responsible for the clinical features of sars and might result in patient death [4] . ding et al. [5] reached the same conclusion. they note that "sars is a systemic disease that injures many organs. the lungs, immune organs, and systemic small vessels are the main targets of virus attack so that extensive consolidation of the lung, diffuse alveolar damage with hy-aline membrane formation, respiratory distress, and decreased immune function are the main causes of death" [5, p. 282 ]. at the time of writing, there is still no evidence to support the view of zhou and chen [1] that lymphopenia is due to the redistribution and migration of lymphocytes. with respect to the expression of lymphocytes and their subsets, li et al. [6] arrived at a conclusion identical to the one we reported [7] . therefore, we believe that the hypothesis that sars-cov may damage the immune system might be supported by recently published studies. we believe that zhou and chen [1] need more data to support their conclusion that the immune function of b cells in our patients appeared not to be impaired because specific anti-sars-cov could be detected as early as 10 days after the onset of illness. although anti-sars-cov was observed in patients with sars, it was not proper to conclude that b cells in these patients were unimpaired, because the extent of impairment might be different and because the function of b cells was not limited to the production of antibody. the fact that 76% of our patients with sars had low b cell counts could imply that b cells were damaged by sars-cov, although the damage was not too severe to affect the production of antibody. there are still too many unanswered questions about sars. although the incidence of sars has subsided in beijing, more investigations are underway. we welcome different opinions and suggestions as we continue our research. expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis pathological study on severe acute respiratory syndrome sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts clinical features and short-term outcomes of 144 patients with sars in the greater toronto area clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study development of a standard treatment protocol for severe acute respiratory syndrome profile of specific antibodies to the sars-associated coronavirus is the immune system impaired in patients with severe acute respiratory syndrome sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis pathological study on severe acute respiratory syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china rapid loss of both cd4 + and cd8 + t lymphocyte subsets during the acute phase of severe acute respiratory syndrome expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome beijing, people's republic of china key: cord-265006-m1dmgcd1 authors: kow, chia siang; hasan, syed shahzad title: do the meta-analyses provide a clean bill of health to the use of renin-angiotensin system inhibitors in covid-19? date: 2020-08-08 journal: clin infect dis doi: 10.1093/cid/ciaa1167 sha: doc_id: 265006 cord_uid: m1dmgcd1 nan m a n u s c r i p t dear editor, we agree with de feria et al. [1] which commented that current observational studies on the effects of renin-angiotensin system (ras) inhibitors use in coronavirus disease 2019 (covid-19) are with marked limitations. in fact, we are aware of the publication of few systematic reviews and metaanalyses [2] [3] [4] [5] [6] [7] [8] which included these observational studies with questionable quality to determine the association between renin-angiotensin system (ras) inhibitors use, including angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs), and mortality/severity of covid-19. therefore, we demand caution when adopting the findings from these meta-analyses. table 1 summarizes the characteristics of systematic reviews and meta-analyses investigating on the use of aceis/arbs on covid-19 mortality and/or severity. firstly, these systematic reviews and meta-analyses suffer from a common methodological flaw where the authors pooled mostly the unadjusted odds ratio in their meta-analyses to determine the risk of mortality or severe/critical illness from covid-19, especially when the adjusted odds ratios were not provided in the included original studies. the pooling of unadjusted estimates can be misleading, since there are many factors that could influence the clinical outcomes of patients with covid-19. without adjustment of the covariates or covariables which could modify the association between ras inhibitors use and mortality/severity of covid-19, the true effect on the use of ras inhibitors in covid-19 cannot be revealed even through a meta-analysis. secondly, some of these systematic reviews and meta-analyses included studies or only involved findings on covid-19 patients with concurrent hypertension. selective inclusion of only hypertensive individuals may not reflect the association between ras inhibitors use and mortality/severity of covid-19 since ras inhibitors are also prescribed for indications other than hypertension, including congestive heart failure, diabetic nephropathy, coronary artery disease, acute coronary syndrome, raynaud phenomenon, amongst others. patients with these conditions too need long-term usage of ras inhibitors, where increased expression of ace2 receptor, which is the hypothesized pathological a c c e p t e d m a n u s c r i p t mechanism leading to worse outcomes among covid-19 patients receiving ras inhibitors, could also occur in the users of ras inhibitors for indications other than hypertension [9] . thirdly, at least half of the studies pooled in these systematic reviews and meta-analyses (table 1) are originated from china. this presented another source of selection bias since patient outcomes may be different across continents or even across different countries, as demonstrated in the wide interval of case fatality rates of covid-19 among countries. in fact, while all studies from china reported either no difference or significant reduced risk of mortality and/or severe/critical disease from covid-19 among users of aceis/arbs compared to non-users, a single-center study [10] from france reported otherwise, in which hospitalized patients with covid-19 receiving ras inhibitors at baseline had significantly increased odds of being admitted to an intensive care unit or death before admission to an intensive care unit (odds ratio 1.73, 95% confidence interval 1.02-2.93). therefore, there may be regional differences in the clinical outcomes from covid-19 among patients receiving aceis/arbs which the currently available systematic reviews and meta-analysis failed to address. we agree with de feria et al. [1] that only through randomized controlled trials that a cause-andeffect relationship can be established. nevertheless, future retrospective or prospective studies should adjust for covariates or covariables in their analysis to provide more clarity on the association between ras inhibitors use and clinical outcomes of covid-19. a c c e p t e d m a n u s c r i p t m a n u s c r i p t ace inhibitors/arb use and covid-19. time to change practice or keep gathering data? a systematic review and meta-analysis to evaluate the clinical outcomes in covid-19 patients on angiotensin-converting enzyme inhibitors or angiotensin receptor blockers decreased mortality of covid-19 with renin-angiotensin-aldosterone system inhibitors therapy in patients with hypertension: a meta-analysis estimation of renin-angiotensin-aldosterone-system (raas)-inhibitor effect on covid-19 outcome: a meta-analysis acei/arb use and risk of infection or severity or mortality of covid-19: a systematic review and meta-analysis outcomes of renin-angiotensin-aldosterone system blockers in patients with covid-19: a systematic review and meta-analysis a meta-analysis of the relationship between renin-angiotensin-aldosterone system inhibitors and covid-19 the use of renin angiotensin system inhibitor on mortality in patients with coronavirus disease 2019 (covid-19): a systematic review and meta-analysis cardiovascular disease and use of renin-angiotensin system inhibitors in covid-19 association between renin-angiotensin system inhibitors and covid-19 complications key: cord-271014-xzpvupms authors: erikstrup, christian; hother, christoffer egeberg; pedersen, ole birger vestager; mølbak, kåre; skov, robert leo; holm, dorte kinggaard; sækmose, susanne gjørup; nilsson, anna christine; brooks, patrick terrence; boldsen, jens kjærgaard; mikkelsen, christina; gybel-brask, mikkel; sørensen, erik; dinh, khoa manh; mikkelsen, susan; møller, bjarne kuno; haunstrup, thure; harritshøj, lene; jensen, bitten aagaard; hjalgrim, henrik; lillevang, søren thue; ullum, henrik title: estimation of sars-cov-2 infection fatality rate by real-time antibody screening of blood donors date: 2020-06-25 journal: clin infect dis doi: 10.1093/cid/ciaa849 sha: doc_id: 271014 cord_uid: xzpvupms background: the pandemic due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has tremendous consequences for our societies. knowledge of the seroprevalence of sars-cov-2 is needed to accurately monitor the spread of the epidemic and to calculate the infection fatality rate (ifr). these measures may help the authorities to make informed decisions and adjust the current societal interventions. the objective was to perform nationwide real-time seroprevalence surveying among blood donors as a tool to estimate previous sars-cov-2 infections and the population based ifr. methods: danish blood donors aged 17–69 years giving blood april 6 to may 3 were tested for sars-cov-2 immunoglobulin m and g antibodies using a commercial lateral flow test. antibody status was compared between geographical areas and an estimate of the ifr was calculated. the seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (ci). results: the first 20,640 blood donors were tested and a combined adjusted seroprevalence of 1.9% (ci: 0.8-2.3) was calculated. the seroprevalence differed across areas. using available data on fatalities and population numbers a combined ifr in patients younger than 70 is estimated at 89 per 100,000 (ci: 72-211) infections. conclusions: the ifr was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. the ifr is likely several fold lower than the current estimate. we have initiated real-time nationwide anti-sars-cov-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic. humanity is suffering from a pandemic due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the local severity of the epidemic and experiences from other countries are used by the health authorities to calibrate societal interventions. these interventions, e.g. the closing of schools, public institutions, prohibition of group gatherings, and even curfew, have tremendous consequences. the authorities rely on accurate real-time data to make informed decisions. thus, numbers of patients tested positive for sars-cov-2, admitted to hospital, needing respiratory assistance or deceased from coronavirus disease 2019 (covid-19) are updated on a daily basis. in contrast, little information exists on the percentage of the population with previous mild or asymptomatic . the proportion of the population who have overcome the infection can probably be approximated by testing for antibodies against sars-cov-2. antibodies may confer immunity to repeat infection and a high proportion of immune individuals can attenuate the epidemic. measures of anti-sars-cov-2 seroprevalence can also be used to estimate the clinical impact of covid-19. statistics on covid-19 morbidity and mortality vary greatly due to varying testing strategies and e.g. the capacity of the health care system to treat infected patients [1] . countries that diagnose mild infections will report lower morbidity and mortality compared to those with a less comprehensive testing strategy. an accurate measure of seroprevalence can be used to estimate the accumulated number of sars-cov-2 infections and thus the infection fatality rate (ifr) in the underlying population. blood donors comprise approximately 4.7% of the danish population in the same age group [2]. healthy volunteers donate blood in all areas of the country ensuring wide geographical coverage. we have initiated a prospective screening of all blood donations for sars-cov-2 antibodies to establish a real-time nationwide overview of antibody status. the objective of this study is to perform a seroprevalence survey among blood donors as a tool in the monitoring of the sars-cov-2 epidemic. a c c e p t e d m a n u s c r i p t 6 in denmark, approximately 270,000 blood donations are given annually. all danish blood donation facilities participated in this survey. from april 6 to may 3, 2020 a total of 20,640 blood donations were given by 17-69-year-old donors. blood donors are healthy and must comply with strict eligibility criteria [3] . currently, donors must self-defer for two weeks if they develop fever with upper respiratory symptoms. the first patient with covid-19 in denmark was diagnosed on february 26, 2020. subsequently, diagnostic testing for sars-cov-2 viral rna was primarily performed in symptomatic individuals returning from high risk areas. on march 3, the government recommended home-quarantine for 14 days in case of exposure to covid-19 including traveling to high-risk areas and on march 6, events with more than 1,000 individuals were recommended to be postponed or cancelled. on march 11, the government established a partial lockdown of the country: for public employees only persons with critical functions were allowed to work, schools and child care facilities were closed except when parents served in critical functions, workers in the private sector were recommended to work from home, and gatherings of more than 100 were prohibited. validation and testing were performed by experienced staff in five regional blood establishments. we retrieved data on population numbers as of january 1 st 2020 [5] and the number of infected and deceased due to covid-19 using daily updated data [6] . statistical analysis was performed in rstudio 1.2 and r 3.6.0. results were reported as percentages with 95% confidence intervals (ci). the epir package was used to adjust seroprevalence for sensitivity and specificity. we used the rogan gladen estimate to calculate the true prevalence. ci were derived by 10^8sample percentile bootstrapping independently sampling sensitivity, specificity and apparent prevalence using posterior binomial distributions. a c c e p t e d m a n u s c r i p t 8 ethics sars-cov-2 antibody testing was performed as a routine screening of all blood donations. only consenting donors were tested and informed about their result. anonymized data was used in this study. the regional scientific ethical committees for the zealand region of denmark approved the investigation as a register project (20-000013). we included blood donors aged 17 to 69 years and a total of 20,640 blood donors were informed and all consented to testing; see table 1 for characteristics. the distribution between seropositivity for igm and igg appears in table 2 . the estimated number of infected individuals was calculated per area in the relevant age group ( table 3) . the overall unadjusted seroprevalence was 2.0% (ci: 1.8-2.2). after adjusting for assay sensitivity and specificity including their ci, the overall seroprevalence was 1.9% (ci: 0.8-2.3). the seroprevalence in the capital region was higher than in the other four regions combined (3.2% vs 1.2%, difference: 2.0 percentage points, ci: 1.4-2.6). there was an insignificant increase in seroprevalence from the first two weeks to the last two weeks (1.7% vs 2.0%, difference: 0.28 percentage points, ci: -0.15-0.78). in this survey of sars-cov-2 antibodies in danish blood donors we found a seroprevalence of 1.9 (ci: 0.8-2.3) adjusted for the assay performance and a low ifr of 89/100,000 (ci: 72-211). this ifr of 0.089% is slightly lower than a recently published covid-19 ifr estimate of 0.145% (ci: 0.088-0.317, individuals below 60 years) not including seroprevalence data [7] . the ratio between estimated antibody-positive individuals and confirmed covid-19 cases is expected given the targeted early danish sars-cov-2 testing strategy. the lack of large seroprevalence surveys prevents a comparison with other areas/countries. the low ifr is encouraging, but several caveats exist. although blood donors represent a very broad population base, they are selected healthy and self-defer for two weeks after signs of covid-19. conversely, blood donor prevalence increases with income [8] and we speculate that this leads to higher risk of exposure through travel and social activity. we may therefore either under or overestimate the true population immunity. we validated the antibody assay primarily in individuals diagnosed with clinical covid-19. if silent and mild infections lead to weaker antibody responses, we will underestimate the population immunity. conversely, we found that 42.7% of donors testing positive were igm-only reactive. it is possible that some of these individuals had asymptomatic infection and we cannot rule out that some were infectious while reporting for donation. also, screening only for antibodies may underestimate the prevalence of infections, if cellular cytotoxicity is able to eradicate virally infected cells, as for sars-cov, before eliciting a humoral response [9] . finally, this study only addresses the ifr in 17-69-year-old individuals. the ifr in other population strata, e.g. among individuals above 80 or with comorbidity is higher [7, 10] . a c c e p t e d m a n u s c r i p t 10 currently, the governments in most countries are trying to balance the economic consequences of a societal lockdown against the risk of an uncontrolled epidemic. our results underpin that social distancing in a healthy population predominately acts as a means to protect vulnerable individuals. it would be challenging to perform an unbiased seroprevalence survey in the background population. as blood donation facilities are located nationwide and operate continuously the screening is suited to monitor regional differences and temporal changes. with greater knowledge of the seroprevalence in other population strata the continued monitoring may also be used to effectively model the activity of the sars-cov-2 epidemic. we undertook a validation and found a less than perfect sensitivity of 82.6% (75.7-88.2) when previous pcr-confirmed covid-19 patients were tested. however, it is known that not all infected individuals produce antibodies. the specificity was acceptable at 99.5% (98.7-99.9) but leads to a low positive predictive value in low-prevalence areas. we used a conservative method to estimate the confidence interval and thus took not only the sample variation but also the uncertainty in the sensitivity and specificity into account. this is necessary because we, unlike most diagnostic and screening tests, do not have a gold standard to confirm positive or negative results. the confidence interval for the regions with lowest antibody prevalence thus reached a lower limit seroprevalence of 0%. we chose to use the current lateral flow test because of early availability enabling us to produce the first sars-cov-2 antibody seroprevalence estimate in denmark. while the assay performed acceptably, we recommend to use a well-validated lab-based assay, which are now available, for subsequent seroprevalence studies. a c c e p t e d m a n u s c r i p t the estimates for the ifr should allow for the lag time from infection to death. based on current literature time from infection to death in non-survivors is 23-30 days [11, 12] . similarly, the lag time from infection to the detection of antibodies may be 16 days [11, 13] . donor self-deferral due to respiratory symptoms will add to the lag time for the detection of antibodies. we used the last available total of deceased citizens due to covid-19 on the last date of the study (may 3, 2020). using earlier values would result in a lower ifr estimate while waiting for later death tolls would result in a higher ifr. the death toll among all citizens below 70 years was used even though only 20 of 65 deaths appeared among individuals with no comorbidity. this was chosen because the denominator included all citizens in the age strata, thus, also individuals with comorbidity. the ifr including only individuals with no comorbidity is thus likely several fold lower than the current estimate. rapid tests are read by individuals and inter-observer variation often exist. furthermore, there is uncertainty regarding cross reactivity of sars-cov-2 and other coronavirus antibodies. the results included in this article will be updated and freely accessible at http://www.bloddonor.dk/antisarscov2. our results indicate that the ifr among individuals aged 17 to 69 years is 89/100,000 (ci: 72-211). this may have implications for risk mitigation. the ifr in older population strata may be considerably higher. nationwide continuous seroprevalence surveying of blood donations may be a tool in monitoring the sarsa c c e p t e d m a n u s c r i p t 12 m a n u s c r i p t 14 tables table 1 female male age and sex stratified seroprevalence of anti-sars-cov-2. likelihood of survival of coronavirus disease 2019 blood donation and blood donor mortality after adjustment for a healthy donor effect spring 2020 -transcripts in danish statistics denmark -population data covid-19 i danmark epidemiologisk overvågningsrapport 2020-04-13 estimates of the severity of coronavirus disease 2019: a modelbased analysis socio-demographic characteristics of danish blood donors t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice severe outcomes among patients with coronavirus disease 2019 (covid-19) -united states the incubation period of coronavirus disease from publicly reported confirmed cases: estimation and application clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study antibody responses to sars-cov-2 in patients of novel coronavirus disease interpreting diagnostic tests for sars-cov-2 we thank laboratory technicians from the departments of clinical immunology at aarhus university hospital, copenhagen university hospital, odense university hospital, zealand university hospital, and aalborg university hospital for their excellent work during the validation of the assay and testing of the samples. the livzon tests were donated by bestseller foundation. bestseller foundation had no influence on the study. the authors report no conflict of interest. a c c e p t e d m a n u s c r i p t 13 m a n u s c r i p t 15 a c c e p t e d m a n u s c r i p t 16 [14] . key: cord-268233-ibxufjrv authors: nagappa, bharathnag; marimuthu, yamini title: seroconversion rate and diagnostic accuracy of serological tests for covid-19 date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa676 sha: doc_id: 268233 cord_uid: ibxufjrv nan a c c e p t e d m a n u s c r i p t we read the recent article by zhao j et al which studied the antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 [1] . this study is the need of the hour which might help in planning the testing strategy for covid-19 in many countries. the study provided valuable information about immunological response in hosts, diagnostic accuracy of antibody assays and rt-pcr in diagnosing covid 19. the findings of the study are very crucial in understanding the natural history of disease. however we have few concerns regarding the study. first, in methodology it was mentioned that all enrolled participants were confirmed to be infected with sars-cov-2 by rt-pcr. however in results, the authors have mentioned that only 112 out of 173 participants tested positive for rna over complete duration of the study, which contradicts the methodology. this finding raise the question about what was the gold standard test considered if not rt-pcr since rt-pcr is the gold standard test for covid-19. second, in table 2, the sensitivity of rna+ab was calculated as 78.7% (74/94) during one to seven days after symptom onset. denominator was considered to be 94 whereas only 87 participants have undergone rt-pcr and the same issue is repeated in all the time periods. third, antibody tests in this study population should be interpreted cautiously since 1) there is plausibility to have cross reaction to antibodies related to other corona virus strains and 2) also diseases caused by other corona virus present with similar symptoms as covid-19, and symptomatic patients might have antibodies which may lead to increased false positivity rate. therefore present study should carefully interpret the serological tests and sensitivity should be calculated only in participants tested positive for rna. fourth, the study reported that median duration for seroconversion was 11 days. however, this should be interpreted cautiously since nearly half of the participants were tested for antibody only after 8 days. it is not clear about whether all participants who tested positive for antibody have a c c e p t e d m a n u s c r i p t given their first sample to be tested for antibodies, before 11 days (25% of them given first sample after 10 days). if not, the results might be biased. to reinforce this point, figure 2b shows that around 58% of the participants were positive for antibodies on day 8 and increased afterwards. with first assumption of kaplan meier method (censored participants will have same probability of experiencing event as uncensored), median time for seroconversion might be lesser than reported [2] . we would also like to know the clinical outcomes of the patients who had undetectable rna in respiratory samples and had detectable antibodies in antibody assay since they could be cured of the disease and recovered with undetectable rna. if all of the participants tested positive for antibody in later stages and negative for rna, were declared as cured and discharged, serological tests will have negligible role in management of such cases in later stage. all the authors declare no conflicts of interest. antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 kaplan-meier method and the log-rank test a c c e p t e d m a n u s c r i p t key: cord-277307-wabruzfs authors: gu, wei; deng, xianding; reyes, kevin; hsu, elaine; wang, candace; sotomayor-gonzalez, alicia; federman, scot; bushnell, brian; miller, steve; chiu, charles title: associations of early covid-19 cases in san francisco with domestic and international travel date: 2020-05-21 journal: clin infect dis doi: 10.1093/cid/ciaa599 sha: doc_id: 277307 cord_uid: wabruzfs in early-to-mid march 2020, 20 of 46 (43%) covid-19 cases at a tertiary care hospital in san francisco, california were travel-related. cases were significantly associated with travel to europe or new york (odds ratio 32.9). viral genomes recovered from 9 of 12 (75%) cases co-clustered with lineages circulating in europe. m a n u s c r i p t as of april 4 th , 2020, the covid-19 pandemic, caused by the novel sars-cov-2 coronavirus 1 , has infected more than 1.2 million people worldwide and the rise in cases has been exponential. in particular, new york cases in the united states quickly surged from 22 to >10,000 between march 10 and 22 2 . by april 4 th there are >150,000 cases in new york and nearby new jersey, threatening to overwhelm hospitals and other regional health care systems in the city. in san francisco, we validated a qrt-pcr test to detect sars-cov-2 infection from nasopharyngeal swab samples based on the eua (emergency use authorization)approved us cdc assay 3 . during the first 10 days since launch, we performed sars-cov-2 testing on 947 samples collected from march 10 through march 20 from patients with suspected sars-cov-2 infection at university of california, san francisco. we reviewed the electronic medical records from the first 46 consecutive sars-cov-2 positive cases admitted to university of california, san francisco hospitals or seen in outpatient clinics from march 10 through march 20. data from these covid-19 patients were matched with 102 randomly selected negative controls who were patients who tested negative for sars-cov-2 over the same time period. documented history was recorded by a physician or nurse practitioner and included sick contacts, health care worker status, and travel history. among the 46 covid-19 positive patients, the median age was 44 years, 46% were female, and 65% were outpatients (table s1) . we noted that a travel history within 2 weeks of symptom onset (median date mar 11, 2020) table s2 , s3). the association with travel may be due to direct exposure to sars-cov-2 while in high prevalence regions (e.g. ny) or exposure while traveling (close contact with fellow travelers or airport personnel). one cluster of 3 positive cases associated with covid-19 infection in an airport worker was categorized as a case of community rather than travel-associated transmission. no significant associations were found with regards to close contacts with known covid-19 infected persons or frontline healthcare workers. those who did not have a recent travel history, a close contact who was covid-19 positive, or were not a frontline healthcare worker were categorized as community transmission with an unknown source of infection and comprised 39% of cases. we conducted viral genomic sequencing and phylogenetic analysis of sars-cov-2 viruses from 12 of 20 travelers for whom the breadth of coverage of the viral genome was >90% 4-6 . these viral genomes were aligned using mafft v7.427 7 with 762 high-coverage viral genomes deposited in the gisaid database 8, 9 as of march 20, 2020, in addition to the most recent viral genomes sequenced in california as of may 3, 2020 4 , for a total of 983 sequences. a maximum likelihood phylogenetic tree was constructed using iqtree (version 2) using an hky substitution model 10 ( figure 2 ). we defined genomic clades through the gisaid nomenclature found at that point in time on march 20, 2020 8, 9 . the majority (9 of 12) of all travel cases clustered in the g a c c e p t e d m a n u s c r i p t clade as defined by the spike protein d614g variant marker (figure 2 , s1, s2), including 3 cases from europe (uc40, uc45, uc46), 4 cases from new york (uc27, uc36, uc44, uc47), 1 case from los angeles (uc26), and 1 case from chicago (uc48). viruses in the g clade comprise most of the genomes sequenced from patients in europe 8, 9 , but notably have also been identified in the vast majority of cases associated with the new york sars-cov-2 outbreak in march to april of 2020, which occurred after the timeline of this study 11, 12 viruses from two additional travel-associated cases from europe (uc43) and new york (uc41) were mapped to other clades circulating in europe (figure 2) . the additional case from europe was found to be part of the v clade, defined by a g251v mutation in the ns3 protein 8,9 . limitations of our study include the use of epidemiological data from only the first 10 days of testing at a single institution. nevertheless, in the setting of an emergent pandemic with shifting epidemiology, the results of our study reached statistical significance over 4 categories of travel (all travel, new york, usa, and europe), and yielded data that may have presaged the exponential rise of new york cases and subsequent large-scale outbreak in the new york metropolitan area 11,12 . a c c e p t e d m a n u s c r i p t m a n u s c r i p t genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding real-time rt-pcr panel for detection 2019-ncov a genomic survey of sars-cov-2 reveals multiple introductions into northern california without a predominant lineage metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform disease and diplomacy: gisaid's innovative contribution to global health nextstrain: real-time tracking of pathogen evolution iq-tree: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies introductions and early spread of sars-cov-2 in the new york city area sequencing identifies multiple, early introductions of sars-cov2 to new york city region cryptic transmission of sars-cov-2 in washington state substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) epidemiology of covid-19 in a long-term care facility a c c e p t e d m a n u s c r i p t key: cord-015493-vf4et613 authors: deresinski, stan title: in the literature date: 2007-10-15 journal: clin infect dis doi: 10.1086/522526 sha: doc_id: 15493 cord_uid: vf4et613 nan after failing to detect nucleic acid of known respiratory viruses in a nasopharyngeal aspirate specimen obtained from a 3-year-old child who had been hospitalized in brisbane, australia, with pneumonia, gaynor and colleagues, in a search for novel viral pathogens, randomly amplified and cloned total nucleic acid from the specimen. they then sequenced a 384well plate of clones using a universal primer. this yielded multiple reads, 6 of which were of sequences of a single virus. further evaluation of these sequences demonstrated homology (albeit limited) to known polyomaviruses, with the closest relationship being to ki, a polyomavirus that had recently been recovered from respiratory tract samples from individuals with respiratory symptoms [1] . the newly discovered virus was designated wu, in keeping with the practice of naming polyomaviruses with 2-letter designations (e.g., ki, jc, and bk). phylogenetic analysis demonstrated that wu and ki are divergent from other known polyomaviruses and define a novel branch within this family. wu was detected by pcr in 37 (3.0%) of 1245 respiratory specimens collected in brisbane in 2003 and in 5 (1.2%) of 410 upper respiratory samples collected in st. louis, missouri, as well as in 1 of 480 bronchoalveolar lavage samples obtained mostly from adults, also in st. louis. in brisbane, the samples yielding wu were obtained from individuals aged from 4 months to 53 years, although 33 of the 37 specimens were obtained from persons aged р3 years. at least 3 of the 4 adults in whom wu was detected were signifi-cantly immunocompromised, and the fourth had cirrhosis and was undergoing mechanically ventilation. wu was the only virus recovered from 12 patients, whereas at least 1 additional virus was detected in the other 25 patients; these included rhinovirus (in 15 patients) and human bocavirus (in 10 patients). three viruses were detected in 6 samples, and 1 sample contained 4 viruses (wu, bocavirus, rhinovirus, and adenovirus). all 6 samples from st. louis that contained wu yielded evidence of the presence of an additional virus. overall, 72% of the samples that contained wu had evidence of coinfection with у1 additional virus. because the bk and jc polyomaviruses are commonly found in urine specimens, 501 urine samples obtained from renal transplant recipients in st. louis and 226 samples obtained from patients in brisbane were screened by pcr; none contained wu. these findings suggest-but do not prove-that this novel polyomavirus has a role in respiratory tract infection. the very high frequency of detection of additional viruses, which were detected in almost three-fourths of the patients, complicates the analysis. nonetheless, the frequency of its detection in individuals (especially young children with respiratory symptoms) in 2 geographically distinct areas highly suggests that it is a cause of human disease. in the meantime, we can expect the continued identification of additional novel viruses as candidate human respiratory pathogens and can add wu, along with the recently identified rhinovirus hrv-qpm [2] , to the list of viruses identified in respiratory tract specimens since 2001: human metapneumovirus, severe acute respiratory syndrome coronavirus, coronaviruses nl63 and hku1, human bocavirus, ki polyomavirus, and now hrv-qpm (rhinovirus) and wu polyomavirus. ruhe jj, menon a. tetracyclines as an oral treatment option for patients with community-onset methicillin-resistant staphylococcus aureus skin and soft-tissue infections. antimicrob agents chemother 2007; 51:3298-303. the role of antibiotics in the treatment of uncomplicated ssti due to community-acquired mrsa has been a matter of dispute. early reports suggested that incision and drainage of cutaneous abscesses due to community-acquired mrsa was sufficient, at least for abscesses of relatively small size. newer, more convincing evidence, however, appears to indicate that antibiotic treatment does matter. ruhe and colleagues retrospectively examined the records of 282 patients with 282 episodes of ssti due to mrsa who presented either to the emergency department or to the outpatient clinic at 2 medical centers during the period 2002-2007. three-fourths of patients had abscesses, 13% had furuncles or carbuncles, and 12% had cellulitis with a purulent focus. the median lesion maximum diameter was 4 cm (range, 3-5 cm). incision and drainage procedures were performed for 91% of episodes with abscess, furuncles, or carbuncles and for 66% of the other episodes. for 90 episodes (32%), patients received doxycycline ( ) or n p 87 minocycline ( ), whereas a b-lactam n p 3 antibiotic was provided for the remaining episodes. during the years of the study, 94%-96% of isolates were found to be susceptible to tetracycline. ten percent of patients experienced treatment failure, with 23 requiring a second incision and drainage procedure and 5 requiring hospital admission. in contrast to previous reports, the size of the lesion was not associated with outcome. logistic regression analysis identified receipt of a b-lactam antibiotic as the only clinical predictor of treatment failure (adjusted or, 3.94; 95% ci, 1.28-12.15). subgroup analysis that included only the 225 episodes in which incision and drainage were performed at presentation confirmed that blactam antibiotic therapy was significantly associated with treatment failure. these results are consistent with those of several other recent studies that have found that administration of an antibiotic that inhibits the growth of mrsa improves outcomes, even in patients who have undergone incision and drainage. for example, in a similar retrospective study of 531 episodes of community-acquired mrsa ssti, most of which were treated with incision and drainage, ruhe et al. [1] found treatment failure rates of 5% among patients who received an antibiotic active against the pathogen and 13% among those who received an antibiotic without activity ( ). there has been little p p .01 study of which antibiotic with activity against mrsa is optimum. although ruhe and colleagues provide evidence of the benefit of doxycyline for the treatment of ssti due to antibioticsusceptible mrsa, alternative antibiotic choices may be considered. in a small, prospective, open-label trial, outpatients with ssti were randomized to receive treatment with either trimethoprim-sulfamethoxazole (160 mg/800 mg) or doxycycline (100 mg), each given twice daily for 7 days [2] . although there was not a statistically significant difference in outcome, all 3 treatment failures occurred among the 14 patients who received trimethoprim-sulfamethoxazole, for a treat-ment failure rate of 21%. the optimal antibiotic choice thus awaits results of future clinical trials with sample sizes sufficient to allow for definitive answers. one problem, however, is that community-acquired mrsa is a moving target, and that, in some geographical areas, it has acquired additional antibiotic-resistance determinants. an additional factor that must be considered in future trials is the potential benefit of effective antibiotic therapy on the prevention or delay of recurrences of infection. identification of a third human polyomavirus characterization of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis community-onset methicillin-resistant staphylococcus aureus skin and soft-tissue infections: impact of antimicrobial therapy on outcome prospective randomized trial of empiric therapy with trimethoprim-sulfamethoxazole or doxycycline for outpatient skin and soft tissue infections in an area of high prevalence of methicillin-resistant staphylococcus aureus in the literature: chlamydia trachomatis finds a way to evade diagnosis screening for chlamydial infection: us preventive services task force recommendation statement the most frequently reported notifiable disease in the united states is infection with chlamydia trachomatis, with neisseria gonorrhoeae infection placing second. urine specimens were obtained from 6632 participants (age, 14-39 years) during the period 1999-2002 in the national health and examination survey; ligase chain reaction detected a prevalence of chlamydial infection of 2.2% (95% ci, 1.8%-2.8%) and a prevalence of gonorrheal infection of 0.24% (95% ci, 0.16%-0.38%). the prevalence of chlamydial infection was similar in male and female subjects but peaked at the ages of 14-19 years in female subjects and 14-29 years in male subjects. the prevalence was higher among non-hispanic black sub-jects (6.4%) than among non-hispanic white subjects (1.5%). the prevalence of gonorrheal infection was also higher in non-hispanic blacks (1.2% vs. 0.07%). forty-six percent of individuals with gonorrheal infection were coinfected with c. trachomatis. it should be noted that the test used in this survey targets a portion of a cryptic plasmid in c. trachomatis that has been lost by some isolates in sweden, leading to false-negative results [1] . although this is not likely to have affected the results of this study, it is an issue that may have to addressed in the future.the authors point out that these data support current recommendations for screening sexually active females aged р25 years for chlamydial infection and for coadministration of treatment for c. trachomatis infection in patients with gonorrheal infection, unless the former has been ruled out [2] . the following is a summary of current recommendations for screening [2] :• screen for chlamydial infection in all sexually active, nonpregnant, young women (age, р24 years) and in older, nonpregnant women who are at increased risk.• screen for chlamydial infection in all pregnant women aged р24 years and in older, pregnant women who are at increased risk.• do not routinely screen for chlamydial infection in women aged у25 years, regardless of whether they are pregnant, if they are not at increased risk.• current evidence is insufficient to assess the balance of benefits and harms of screening for chlamydial infection in men. key: cord-011745-dbdtpojs authors: thompson, mark g.; sokolow, leslie z.; almendares, olivia; openo, kyle; farley, monica m.; meek, james; ray, julie; kirley, pamala daily; reingold, arthur; aragon, deborah; hancock, emily; baumbach, joan; schaffner, william; lynfield, ruth; ryan, pat; monroe, maya; cheng, po-yung; fry, alicia m.; shay, david k. title: effectiveness of nonadjuvanted monovalent influenza a(h1n1)pdm09 vaccines for preventing reverse transcription polymerase chain reaction–confirmed pandemic influenza hospitalizations: case-control study of children and adults at 10 us influenza surveillance network sites date: 2013-12-01 journal: clin infect dis doi: 10.1093/cid/cit551 sha: doc_id: 11745 cord_uid: dbdtpojs during 2009–2010, we examined 217 patients hospitalized with laboratory-confirmed pandemic influenza in 9 influenza hospitalization surveillance network sites and 413 ageand community-matched controls and found that a single dose of monovalent nonadjuvanted influenza a(h1n1)pdm09 vaccine was 50% (95% confidence interval, 13%–71%) effective in preventing hospitalization associated with a(h1n1)pdm09 virus infection. during 2009-2010, we examined 217 patients hospitalized with laboratory-confirmed pandemic influenza in 9 influenza hospitalization surveillance network sites and 413 ageand community-matched controls and found that a single dose of monovalent nonadjuvanted influenza a(h1n1)pdm09 vaccine was 50% (95% confidence interval, 13%-71%) effective in preventing hospitalization associated with a(h1n1) pdm09 virus infection. keywords. influenza; influenza vaccines; vaccine effectiveness; hospitalization; pandemic/prevention & control. the 2009 influenza a(h1n1) pandemic, which began in 2009 and continued into 2010, resulted in increased hospitalizations in the united states and globally [1, 2] . a recent review reported that monovalent a(h1n1)pdm09 vaccines were 69% effective in preventing medically attended influenza during the pandemic [3] . studies focused specifically on hospitalization have reported estimates of vaccine effectiveness (ve) that ranged from 49% to 90% [3] [4] [5] [6] ; however, to date, no estimate of ve against these outcomes in the united states has been published. we identified patients with community-acquired, laboratoryconfirmed a(h1n1)pdm09 infections through the influenza hospitalization surveillance network (flusurv-net) within the centers for disease control and prevention's (cdc) emerging infections program. flusurv-net conducts populationbased surveillance of influenza-associated hospitalizations among children and adults from select counties in 9 states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, oregon, tennessee) that represent approximately 7.3% of the us population [1, 7] . although a(h1n1) pdm09 vaccine was initially only available to priority groups [8] , enrollment for this study started after vaccine became available to the general population and at least 10% of the local population was estimated to be vaccinated (november-december 2009; supplementary figure) . cases were patients aged >6 months residing in a flusurv-net catchment area who tested positive for a(h1n1)pdm09 influenza by reverse transcription polymerase chain reaction (rt-pcr) assay from respiratory specimens collected shortly before (1%) or upon (99%) admission to the hospital for an acute respiratory illness; 86% of specimens were collected within 7 days of illness onset. patients in longterm care facilities and those with nosocomial infections were not enrolled. for each rt-pcr-confirmed hospitalized influenza case, 2 community controls matched by age group and county of residence were enrolled. persons who had not been hospitalized for a respiratory illness from 1 october 2009 until the hospital admission date of a corresponding case were eligible for enrollment. control children were matched on differing age bands depending on age (children aged 7-23 months were matched ±2 weeks of the case's birth; those aged 24-59 months were matched ±2 months of the case's birth; children aged 5-13 years were matched in 3-or 4-year age bands, ie, 5-7 years, 8-10 years, 11-13 years, and 14-17 years; and adults were matched in 5-year age bands, with the exception of a single 18-24 years age band). controls were recruited from individuals identified through birth certificate registries (for children aged <5 years) or lists of random landline telephone numbers (for those aged ≥5 years). structured telephone interviews (forms available upon request) were conducted with cases after hospital discharge and their matched controls; guardians were interviewed for participants aged <18 years. participant characteristics, vaccination status and date, and the presence of medical conditions associated with increased risk of influenza complications [9] were collected from telephone interviews, from reviews of medical records at primary care providers, and through review of hospital records for hospitalized cases. receipt of at least 1 dose of vaccine was documented by medical record or by self-report (if date and location of vaccination could be provided). high-risk conditions and vaccination status were documented by self-report only for the 35% of cases and 34% of controls for whom medical records were unavailable. immunization was defined as receipt of any monovalent a (h1n1)pdm09 vaccine ≥14 days before illness onset for the case; for matched controls, the illness onset date of their corresponding case was used. ve was estimated as 100% × (1odds ratio [ratio of odds of being immunized among the cases to the odds of being immunized among the controls]) using conditional logistic regression models. the following covariates in our adjusted models were similar to those used in other recent pandemic ve studies [5, 6, 8] : age, race, ethnicity, region, highrisk medical condition, and month of index-case illness onset. other covariates were considered for inclusion in the model as confounders if they were related to both vaccination and influenza status or if they affected the ve point estimate by >5%. we estimated that 78 cases were needed to achieve 80% power (α = .05) to detect a ve of 60% with 2 controls per case and a vaccination rate of 30% among controls. each participating site submitted a protocol for evaluation by their state institutional review board (irb), and irb approval was obtained if required. april 2010, a total of 329 hospitalized patients with rt-pcr-confirmed a(h1n1)pdm09 infection were identified as potential study cases; 24 pediatric and 88 adult patients were excluded either because they did not give consent (n = 107) or because vaccination status could not be confirmed (n = 5). enrolled cases were similar to all eligible cases with respect to age and onset month (data not shown). only 1 matched control was enrolled for 10% of the cases (21 of 217). compared with community controls, hospitalized influenza cases were more likely to be non-white, hispanic, and unmarried; have less education and lower incomes; lack private healthcare insurance; be obese; and have high-risk medical conditions (table 1) . among cases, the proportion immunized differed by age; among controls, hispanics and those with a high-risk medical condition were more likely to be immunized; no other significant association between participant characteristics and immunization status was observed. among 217 hospitalized influenza cases, 14% were immunized compared to 22% immunized among 413 community controls. the crude ve against a(h1n1)pdm09 for prevention of hospitalization was 38% (95% confidence interval [ci], 4%-61%) for all ages. the ve adjusted for age, race, ethnicity, region, high-risk respiratory condition, and month of index case illness onset was 46% (11%-67%). potential confounding was observed for education, insurance status, and presence of a nonpulmonary high-risk medical condition. adding these to the multivariate model resulted in a fully adjusted ve of 50% (95% ci, 13%-71%). hispanic ethnicity, lower education, lack of healthcare insurance, and pulmonary and nonpulmonary high-risk medical conditions were all statistically significant contributors in the final multivariate model (data not shown). in secondary stratified analyses, the adjusted ve for those with 1 or more high-risk medical condition was 54% (95% ci, 16%-75%), and the ve point estimate for those without a high-risk condition was similar at 46% (95% ci, −89% to 84%), although confidence intervals were wide given the small number of cases without a high-risk condition. in sensitivity analyses, ve estimates were unchanged when those vaccinated 7-13 days prior to the illness onset of the index case were considered immunized. we estimated that during the winter wave of the 2009 influenza a(h1n1)pdm09 pandemic in the united states, a single dose of monovalent nonadjuvanted a(h1n1)pdm09 vaccine was 50% (95% ci, 13%-71%) effective in preventing hospitalization associated with a(h1n1)pdm09 virus infection. this finding is similar to the adjusted ve of 56% (23%-75%) against medically attended a(h1n1)pdm09 influenza reported by a us study with the same vaccine options in which 9% of the cases were hospitalized [8] . our ve point estimate of 50% was consistent with ve estimates in an australian study of hospitalization and unadjuvanted pandemic vaccine that used a test-negative control design (ve = 49%; 95% ci, 13%-70%) [6] , but was substantially lower than the inpatient ve reported by a european study of adjuvanted pandemic vaccine that used a test-negative control design (ve = 90%; 95% ci, 48%-100%) [5] . the strengths of this study include case ascertainment through population-based surveillance and confirmation of abbreviations: bmi, body mass index; ca, california; co, colorado; ct, connecticut; ga, georgia; md, maryland; mn, minnesota; nm, new mexico; ns, not statistically significant (p > .05); or, oregon; tn, tennessee. a two controls who had not been hospitalized for a respiratory illness since 1 october 2009 were recruited matched by age and community. for cases aged 7-23 months, controls were within plus or minus (±) 2 weeks of the case's age at illness onset; for cases aged 24-59 months, controls were ±2 months of the case's age. potential controls for cases aged <5 years were selected at random from birth certificate registries and matched to the case's zip code. for cases aged >5 years, controls were within ±7 years of the case's age, resided in the case's county, and were contacted from a telephone sampling list generated by survey sampling international (shelton, connecticut). b pearson χ 2 test was used to assess differences between cases and controls in the distribution of participant characteristics. c pearson χ 2 test was used to assess differences between participant characteristic groups (rows) in the percentage vaccinated; differences in vaccination by participant characteristics were tested separately with cases and then controls. d characteristic describes interviewed parent/caregiver when participant is aged <18 years. e high-risk respiratory conditions included lung disease and asthma as indicated by self-report of physician diagnosis and/or a medical encounter with international classification of disease (icd-9) codes available upon request. other high-risk medical conditions were indicated by self-report of physician diagnosis and/or 1 or more medical visits for a condition associated with increased risk of influenza complications, including cancer, diabetes, and neurological disorders as well as heart, immune, and kidney disease (icd codes available upon request). medical conditions were determined by self-report for 35% of cases and 34% of controls with missing or unavailable medical records. f vaccination is defined as receipt of at least 1 dose of vaccine documented by medical record or self-report (if date and location were also provided); immunization is defined as receipt of any a(h1n1)pdm09 vaccine ≥14 days before illness onset of the case or index case if a matched control. the difference between the number vaccinated and the number immunized reflects the fact that 15 cases and 24 controls were vaccinated after the illness onset date (of the index case) and 8 cases and 11 controls were vaccinated 1-13 days before the illness onset date. vaccination status and dates were determined by self-report for 35% of cases and 34% of controls with missing or unavailable medical records. influenza by rt-pcr. the study addresses a gap in knowledge regarding ve against serious influenza complications during the pandemic and the ve of nonadjuvanted vaccines, which may have fewer local and systemic reactions but can be less immunogenic in some populations [10] . our study also has limitations. first, cases and matched controls differed on multiple characteristics. although adjusting for potential confounders increased the ve point estimate from 38% to 50%, residual or unmeasured confounding may have biased our results in unknown ways. second, our limited sample size resulted in ve estimates with wide confidence intervals and precluded stratification by age or consideration of site differences in a mixed-effects model. third, our findings may have been influenced by information and selection biases, as only residents with telephone landlines could be enrolled as controls (for those aged ≥5 years) and medical history was incomplete for one-third of cases and controls. because the proportion with missing information was similar for cases and controls, we do not expect this was a significant source of bias, but bias could have been introduced if the likelihood of recalling vaccination differed for cases vs controls with only selfreport data. fourth, information on vaccine type (inactivated vs live attenuated) and on receipt of a second recommended dose (among children aged <10 years) was not available for every subject, which likely resulted in underestimation of ve, as other studies found that ve improved after accounting for these differences [8] . finally, similar to other ve estimates [5, 6, 8] , we lacked information on medical utilization or possible infections during earlier waves of the pandemic. natural immunity acquired from infection prior to the availability of vaccine would lead to over-or underestimating ve if those immune were more or less likely to be vaccinated, respectively. in conclusion, our results suggest that a single dose of monovalent nonadjuvanted a(h1n1)pdm09 vaccine prevented onehalf of the potential hospitalizations associated with a(h1n1) pdm09 virus infection. this finding from the flusurv-net, which serves geographically and economically diverse communities across the united states, confirms previous reports of the preventive benefit of the vaccine [8, 11] and adds to the evidence indicating that influenza vaccines have the potential to prevent a substantial proportion of influenza-associated hospitalizations [12] . increase in rates of hospitalization due to laboratory-confirmed influenza among children and adults during the 2009-10 influenza pandemic epidemiology of 2009 pandemic influenza a (h1n1) in the united states efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis estimating the effect of influenza vaccines and pandemic vaccines, to prevent influenza hospitalizations during the autumn 2009 influenza pandemic wave in castellon, spain. a test-negative, hospital-based, case-control study effectiveness of h1n1/09 monovalent and trivalent influenza vaccines against hospitalization with laboratory-confirmed h1n1/09 influenza in australia: a test-negative case control study adult hospitalizations for laboratory-positive influenza during the effectiveness of non-adjuvanted pandemic influenza a vaccines for preventing pandemic influenza acute respiratory illness visits in 4 u.s. communities prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) immunogenicity and safety of as03-adjuvanted 2009 influenza a h1n1 vaccine in children 6-35 months effectiveness of 1 dose of influenza a (h1n1) 2009 monovalent vaccines in preventing reverse-transcription polymerase chain reaction-confirmed h1n1 infection among school-aged children in maine effectiveness of seasonal vaccine in preventing confirmed influenza-associated hospitalizations in community dwelling older adults disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the cdc.potential conflicts of interest. w. s. serves as a consultant for pfizer, inc, and dynavax technologies; has received an honorarium from sanofi pasteur; and is a member of the advisory board for the data safety monitoring board for merck & co, inc. r. l. has received royalties from a book chapter published by blackwell-wiley and travel expenses partially paid by the global pertussis initiative (parexel international). all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-291363-re45w37d authors: sanville, bradley; corbett, rebecca; pidcock, wesley; hardin, kaitlyn; sebat, christian; nguyen, minh-vu; thompson, george r; haczku, angela; schivo, michael; cohen, stuart title: a community transmitted case of severe acute respiratory distress syndrome due to sars cov2 in the united states date: 2020-03-30 journal: clin infect dis doi: 10.1093/cid/ciaa347 sha: doc_id: 291363 cord_uid: re45w37d nan the current novel coronavirus (sars cov2) outbreak, which was identified in december 2019 in wuhan, hubei, china has spread rapidly causing a significant public health crisis worldwide 1 . the focus of public health measures in the united states has been on individuals with known at risk travel or contacts with at risk individuals 2 . while person-toperson transmission without clear exposure has been observed in other countries 3 , we present a case of the first community transmission of sars cov2 in the united states. a patient in their 40s presented to an outside facility with 3-4 days of flu-like symptoms. chest x-ray upon admission showed a right upper lobe consolidative process with airbronchograms ( figure 1) . computed tomography revealed a dense consolidation with airbronchograms in the right upper lobe with minimal areas of ground glass, primarily in the right middle lobe. the remaining lung parenchyma was normal in appearance. within 24 hours of admission, her respiratory status deteriorated, and she required intubation. followup imaging showed bilateral dense alveolar filling. she progressed to develop the acute respiratory distress syndrome (ards) with refractory hypoxemia requiring a fraction of inspired oxygen (fio2) as high as 100% with a positive end expiratory pressure (peep) of 16 despite intermittent neuromuscular blockade. she developed septic shock requiring large volume crystalloid resuscitation and high dose norepinephrine. antibiotics were expanded to linezolid, piperacillin-tazobactam, and azithromycin. she underwent bronchoscopy with report from the treatment team of negative bacterial cultures after two days. a respiratory viral panel was in process at time of transfer. the patient was transferred to our facility for consideration of extracorporeal life support (ecls) given refractory hypoxemia on hospital day 5. upon arrival the patient had a heart rate of 123 beats per minute and a temperature of 36.9°c. the blood pressure was 117/61, which was maintained by a norepinephrine drip at 0.5 mcg/kg/min. peripheral saturation of oxygen was 91% on an fio2 of 90%, respiratory rate of 26 breaths per minute, a peep of the patient was immediately placed on droplet and contact precautions, and a respiratory viral panel, respiratory culture, and blood cultures sent. she underwent prone positioning and therapeutic paralysis with cisatracurium to maintain ventilator synchrony for treatment of her severe ards. a vasopressin infusion was started for further blood pressure support. given her critical condition in light of the current outbreak, a suspicion for a potential sars cov2 infection was raised. but the patient had no travel to high-risk countries and no contact with an individual with high-risk travel thus, as she did not meet the current centers for disease control (cdc) criteria, testing was not pursued by public health officials. a sars cov2 infection was therefore low on our initial differential diagnosis and a typical bacterial infection was thought to be more likely. to date, no source of exposure has been identified for this patient. therefore, we conclude that she must have had community acquired disease. although her course was complicated by acute renal failure, dialysis was never required and she did not require ecls. she improved clinically following a week of therapy, with a significant reduction in ventilator support and interval improvement in her oxygenation and chest radiograph findings ( figure 3 ). she was successfully extubated 14 days after her hospital transfer and is currently in as a result of the positive sars cov2 test, healthcare workers who were exposed to the patient were required to undergo home quarantine with symptom and fever monitoring. two healthcare workers in contact with the patient at the outside hospital have subsequently tested positive for sars cov2. no transmission has been noted to healthcare workers at our institution. while the majority of covid-19 cases were identified within our patient's age group years), only 18.5% of severe cases were from this age group. those over the age of 65 comprised 49.2% of severe cases 6, 9 . in a large review of 73,314 cases from china the authors reported 5% of all cases meeting critical illness (i.e. shock, respiratory failure, and/or multi-organ failure) with a case-fatality rate of 49% in that subgroup 9 . health care personnel that became infected suffered a similar amount of severe and critically ill cases as the general population. overall, these reviews note a case fatality rate of 1.40-3.46%, though this may be considerably lower when accounting for a likely large number of mild or asymptomatic patients that were not tested 6, 9, 10 dewit and colleagues from the nih, gilead, and columbia university successfully treated rhesus macaques against a model of mers 13 . whether remdesivir is effective against human covid-19 is not known. the ability to provide early testing and diagnosis would certainly accelerate clinical trial efforts to investigate this compound. as will any novel infection, our knowledge evolves over time as more data from affected individuals is collected and analyzed. given changes in epidemiologic risk and potential for atypical presentations, the challenge for clinicians to identify whom to test becomes more difficult. this virus has a prolonged incubation time (2.1-11.1 days in the most optimistic 8 estimate of 88 chinese patients) with a wide range of disease severity 16 . as we have reviewed, its radiographic appearance can be quite varied both in time from symptom onset and overall appearance. a wide variety of symptomology and radiographic appearances create difficulty for providers to identify covid-19 and differentiate it from other more common respiratory infections. it has also been shown that human coronaviruses persist on inanimate surfaces for up to 9 days, thus, can be picked up without close contact to an infected person 17 . as noted in a recent editorial, diagnosis becomes even more difficult considering the likelihood of a large number of mild or asymptomatic patients who are not formally identified with a sars cov2 infection 18, 19 . because there are individuals in the community that are not manifesting severe enough symptoms to warrant presenting to healthcare providers, we should expect community spread to occur more frequently, challenging our ability to adequately contain the spread of sars cov2. our case has influenced national health policies for revising screening criteria. nonetheless, to tackle the outstanding burning issues (true prevalence and mortality rate, impact on update and interim guidance on outbreak of 2019 novel coronavirus (2019-ncov) a locally transmitted case of sars-cov-2 infection in taiwan evaluating and reporting persons under investigation (pui) ct imaging features of 2019 novel coronavirus (2019-ncov) clinical characteristics of coronavirus disease 2019 in china time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia covid-19): relationship to duration of infection characteristics of and important lessons from the covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures the 2019 novel coronavirus outbreak -a global threat epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study prophylactic and therapeutic remdesivir treatment in the rhesus macaque model of mers-cov infection coronaviruses -drug discovery and therapeutic options the nucleoside analog gs-441524 strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents covid-19 -navigating the uncharted presumed asymptomatic carrier transmission of covid-19 key: cord-010578-uib9h1lb authors: mawle, alison c.; nisenbaum, rosane; dobbins, james g.; gary, howard e.; stewart, john a.; reyes, michele; steele, lea; schmid, d. scott; reeves, william c. title: seroepidemiology of chronic fatigue syndrome: a case-control study date: 1995-12-17 journal: clin infect dis doi: 10.1093/clinids/21.6.1386 sha: doc_id: 10578 cord_uid: uib9h1lb we performed serological testing for a large number of infectious agents in 26 patients from atlanta who had chronic fatigue syndrome (cfs) and in 50 controls matched by age, race, and sex. we did not find any agent associated with cfs. in addition, we did not find elevated levels of antibody to any of a wide range of agents examined. in particular, we did not find elevated titers of antibody to any herpesvirus, nor did we find evidence of enteroviral exposure in this group of patients. we performed serological testing for a large number of infectious agents in 26 patients from atlanta who had chronic fatigue syndrome (cfs) and in 50 controls matched by age, race, and sex. we did not find any agent associated with cfs. in addition, we did not find elevated levels of antibody to any of a wide range of agents examined. in particular, we did not find elevated titers of antibody to any herpesvirus, nor did we find evidence of enteroviral exposure in this group of patients. chronic fatigue syndrome (cfs) is an illness of unknown etiology, characterized by debilitating fatigue lasting longer than 6 months and a variety ofnonspecific symptoms, including myalgia, arthralgia, lymphadenopathy, low-grade fever, sleep disorders, and inability to concentrate [1] . an infectious etiology has been suggested for cfs, although the evidence is not compelling [2] . many patients report the sudden onset of a flulike illness that presaged their fatiguing illness, and a number of infectious agents are known to cause postinfection fatigue. reports that viral antibody titers are elevated in cfs cases has led to the speculation that latent viruses may be reactivated in this illness as a result of an underlying perturbation of immune function, and that elevated titers of antibody to common agents may be a reflection of this disturbance. we conducted serological tests for a large number of infectious agents as part of a case-control study assessing risk factors for cfs. our goals were (1) to determine whether we could detect a common exposure history among cfs patients who met a stringent research case definition and (2) to determine whether levels of antibody to any of the agents examined were elevated in cfs cases. patients with cfs were recruited from the atlanta component of the centers for disease control and prevention's (cdc's) surveillance study ofcfs [3] . cases ofcfs in atlanta received who currently met the 1988 cfs research case definition [4] and who had been sick for~10 years were eligible for the study; 26 cases were recruited to participate in the study. we selected two controls for each case. they were matched for age (±5 years), race, and sex. controls were selected by random-digit dialing in the five-county area of atlanta covered by the surveillance system. all controls were screened to eliminate those with medical conditions that could bias the results (reyes et al., unpublished data). we collected blood and stool samples from each participant. serum and stool specimens were stored at -70°c. each laboratory was requested to perform serological testing of samples as a single batch to minimize interassay variation. if that was not possible, samples from a case and two matched controls were tested in the same batch. all samples were coded so that the tests were performed in a blinded fashion. retroviruses. serological testing for hiv-1 and hiv-2 was performed with use of a u.s. food and drug administration (fda)-licensed elisa kit (genetic systems, redmond, wa). confirmatory testing was performed with an fda-licensed western blot test (cambridge biotech, rockville, md). antibodies against human t-lymphotrophic virus types i and ii were detected with an elisa, and confirmatory testing was performed by western blotting [5] . to detect the presence of a retrovirus in peripheral blood lymphocytes (pbls) from cases, we cultured 2 x 10 6 pbls with allogeneic phytohemagglutinin-stimulated pbls for 4 weeks. cultures were fed weekly and culture supernatants were screened weekly for reverse transcriptase activity [6] . igm with use of a monoclonal antibody-capture elisa similar to that described previously [7] . to detect active infection, we prepared rna from stool samples and performed a reverse transcriptase pcr to detect enteroviral sequences [8] . for four cases and three controls, materials were insufficient for enteroviral rna testing. arboviruses. sera were screened with a hemagglutinationinhibition assay [9] against a panel of 10 arboviruses. antibodies against colorado tick fever virus and vesicular stomatitis virus were detected by means of a cf assay [10] . titers of 1:10 were considered positive. herpesviruses. titers of antibody to cytomegalovirus, human herpesvirus 6, and varicella zoster virus were determined by means of an elisa against the whole virus. optical density readings were converted into antibody units with use of known standards [11] . cytomegalovirus and varicella zoster virus values were considered positive at~100 antibody units, and human herpesvirus 6 values were considered positive at~200 antibody units. epstein-barr virus (ebv) early antigen antibody titers were determined with a commercial elisa (gull laboratories, salt lake city), and a titer of~i: 10 was considered positive. antibodies specific for ebv viral capsid antigen (vca) and nuclear antigen were titrated by indirect immunofluorescence assay (ifa) [12] . a titer of~1:10 was considered positive. type-specific herpes simplex virus serology was performed with a glycoprotein g-based western blot assay [13] . respiratory viruses. sera were tested for the presence of antibodies to adenovirus; parainfluenza viruses i, 2, and 3; and respiratory syncytial virus. testing was performed by elisa with a 1:100 serum dilution [14] . antibodies to coronavirus subtypes oc43 and sn229e were detected with a microneutralization assay [15] . hepatitis viruses. sera were screened for antibody to hepatitis b or hepatitis c by elisa, with use of commercially available kits (abbott laboratories, abbot park, il). elisapositive sera were subjected to confirmatory testing by western blotting (abbott laboratories). other viruses. antibody to measles nucleoprotein [16] , rubella (rubestat g, bio whittaker, walkersville, md), and parvovirus b19 [17] was detected by elisa. measles neutralization titers were also determined [18] . rickettsiaceae. serological testing for antibodies to rickettsia typhi, rickettsia ricketts ii, coxiella burnetii, and ehrlichia chaffeensis was performed with use of ifa against fixed whole organisms [19] . a titer of~1:32 was considered positive. bartonella (formerly rochalimaea species). sera were screened for antibodies to bartonella henselae, b. quintana, and b. elizabethae by means ofifa with fixed whole organisms [20] . a titer of~1:32 was considered positive. borrelia burgdorferi. sera were screened by elisa for antibodies against flagellin. a positive result was confirmed by western blotting (robbins et ai., manuscript in preparation). candida albicans. precipitating antibodies were detected by both latex agglutination and immunodiffusion. the result was considered positive if either the immunodiffusion assay was positive or the latex agglutination titer was~1:4. we also screened for the circulating candida antigens mannan a and mannan b [21] . chlamydia. sera were screened by ifa at 1:32 dilution against chlamydia trachomatis, serotype l2. this contains the lipopolysaccharide antigen specific for the genus chlamydia and thus detects antibodies to all chlamydia species [22] . we analyzed our data using matched analysis procedures. we used a nonparametric test (cochran-mantel-haenzel statistic [23] with modified ridit scores [24] ) to assess differences in continuous variables between cases and controls. the logarithm in base 2 of the titers of antibody to ebv-vca, ebvearly antigen, ebv -nuclear antigen, and coronavirus oc43 as well as the measles neutralization titer were used in the analysis, since they have a natural interpretation when titers are considered (each dilution represents 1 unit in the log base 2 scale). in all statistical testing we carried out, a p value of~.05 was considered significant. since the design of the study was exploratory and not hypothesis-testing, we did not correct for multiple comparisons [25] . statistical analyses were performed with the statistical software sas (sas institute, cary, nc). we recruited 26 patients with cfs (23 female and 3 male) and 52 matched controls. two controls withdrew, leaving 2 cases with only 1 matched control. all participants were white (this finding is consistent with those of other studies). the median duration of illness for the cases was 5.3 years (range, 2.2-10.5 years), and the median age at onset of illness was 33.5 years (range, 16-49 years). occupation, income, and education were comparable between cases and controls. agents infrequently detected in or absent from cfs cases. reverse transcriptase pcr analysis of stool specimens revealed the presence of enteroviral rna in three cases (13.6%) and four controls (8.5%). there was no overlap between those who had enterovirus-specific igm and those who had detectable viral rna in their stool. there were no statistically significant differences between cases and controls for any of these agents. agents frequently detected in cfs cases. all other agents tested were detected in ;;:::25% of cfs cases, and antibody levels were compared between cases and controls. these agents included the herpesviruses cytomegalovirus, varicella zoster virus, human herpesvirus 6, ebv (early antigen, nuclear antigen, and vca), and herpes simplex virus 1; the respiratory viruses adenovirus, respiratory syncytial virus, coronavirus, and parainfluenza viruses 1,2, and 3; measles virus; parvovirus b19; and rubella (tables 1 and 2). the seropositivity rates in both cases and controls were ;;:::50%. there were no differences between the two groups in terms of seropositivity rates or virusspecific antibody levels, as measured by titer, by antibody units, or by optical density in an elisa. we have screened a well-characterized group of patients with cfs for evidence of infection with a wide range of agents and have compared percentages of infection with age-, race-, and sex-matched controls. we found no evidence of a common infectious agent in the patients studied. there were no differ-ences in prevalence of current enteroviral infection between cases and controls, as determined by levels of circulating enterovirus-specific igm, nor in the frequency of detectable enterovirus in stool samples. thus, in the group of patients from atlanta, enteroviral infection was not more common in cfs cases. we were unable to detect evidence of infection with any known human retrovirus, and our inability to detect reverse transcriptase activity in cocultures suggests that there is no common, unknown retrovirus in these patients. this is consistent with other studies that have been unable to detect retroviruses in cfs [26, 27] . we found no evidence of infection withb. burgdorferi, the causative agent oflyme disease. infection with b. burgdorferi can lead to a cfs-like illness, and in areas where lyme disease is endemic, it can contribute to cfsrelated estimates [28] . in this group of patients from atlanta, there was no evidence that lyme disease is a factor in cfs. other agents that had <25% seroprevalence in these patients included arboviruses, hepatitis b and c, rickettsiaceae, bartonella species, chlamydia species, and precipitating antibodies to c. albicans. we also looked for circulating candida antigens, since these are frequently found in severely immunodeficient patients. none of our patients with cfs had detectable mannan a or mannan b, which suggests that these patients were not immunodeficient. the lack of precipitating antibodies to c. albicans argues against the occurrence of a chronic candidal infection in patients with cfs. herpesviruses-in particular, ebv and human herpesvirus 6-have been associated with cfs. although elevated titers to ebv-vca and ebv early antigen were initially described as occurring in patients with cfs [29, 30] , subsequent studies made it clear that these were not specific to cfs [31] [32] [33] . in this study, we found no differences in titers to ebv-vca or ebv nuclear antigen between cases and controls. however, the titer of early antigen was slightly elevated in the cases compared with that in controls, although the difference was not statistically significant. in conclusion, we were unable to find evidence of a single infectious agent associated with cfs in this patient population, nor did we find evidence of elevated antibody titers consistent either with reactivation of a latent virus or with generalized immune activation. the chronic fatigue syndrome: a comprehensive approach to its definition and study is chronic fatigue syndrome an infectious disease? epidemiology of chronic fatigue syndrome: the centers for disease control study chronic fatigue syndrome: a working case definition simultaneous confirmation and differentiation of human t-lymphotropic virus types i and ii infection by modified western blot containing recombinant envelope glycoproteins isolation, characterization, and transmission of human t-lymphotrophic virus types i and ii in culture echovirus 30 infection and aseptic meningitis in parents of children attending a child care center enteroviral rna sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses adaptation oflecf microtechnique in standardized diagnostic complement fixation method and adaptation to micro test prior herpes simplex type 2 infection as a risk factor for hiv infection immunofluorescence in cells derived from burkitt's lymphoma evaluation of a test based on baculovirus-expressed glycoprotein g for detection of herpes simplex virus type-specific antibodies viral antibody screening system that uses a standardized single dilution immunoglobulin g enzyme immunoassay with multiple antigens antigen detection in human respiratory coronavirus infections by monoclonal time resolved fluoroimmunoassay baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays human parvovirus bi9-specific igg, iga, and igm antibodies and dna in serum specimens from patients with erythema infectiosum evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus a comparison of the complement fixation, indirect immunofluorescent antibody, and microagglutination tests for the serological diagnosis of rickettsial diseases serological response to rochalimaea henselae antigen in suspected cat-scratch disease serodiagnosis of fungal diseases single antigen fluorescence test for chlamydia antibodies average partial association in threeway contingency tables: a review and discussion of alternative tests on the combination of independent two-sample tests of wilcoxon no adjustments are needed for multiple comparisons search for retrovirus in the chronic fatigue syndrome lack of evidence for infection with known human and animal retroviruses in patients with chronic fatigue syndrome jennekens fgl chronic muscle weakness caused by borrelia burgdorferi meningoradiculitis evidence for active epstein-barr virus infection in patients with persistent unexplained illness: elevated anti-early antigen antibodies persisting illness and fatigue in adults with evidence of epstein-barr virus infection frequency of chronic active epstein-barr virus infection a cluster of patients with a chronic mononucleosis-like syndrome: is epstein-barr virus the cause? antibodies to epstein-barr virus in patients with chronic fatigue the authors gratefully acknowledge drs. l. schonberger, w. gunn, a. khan, and g. holmes (cdc), whose initial work on cfs led to this study. they are indebted to drs. w. bellini, c. black, r. craven, d. erdman, t. folks, 1. hierholzer, n. karabatsos, r. lal, s. lambert, m. pallansch, r. regnery, e. reiss, and t. tzianabos (cdc), in whose laboratories many of the reported assays were performed, and to dr. 1. gow (institute of neurological science, scotland) for performing the enteroviral rtpcr. they also acknowledge d. connell, b. randall, and r. jacobs (abt associates) as well as e. parris (cdc), who were responsible for study logistics and data processing. key: cord-282539-skzosh6u authors: casadevall, arturo; joyner, michael j; pirofski, liise-anne title: implications of coronavirus disease 2019 (covid-19) antibody dynamics for immunity and convalescent plasma therapy date: 2020-08-17 journal: clin infect dis doi: 10.1093/cid/ciaa1213 sha: doc_id: 282539 cord_uid: skzosh6u nan the article by wang et al in this issue of clinical infectious diseases reports that neutralizing antibody titers to the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus peak 4-5 weeks after the onset of symptoms and decline by 3 months [1] . a lentiviral pseudovirus expressing the sars-cov-2 spike protein was used as a surrogate for measurement of sars-cov-2 neutralizing antibodies. although this assay does not use live sars-cov-2 virus, it has a good correlation with assays that use native virus [2] . the results reported by wang et al [1] match those of plasma donors in the united kingdom, in whom antibody titers declined over the first 3 months after resolution of covid-19 [3] . these findings have important implications for immunity and ongoing efforts to deploy convalescent plasma for prevention and therapy of covid-19, as well as for the generation of antibody products from plasma, such as specific immunoglobulins. the implications of the results of this study for immunity to sars-cov-2 are not surprising: for most, if not all infectious diseases, immunity, as reflected by antibody levels, wanes with time. should this be the case for covid-19, what will matter is the slope by which immunity declines over time, something that varies for different pathogens. for example, measles confers lifelong immunity [4] , whereas a bout of norovirus disease results in immunity that lasts from 6 months to 2 years [5] . as discussed by wang et al [1] , coronaviruses are notorious for eliciting short-lived immunity. the decline in neutralizing antibody titers measured by wang et al [1] in 93.5% of the patients studied is concerning for it raises the possibility that like other coronaviruses, covid-19 may not result in the establishment of long lasting immunity. however, such a conclusion is premature at this time because the relationship between different components of the immune response and resistance to re-infection has not been established. furthermore, it is very early in the covid-19 epidemic and conclusions about long-term immunity will have to wait for longitudinal studies of susceptibility as a function of time. at present, it is not known if sars-cov-2 antibody titers will plateau at a lower level sufficient to prevent reinfection. we also do not know the specificity or functional activity of antibodies that may prevent reinfection. for example, antibodies to sars-cov-2 determinants that are not identified by current assays and/or nonneutralizing antibodies may prevent infection by potentiating other antiviral mechanisms such as antibody-dependent cellular cytotoxicity, as has been described for other viruses. furthermore, antibody-mediated humoral immunity is only one component of successful immune responses that also include t-cell immunity, which can exert antiviral effects. in addition, together with b cells, t cells help provide memory for future encounters with sars-cov-2. importantly, the amount of antibody needed to protect a recovered person from reinfection in the setting of immunological memory of sars-cov-2 is likely to be only a small fraction of the amount of antibody generated in the immediate convalescent period. therefore, the conclusion that there is a causal relationship between declining titers of neutralizing antibody and susceptibility to reinfection is currently premature. medicine has known of sars-cov-2 for less than 1 year and correlates of immunity are not well understood. more time and research are needed to inform our knowledge of the duration and durability of immunity to sars-cov-2 following asymptomatic infection as well as symptomatic covid-19. it is also difficult to extrapolate the results of wang et al [1] to the type of immune response that should be elicited by sars-cov-2 vaccines. although the goal of most current vaccine formulations is to elicit neutralizing antibodies to sars-cov-2 that might recapitulate the response to natural infection, it is important to note that protective immune responses elicited by vaccines and natural infection can differ [6] . perhaps the most extreme example of this phenomenon are vaccines for diphtheria and tetanus, whereby toxoids produced from their toxins elicit long-lasting protection, but these diseases do not induce immune responses that confer immunity [6] . any conclusions concerning sars-cov-2 vaccine design or efficacy drawn from the findings of the work of wang et al should be cautious yet optimistic. wang et al [1] . show that infection with sars-cov-2 does elicit neutralizing antibodies with some durability, and this is good news for the prospect of a successful vaccine. whereas implications of the kinetics of the antibody response to sars-cov-2 for long-lasting and vaccine-mediated immunity are uncertain, the results of wang et al [1] are important for the development and use of antibody therapies. antibody therapies rely on donor b cells, which are used to isolate monoclonal antibodies (mabs), and plasma, which is used for therapy and production of hyper-immune globulin. convalescent plasma has emerged as a promising therapy for covid-19, and there is evidence that its administration early in the course of hospitalization is associated with reduction in viral load, clinical improvement, and reduction in mortality [7] [8] [9] . studies reporting a reduction in viral load following plasma administration have used plasma with high titer neutralizing antibody, with the caveat that these studies were observational and dose-response data are not available. hence, the finding by wang et al [1] that neutralizing antibody titers can drop rapidly after recovery from covid-19 means that there may be a narrow window when a recovered patient is a suitable convalescent plasma donor. similar concerns apply to donations of plasma for the generation of hyper-immune globulin preparations, which are produced from human convalescent plasma. although not analyzed in the wang et al [1] study, similar kinetics apply to circulating b cells that are used to isolate human mabs or serve as the source of variable region genes for construction of human antibodies. the finding that sars-cov-2 neutralizing titers declined relatively quickly in the wang et al study [1] means that efforts to collect convalescent plasma with high titers of neutralizing antibody for therapy and hyper-immune globulin preparation need to be highly organized such that potential donors are contacted early in the weeks following covid-19. a prior report from china found that antibodies to the sars-cov-2 protein peaked at 4 weeks [2] . given that current recommendations for plasma donation advise waiting 4 weeks after the resolution of symptoms to ensure viral clearance and a rise in convalescent antibody titer, the preferred window for plasma collection begins at 4 weeks and could narrow rapidly by 12 weeks. this short collection window means that ensuring a plentiful supply of high-quality plasma requires making it a priority that individuals diagnosed with covid-19 recruited for donation during the relatively short time window between resolution of symptoms, clearance of virus (to eliminate chance of infecting transfusion personnel), and the decline in antibody titer. this is important information for the blood banking community that is producing and supplying convalescent plasma for therapeutic use and the hyper-immune globulin purveyors in the pharmaceutical industry. in addition to studies of the kinetics and durability of sars-cov-2-binding b cells, an important issue that should be addressed by future longitudinal studies is the quality of the antibody response as a function of time. the observation that covid-19 patients who received convalescent plasma late in their course of disease exhibited a reduced viral load [7] , even though most individuals make their own neutralizing antibody by day 10-12 of disease [2] , suggests that there are qualitative differences between the type of antibodies made during the endogenous response and convalescent plasma [10] . in summary, wang et al [1] show that immunity as measured by neutralizing antibody wanes rapidly in the months after infection. at present, the implications of this finding for individual susceptibility to re-infection are unknown. nonetheless, this report has major implications for the timing of harvesting of convalescent plasma for therapeutic use and hyperimmune globulin production because it implies there is a narrow window of time between recovery from covid-19, viral clearance, and still having high levels of neutralizing sars-cov-2 antibody. hence, convalescent plasma collection efforts for covid-19 need to be organized around the temporal dynamics of the immune response to ensure that optimal plasma is obtained from donors. financial support. ac was supported in part by nih grants ai052733, ai15207 and hl139854. mj was supported by nih grant hl139854. lp was supported in part by nih grants ai23654, ai143153, x and a grant from the g. harold and leila y. mathers charitable foundation. longitudinal dynamics of the neutralizing antibody response to sars-cov-2 infection kinetics of viral load and antibody response in relation to covid-19 severity convalescent plasma treatment for sars-cov-2 infection: analysis of the first 436 donors in england immune responses during measles virus infection duration of immunity to norovirus gastroenteritis exploiting the redundancy in the immune system: vaccines can mediate protection by eliciting 'unnatural' immunity effectiveness of convalescent plasma therapy in severe covid-19 patients effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid-19: a randomized clinical trial mortality reduction in 46 severe covid-19 patients treated with hyperimmune plasma: a proof of concept single arm multicenter trial sars-cov-2 viral load and antibody responses: the case for convalescent plasma therapy key: cord-252423-ojmt4k2w authors: qu, bing; yang, jing title: monologue of a physician who tested persistently positive for covid-19 date: 2020-05-11 journal: clin infect dis doi: 10.1093/cid/ciaa551 sha: doc_id: 252423 cord_uid: ojmt4k2w nan m a n u s c r i p t 2 dear editor: life is doomed to be dramatic. as human beings, we tend to think that bad things, such as earthquake, fires, car accident, and severe illness, never occur to us. we believe that these things never happen, even though we have seen or heard about such tragedies that others experienced. when a major medical event hits, the general public is prone to judge it based on their own experience without sufficient specific medical knowledge. healthcare professionals are sometimes no exception, particularly when a new/unknown disease emerges. the covid-19, as other new infectious diseases, has presented us unprecedented challenges and put us into huge dilemma. as a physician on clinical frontline, i had been treating patients actively and wishing them an early recovery, but never expecting that i would be infected. however, i suddenly turned into a patient, an ill person in need of intensive care. as in the early stage we knew little about covid-19 and the possibility that someone with covid-19 can transmit the virus even if they are not showing symptoms, many healthcare professionals were infected. the situation became even worse later due to the shortage of medical supplies, inadequate protective equipment, and accumulated fatigue of medical staff as a result of high intensity work. according to a press conference of the who-china joint mission on covid-19 on february 24, 3387 medical staff have been reported to be infected, among which over 90% were from hubei province. 1 similar to other covid-19 patients, i had dry cough, fever, body aches, and fatigue during the first week. my chest ct examination showed features of ground-glass opacities, and, of course, my rt-pcr test was positive. it has been reported that clinical deterioration/improvement occurs during the second week of illness. 2 on the 10th day after the onset, i developed chest tightness and palpitations, which became worse after activities. the ct re-examination showed increased and enlarged lesions in both lungs. with the additional administration of small doses of glucocorticoids, the clinical symptoms completely disappeared in the third week. unexpectedly, the ct scan in the fourth week revealed new lesions in the right lung which showed lesions completely absorbed the week before. moreover, my rt-pct retests (eight times) were all positive after hospital admission. all of these indicated that the disease remained m a n u s c r i p t 3 uncontrolled. as a potential "persistently positive" patient, i was not alone. the corriere della sera, a daily newspaper in milan, reported that a 23-year-old model had tested positive for novel coronavirus six times during the 57 days after her diagnosis [reference]. this new group of patients have brought new challenge to covid-19 treatment. however, the cause of "persistent positiveness" remains unknown, and no treatment has so far been proven to efficiently help patient turn positive to negative. as time went by, my condition neither worsened nor improved. the courage to win the battle was gone. unstable mood swing started, accompanied by endless imagination of gain and loss. was it due to that covid-19 is severer than other infectious diseases such as seasonal flu? or was it due to a lack of proper immune responses? is it possible that i would become a long-term carrier of this new coronavirus？would i be discriminated against? there is no existing/specific medication for the disease. my heart rate was high (the highest was 130). given a high angiotensin-converting enzyme 2 (ace2) level in heart, would the virus attack the heart and cause viral myocarditis? would it lead to cardiopulmonary sequelae? the china's diagnosis and treatment protocol for novel coronavirus pneumonia has been renewed six times, but various treatment options still exist and agreement has yet to be reached. singing is my favorite way to relieve pressure. when i sang my favorite folk"chengdu" in the hospital room, my daughter played piano accompaniment for me at home through online video, which made us immersed into her piano sound. it is also as if we casually went from street to street in search of local various food with my daughter holding my right cuff while i putting my left hand in my trouser pocket. "daddy, when are you going home?" my daughter repeated in every video call. "a few more days." "a few more days? how many more days exactly?!" she pouted, and i knew she was upset. my friends sent me photos of food, boasting as if they were chefs and gourmets. i knew they just tempted me with food, and encouraged me to carry on. relatives, colleagues, and friends sent me wishes to keep me warm. doctors and nurses encouraged me to stay strong. it seemed that my life was back to normal and full of trivial things: chatting with family and neighbors, and strolling on the street or in the campus. i stopped distorted thinking but gradually calmed down. finally, after 53 days of hospitalization with treatment of integrated chinese and western medicine, i was declared cured and discharged! i wanted to thank every person who offered me support and helpyou are the torch carriers, lighting my way home. m a n u s c r i p t 4 the epidemic in china is at present under control and being improved, but covid-19 has become a pandemic and spread rapidly across the whole world. although the disease was first reported in china, the source of the virus has not been confirmed. many issues regarding prevention, control and treatment of the disease need to be addressed by medical scientists and sociologists all together in the world. as a doctor infected with this highly infectious disease, i sincerely exhort that all of us stay in awe of the virus but do not despise or panic. we should not only maintain proactive health and hygiene regiments, but also cooperate and study actively to fully understand and defeat covid-19. m a n u s c r i p t 5 exploring the reasons for healthcare workers infected with novel coronavirus disease 2019 (covid-19) in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china we thank dr. ruoqing chen for her help with language editing. a c c e p t e d m a n u s c r i p t 6 key: cord-032234-pfr4l1v9 authors: poloni, chad; tsoukas, chrisos title: evaluating immune dysregulation in patients with covid-19 requires a more accurate definition of the cd45ra(+) t-cell phenotype date: 2020-06-04 journal: clin infect dis doi: 10.1093/cid/ciaa664 sha: doc_id: 32234 cord_uid: pfr4l1v9 nan to the editor-the coronavirus disease 2019 (covid-19) pandemic has disproportionally affected the elderly. the recently published study conducted in wuhan, china, by qin et al indicated dysregulation of the immune response specifically related to t lymphocytes, suggesting that they are highly involved in the pathophysiology of covid-19 [1] . t-cell dysregulation is a major contributor to age-related changes of the immune system in the elderly, where t-cell responses become defective. the causes of immunodeficiency are multifactorial, including t-cell phenotypic changes, signal transduction failure, and thymic involution [2, 3] . dysregulated t-cell responses have been linked to a variety of different diseases typically seen in the elderly, notably cardiovascular disease and alzheimer's [4, 5] . furthermore, an immune phenotype known as the immune risk phenotype (irp) has been used as a marker to track these changes, and is defined by a low cd4:cd8 t-cell ratio and an expansion of cd8 + cd28 − t cells in those cytomegalovirus seropositive [6] . it has been shown that irp-positive individuals have an expansion of cd8 + effector memory t cells (t em cells) that are low functioning and late-differentiated, causing memory inflation [7] . the recent covid-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has disproportionately impacted the elderly, with severe cases being linked to increases in proinflammatory cytokines in serum [1] . qin et al sought to characterize the t-lymphocyte responses in covid-19, with aims to differentiate between nonsevere and severe cases. the severe cases had a significantly higher average age compared to the nonsevere cases, indicating a worse outcome in the elderly. additionally, the severe group had an increased incidence of cardiovascular disease as compared to the nonsevere population. as the irp is seen at increased levels in the elderly and has been associated with increased incidence of cardiovascular disease, it would be interesting to see the significance of the irp in terms of covid-19 response. the wuhan study also identified several differences in t-cell populations between the severe and nonsevere covid-19 cases. most notably, there were significantly higher levels of cd3 + cd4 + cd45ra + t cells in the severe cases, which was attributed to increases in naive cells. although naive t cells are characterized by the presence of a combination of surface markers including cd45ra, this marker alone cannot be used to define naive subsets. furthermore, cd45ra is re-expressed during late differentiation and is part of a proinflammatory phenotype identified in the elderly [8] . this terminally differentiated t-cell population has been associated with immune dysregulation in the elderly and is further characterized by low cd28 and increased cd57 expression [9] . the wuhan study failed to further characterize the cd45ra + t-cell subset, making it impossible to attribute the increase specifically to the naive t-cell subset. furthermore, the study did not report on cd8 + cd45ra + t-cell subsets, which are thought to play an important role in the inflammatory aging process. improved characterization of terminally differentiated cd45ra + t cells, along with screening for irp positivity, may be beneficial in identifying those with potential for severe covid-19. dysregulation of immune response in patients with covid-19 in wuhan, china t cell aging: a review of the transcriptional changes determined from genome-wide analysis immunosupportive therapies in aging cytomegalovirus infection and relative risk of cardiovascular disease (ischemic heart disease, stroke, and cardiovascular death): a meta-analysis of prospective studies up to cytomegalovirus infection and risk of alzheimer disease in older black and white individuals comprehensive evaluation of the immune risk phenotype in successfully treated hiv-infected individuals t cell responses to cytomegalovirus from "truly naïve" to "exhausted senescent" t cells: when markers predict functionality multiparameter flow cytometric analysis of cd4 and cd8 t cell subsets in young and old people key: cord-015516-hx7ktq8j authors: nan title: in the literature date: 2005-10-15 journal: clin infect dis doi: 10.1086/497097 sha: doc_id: 15516 cord_uid: hx7ktq8j nan ace converts angiotensin i to angiotensin ii. the carboxypeptidase ace2 negatively regulates angiotensin ii production by cleaving a single residue from both angiotensin i and ii, thus interfering with the renin-angiotensin cascade. this cascade has recently been demonstrated to play a role in diffuse pulmonary alveolar injury mediated by the interaction of angiotensin ii with angiotensin ii receptor type 1. ace2 has been demonstrated in vitro to be a cell-surface receptor for the surface spike protein of the sars coronavirus. this suggests the possibility that the severe diffuse alveolar damage commonly seen in patients with sars may be, at least in part, the consequence of the effects of angiotensin ii. if so, the down-regulatory effects of ace2 could play a protective role. experimental pulmonary infection with sars coronavirus infection in mice reduced the expression of ace2 in the lungs and, as also occurs in humans, caused diffuse lung injury. these findings were reproduced by exposure to sars coronavirus spike protein in the absence of replicative virus. in a murine model of acute lung injury due to acid aspiration, sars coronavirus spike protein exacerbated lung failure by down-regulation of the renin-angiotensin system. sars coronavirus spike protein-induced lung failure could be mitigated by inhibition with angiotensin ii receptor type 1, the receptor that mediates angiotensin ii-induced vascular permeability and lung injury. in a separate publication, the same investigators demonstrated that recombinant ace2 protects mice from acid aspiration-induced lung injury [1] . the investigators also note that ace1 polymorphisms have been associated with progression of sars [2] . on the basis of these findings, the investigators proposed that the severe pulmonary alveolar injury seen in patients with sars is the consequence of sars coronavirus spike surface protein-associated down-regulation of ace2. the decreased availability of ace2 impairs the ability to down-regulate the renin-angiotensin system, exacerbating lung injury. the authors suggest that recombinant ace2 protein could be a potential therapeutic agent in this and related pulmonary infections and diffuse alveolar injuries. trim5 is an intracellular retroviral restriction factor that effectively binds to incoming retroviral capsids and inhibits their replication at a stage prior to or concurrent with reverse transcription. trim5a appears to account for the resistance of old world monkeys to hiv-1 infection. human trim5a, in contrast, is unable to abort intracellular hiv-1 infection, despite having 87% homology at the amino acid level with its rhesus monkey counterpart. stoye and colleagues explored the difference between simian and human trim5a by constructing chimeras of human and rhesus monkey trim5a gene se-quences and mapping the regions required for hiv-1 restriction. this approach revealed that the region involved in this restriction is the spry domain of trim5a and that a single amino acid substitution in the human gene product (arginine for proline at amino acid position 332) enables human cells to block hiv-1 replication. these findings suggest that the vulnerability of the human population to hiv-1 is the consequence of mutations in trim5a during our remote evolutionary history. other investigators have provided evidence for positive selection within the relevant spry domain, presumably as a consequence of ancient exposures to retrovirus [1] . finally, these results point to the potential therapeutic value of molecules that mimic trim5a, as well as to the possibility of future targeted gene therapy. the golden pigment produced by s. aureus is compromised of a group of carotenoids. carotenoids produced by fruits and vegetables are capable of scavenging free radicals and quenching singlet oxygen. liu and colleagues devised a set of experiments designed to examine the hypothesis that carotenoids produced by s. aureus provide a survival advantage to the organisms by allowing them to resist the oxygen-dependent bactericidal activity of phagocytic cells. with use of targeted mutagenesis and heterologous expression, they demon-strated that mutant (knockout) s. aureus with impaired synthesis of carotenoids was more susceptible to killing by oxidants than was the wild-type strain. inhibition of the neutrophil oxidative burst eliminated the relative survival advantage of wild-type s. aureus strains that were capable of normal carotenoid biosynthesis. mutant s. aureus strains were incapable of abscess formation after subcutaneous injection in mice. heterologous expression of s. aureus carotenoids in streptococcus pyogenes conferred relative resistance to oxidants and killing by neutrophils, as well as increased virulence in an animal model of infection. a mixed-function oxidase inhibitor known to inhibit pigment formation in s. aureus did so in a dose-dependent fashion that paralleled an increase in the susceptibility of the organism to killing by singlet oxygen, as well as a decreased survival rate in murine whole blood. this observation indicates that targeting the carotenoids of s. aureus may provide a fruitful approach to the development of novel therapeutic agents directed at this organism. schwartz and colleagues identified 81 patients with definite ( ) or prob-n p 48 able ( ) cns aspergillosis who had n p 33 received treatment with voriconazole in either clinical trials or compassionate-use programs. all patients but 3 had received у1 systemic antifungal agent before receiving voriconazole. thirty-one patients (38%) also underwent neurosurgical procedures (14 underwent craniotomy and abscess resection, and 12 underwent abscess drainage), 4 patients underwent shunting, and 1 patient had an ommaya reservoir placed. thirty-two patients were recipients of hematopoietic stem cell transplants (hscts), all but 2 of which were allogeneic, whereas 11 had received solid organ transplants. all but 6 of the 50 isolates speciated were found to be aspergillus fumigatus. a complete or partial response to voriconazole was achieved in 25 patients (35%) overall but in only 16% of hsct recipients. thirty-one percent of patients were alive at the last follow-up visit, but only 22% of hsct recipients and 27% of solid organ transplant recipients had survived. multivariate analysis found that hsct was a significant predictor of a reduced survival rate, whereas neurosurgical procedures were associated with an improved survival rate. however, patients who underwent such procedures were less likely to have multiple brain lesions. the potential role of surgical intervention was previously suggested in a review of 26 survivors of cerebral aspergillosis, 21 of whom had undergone a neurosurgical procedure [1] . as miserable as these results seem at first glance, they are quite superior to previously published data, which have indicated that responses to other therapies occur in as few as 10% of patients and that the fatality rate for cns aspergillosis in solid organ transplant recipients approaches 100%. although affected by the study's observational nature and likely selection bias, these data strongly suggest that voriconazole therapy is effective for some patients with cns aspergillosis. furthermore, although objections can be made with regard to the findings for neurosurgical interventions (i.e., patients were more likely to have single lesions and had to be considered capable of surviving the procedure), the availability of an aggressive neurosurgical colleague may also be invaluable in improving survival rates for this devastating infection. crabtree jh, burchette rj. surgical salvage of peritoneal dialysis catheters from chronic exit-site and tunnel infections. am j surg 2005; 190:4-8. in patients with chronic infections involving the exit site or tunnel portion of peritoneal dialysis catheters, treatment with antibiotics and local care is often unsuccessful. in such cases, removal of the catheter is usually recommended. one less drastic method that allows salvage of the infected catheter-unroofing of the tunnel tract and removal of the superficial catheter cuff-has been used, but published evidence of benefit has been scanty. this procedure was used by crabtree and burchette in 13 consecutive patients with chronic peritoneal dialysis catheter infections that had been present for a mean duration of 3.2 months, with successful results for all 13. the procedure consisted of excision, by shaving, of the superficial cuff after its identification and mobilization through an incision along the course of the catheter. the exit-site skin and all inflammatory tissue were excised. the catheter and shaved tubing segment were moved out of the wound and secured to the adjacent skin surface with tincture of benzoin and sterile adhesive strips. the wound was packed open and allowed to heal. peritoneal dialysis was allowed immediately, and antibiotics were administered orally for 2-4 weeks. wound healing was complete after a mean period of 1.4 months (range, 0.9-2.2 months). during a mean follow-up period of 18.2 months, 2 persons with nasal carriage of s. aureus developed infection involving the deep cuff (at months 8.9 and 33.7). three patients subsequently had their catheters removed because of peritonitis without exit-site or tunnel infection at months 8-11. this procedure-at least, as performed by this team-appears to be a highly effective means of catheter preservation that allows continuation of peritoneal dialysis without interruption. angiotensin-converting enzyme 2 protects from severe acute lung failure ace1 polymorphism and progression of sars aspergillosis case-fatality rate: systematic review of the literature key: cord-259243-1lkzcslx authors: álvarez-aragón, luis miguel; cuesta-muñoz, antonio luis; álvarez-lópez, inmaculada title: inquiring into benefits of independent activation of non-classical renin-angiotensin system in the clinical prognosis and reduction of covid-19 mortality date: 2020-04-08 journal: clin infect dis doi: 10.1093/cid/ciaa402 sha: doc_id: 259243 cord_uid: 1lkzcslx nan a c c e p t e d m a n u s c r i p t we have read with great interest the elegant manuscript by hanff et al [1] proposing a very interesting association between the classical renin-angiotensin system (ras) and angiotensinconverting enzyme 2 (ace2) dysregulation present in cardiovascular disease (cvd) and the high mortality index in patients with cvd and coronavirus disease 2019 (covid-19). the authors state that pharmacological inhibition of classical ras could have two simultaneous and incompatible outcomes. on the one hand, it will decrease the proinflammatory effect of angiotensin ii with its subsequent benefit on decreasing the risk of acute respiratory distress syndrome (ards) observed in these patients, and on the other hand, it will increase ace2 expression and therefore the virulence of sars-cov2. it has been shown that coronaviruses, in order to access the inside of their host cells, first must bind to a host receptor to be able to fuse both viral and host membranes [2] . in humans, the host receptor is ace2 and, like sars coronavirus (sars-cov), sars-cov-2 also needs ace2 to attack human alveolar epithelial cells [3] . therefore, this also makes ace2 crucial in the infectivity and pathogenesis of sars-cov-2. in addition to the heart and kidneys, the classical ras and ace2 are also present in the lungs [4] . it is pertinent to evoke that ace2 is also a fundamental component in the ace2-angiotensin(1-7)-masr axis, also known as non-classical ras, indicating, therefore, the existence of non-classical ras also in the lungs. non-classical ras is a counter-regulatory system of the classical ras in that its end product, angiotensin(1-7), which after binding to the mas receptor, presents important antiinflammatory, anti-proliferative, anti-fibrotic, natriuretic and vasodilator effects [5] , actions completely opposed to those promoted by the end product of the classical ras angiotensin ii. while pharmacological exclusion or initiation of classical ras inhibition as an adjuvant treatment for sars-cov2 in patients with cvd is elucidated, and considering that covid-19 patients present downregulation of ace2 [1] , and hence low angiotensin(1-7), the direct pharmacological activation of the non-classical ras would be an attractive and plausible approach to tackle the reduction of angiotensin(1-7) to lessen the unwanted effects of angiotensin ii. sodium-glucose co-transporter-2 (sglt2) inhibitors are a group of oral medications used to treat type 2 diabetes, however, large epidemiological studies have demonstrated that slgt2 inhibitors present strong nephro-and cardiovascular-protective effects. in addition, in vitro studies in human renal cells treated with slgt2 inhibitors have shown an increment in angiotensin(1-7) due to the independent activation of the non-classical res, leading to important anti-inflammatory and anti-m a n u s c r i p t fibrotic effects [6, 7] . by analogy, it is reasonable to assume that slgt2 inhibitors could also activate the non-classical res in the lungs. a vast majority of diabetic patients also present cvd and many of them are treated with slgt2 inhibitors to both lower blood glucose and protect the kidney and heart. hence, would diabetic patients with cvd and treated with slgt2 inhibitors present a milder ards as compared to those with a different treatment? would they have a better clinical prognosis? and, would the use of slgt2 inhibitors in non-diabetic patients improve clinical prognosis as well? these are interesting questions since the answers might open a new door to counteract the devastating consequences of the proinflammatory cytokine storm present in covid-19 patients. we declare no conflict of interest. is there an association between covid-19 mortality and the renin-angiotensin system-a call for epidemiologic investigations receptor recognition by the novel coronavirus from wuhan: an analysis based on decadelong structural studies of sars coronavirus structure analysis of the receptor binding of 2019-ncov renin-angiotensin-system, a potential pharmacological candidate, in acute respiratory distress syndrome during mechanical ventilation counterregulatory renin-angiotensin system in cardiovascular disease mechanical forces in diabetic kidney disease: a trigger for impaired glucose metabolism beat it early: putative renoprotective haemodynamic effects of oral hypoglycaemic agents a c c e p t e d m a n u s c r i p t key: cord-280958-36ytqapi authors: decker, summer j; goldstein, todd a; ford, jonathan m; teng, michael n; pugliese, robert s; berry, gregory j; pettengill, matthew; silbert, suzane; hazelton, todd r; wilson, jason w; shine, kristy; wang, zi-xuan; hutchinson, morgan; castagnaro, joseph; bloom, ona e; breining, dwayne a; goldsmith, barbara m; sinnott, john t; o'donnell, donna gentile; crawford, james m; lockwood, charles j; kim, kami title: 3d printed alternative to the standard synthetic flocked nasopharyngeal swabs used for covid-19 testing date: 2020-09-10 journal: clin infect dis doi: 10.1093/cid/ciaa1366 sha: doc_id: 280958 cord_uid: 36ytqapi background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus that causes covid-19, can be detected in respiratory samples by real-time reverse transcriptase (rt)-pcr or other molecular methods. accessibility of diagnostic testing for covid-19 has been limited by intermittent shortages of supplies required for testing, including flocked nasopharyngeal (flnp) swabs. methods: we developed a 3d-printed nasopharyngeal (3dp) swab as a replacement of the flnp swab. the performance of 3dp and flnp swabs were compared in a clinical trial of symptomatic patients at three clinical sites (n=291) using three sars-cov-2 eua tests: a modified version of the cdc real-time reverse transcriptase (rt)-pcr diagnostic panel and two commercial automated formats, roche cobas and neumodx. results: the cycle threshold (c(t)) values from the gene targets and the rnase p gene control in the cdc assay showed no significant differences between swabs for both gene targets (p=0.152 and p=0.092), with the rnase p target performing significantly better in the 3dp swabs (p & 0.001). the c(t) values showed no significant differences between swabs for both viral gene targets in the roche cobas assay (p=0.05 and p=0.05) as well as the neumodx assay (p=0.401 and p=0.484). the overall clinical correlation of covid-19 diagnosis between all methods was 95.88% (kappa 0.901). conclusions: 3dp swabs were equivalent to standard flnp in three testing platforms for sars-cov-2. given the need for widespread testing, 3dp swabs printed on-site are an alternate to flnp that can rapidly scale in response to acute needs when supply chain disruptions affect availability of collection kits. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that causes coronavirus disease 2019 (covid19) , was first described in december 2019, in wuhan, china and quickly spread globally. the world health organization (who) confirmed, 282 cases on january 20, 2020 and, as of june 30, 2020, confirmed over seven million cases [1] , causing over ~500,000 deaths according to the database from the center for system science and engineering (csse) at johns hopkins university. [2, 3] sars-cov-2, the seventh coronavirus known to be transmitted between humans, has shown high transmission rates and is stable on surfaces and in aerosols. [4] [5] [6] symptoms of covid-19 range from mild to severe respiratory illness, with cough, fatigue, and fever being common. it is a progressive and severe disease in certain individuals. those with underlying medical conditions and the elderly are most at risk of developing more severe disease. [4] in addition, high levels of sars-cov-2 shedding in the upper respiratory tract has been reported, even in asymptomatic patients, [7, 8] making individual testing to identify viral carriage even more critical for infection control. initially, there were no available therapeutics approved for treatment or prevention of covid-19 [9] , however the investigational drug remdesivir received emergency use authorization on may 1, 2020 by the us food and drug administration (fda). [10] containment depends on wide-spread testing, isolation of positive cases, and contact tracing. [11] the gold standard for testing respiratory viruses, including sars-cov-2, is collection from a patient's nasopharynx (np) using a standard flocked np swab (flnp). [12] a c c e p t e d m a n u s c r i p t covid-19 diagnostic testing has expanded exponentially in an international effort fighting the pandemic. however, disruption in the medical supply chain [13] caused by this crisis resulted in test kit shortages, including flnp swabs. [14] furthermore, initial phases of the sars-cov-2 pandemic coincided with influenza season in many locations, compounding supply chain issues. np swab global utilization relies on a limited number of manufacturers, who require significant lead time to ramp up production, and created major barriers to testing and containment of the disease. to address supply shortfalls, 3d printing is an effective stopgap technology for medical devices and supplies. [15] to combat critical np swab shortages, we designed and clinically evaluated a 3d-printed swab (3dp) alternative to the flnp swab used for nasopharyngeal sample collection. the 3d-printers selected for 3dp swab manufacturing were the form2 and form 3b (formlabs, usa) due to pre-existing validation and fda-cleared workflows using an autoclavable surgical grade resin (surgical guide, formlabs) manufactured in an iso 13485 certified facility. a detailed methodology used to develop the 3dp swab is available in an article in the journal, 3d printing in medicine by ford et al. [16] these printers are relatively affordable and ubiquitous in hospitals that maintain their own 3d printing labs. also critical to the viability of this alternative swab, the surgical guide resin is manufactured in the us by formlabs and the supply was deemed stable to ensure access to raw materials. because clinical sites had difficulties obtaining commercially supplied collection kits, 3dp swabs were usually provided with collection media in collection kits assembled inhouse. amongst the media used were who approved viral transport media, [17] in-hospital manufactured vtm following the centers for disease control and prevention (cdc) a c c e p t e d m a n u s c r i p t recommended procedure for preparation of vtm (cdc sop# dsr-052-03) [18] or, when available, either vtm or universal transport media provided by commercial manufacturers (bd). in-hospital vtms were manufactured and validated on-site and were accepted by our clia laboratories. individual 3dp swabs were placed in peel-packs along with sterilization indicators and autoclaved. if needed, vtm collection media was packaged in sterile aliquots of 1.5 or 3 ml and provided as collection kits. sites were also given detailed protocols so that they could produce collection kits on-site. [16] to validate sample collection, the 3dp swab was first compared against the flnp equivalent using respiratory syncytial virus (rsv) as a surrogate rns virus. 1 ml of synthetic sputum [19] was spiked with 10 6 plaque-forming units (pfu) of rsv. flnp and 3dps were dipped in sputum and rotated for 5 seconds. tips were cut off into 1 ml of bd universal transport media (utm). each tip was incubated in utm for 15 minutes at 22° c. 140 µl utm from each tube was subjected to rna isolation with qiagen viral rna mini kit, elution in 50 µl. 10 µl of rna was used to make cdna (iscript, biorad) in a 20 ml final volume. a qrt-pcr was performed with 2 sets of rsv primers for the n gene (rsv n forward: aagggatttttgcaggattgttt, rsv n reverse: ctccccaccgtagcattacttg) and the sh-g intergenic region (rsv sh-g forward: ttaacatcccaccatgcaaa, rsv sh-g reverse: gcatttgccccaatgttatt). 2 µl of cdna per well, in triplicate wells, was used in a 20 µl qrt-pcr reaction using sensifast sybrgreen kit (bioline). cycle thresholds (c(t)) were recorded for rsv rna detection. leeching tests were performed to ensure 3dps stability after sterilization and to ensure swab materials did not interfere with downstream testing. bd utm was spiked with a c c e p t e d m a n u s c r i p t rsv (10 6 pfu/ml) and aliquoted (1 ml) into 1.8 ml cryotubes. triplicate tubes either contained a sterile 3dps tip or was left swab-less and incubated at 4° c. samples (140 µl) harvested at 24, 48 and 72 hours and subjected to rna isolation and qrt-pcr as above. the clinical trial was approved by western irb for all sites under an umbrella protocol (no. 20200779). participants (n=291) presenting for covid-19 testing were verbally consented by health care providers. three different medical centers participated in this initial study. dual paired swabs (flnp and 3dps) were collected from each participant following cdc guidelines on covid-19 np collections. flnp and 3dp swab order was not systematically tested. one swab, be it flnp or 3dp, was used per nostril. the left or right nostril order was randomly selected. all sites printed and processed their own 3dp swabs. for bench lab testing, measures of central tendency were calculated for swab rsv detection as well as leeching. for clinical testing statistics, wilcoxon signed rank tests were conducted for c(t)s for both covid-19 gene targets for positive cases for every testing platform. for cdc assays, rnase p (rp) were also compared via wilcoxon signed rank tests. a p-value ≤ 0.01 was considered statistically significant. percent agreement and kappa coefficient was also calculated to measure flnp and 3dp agreement. conventional flnp are used for sampling the np, requiring enough flexibility to access the np while collecting sufficient material for diagnostic testing. materials such as wood or cotton inhibit pcr reactions. the material used must be safe for humans, yet not inhibit down-stream diagnostic testing. the 3dp swab underwent several iterations before establishing a final design. flocked swab geometry was referenced to influence ultimate 3dp design. other influences included patient comfort, surface area maximization and the selected 3d-printer's capabilities. initial cad 3dp swab drawings were created in 3-matic (materialise) with a "cattail" tip design, a rounded nose for patient comfort and lateral alternating nubs to maximize surface area. the total swab length is 150 mm, with a tip length of 15 mm and a 3.85 mm diameter. the neck is 20 mm in length with 1.5 mm in diameter and the shaft is 2.45 mm in diameter with a break-point at 70 mm, dimensions which meet most test tube requirements. the swab is printed without supports with 324-380 per batch (figure 1 ). 3dp swab were tested for safety and comfort using volunteers. an independent study compared complications using the standard synthetic flocked swab and the 3dp swab and found they performed equally. [23] a c c e p t e d m a n u s c r i p t to verify the swab material and collection kit vtm did not inhibit pcr reactions, initial lab testing was performed using a validated qrt-pcr assay rsv. it was not possible to directly test the stability of sars cov-2 due to lack of access to either a bsl3 facility or live virus. however, rsv is an enveloped rna virus with a helical nucleocapsid and is biochemically very similar to sars cov-2. we used the standard who vtm recipe for human samples containing viruses, chlamydia and mycoplasma. pilot studies examining the qrt-pcr assay sensitivity on rsv in vtm showed that 10 6 pfu/ml gave the best dynamic range to detect changes in viral rna in the sample. therefore, the 3d printed swab was tested with this concentration. for swab performance, c(t) values for rsv genome (sh-g) respectively. we tested both negative and positive samples at 3 different trial sites in 291 adults (age range 14-94) presenting to our emergency rooms or inpatients requiring sars-cov-2 testing ( 26.74 ± 6.41 c(t), 27.63 ± 6.29) viral gene targets (p=0.401, p=0.484 respectively). these results are graphically displayed in figure 2a , b and c with numeric agreement in tables 2, 3 overall clinical correlation of covid-19 diagnosis between the two swabs was 95.88% (279/291) with kappa = 0.901, a high level of agreement. [24] a detailed breakdown of the combined flnp and 3dp swab agreement is in table 5 . half of cases where swab results disagreed were inconclusive (i.e. a result could not be reported by the lab) and in a clinical setting would require patient resampling (6 times). there were two cases where the flnp sample was positive but the 3d swab was negative. there were four cases where the flnp sample was negative but the 3d swab was positive. in general, discordant cases were ones where c(t) values were high and less viral material was detectable. throughout the trial's course, scant nasal bleeding after completion of both swabs was reported but no clinical intervention was needed in any of those cases and no other adverse reactions or breakage of either flnp or 3dp swabs were reported. the sars-cov-2 pandemic created a burden on healthcare systems worldwide and interrupted supply chains used to fight against covid-19. diagnostic testing is critical to the response to the novel coronavirus. current business models operate on "just-in-time" supply chain models, relying upon readily available supplies and delivery. thus, stockpiling reagents a c c e p t e d m a n u s c r i p t and supplies is atypical. unfortunately, this efficient business model for production of flnp swabs was disrupted by the sars-cov-2 health crisis, and manufacturers were unable to respond to the sudden world-wide surge in demand for flnp. 3d-printed technology provides an alternate strategy for swab shortages by facilitating a local solution to flnp shortages. the 3dps displayed statistically identical results to standard flnp in a head-to-head clinical trial, making it a viable option in testing for sars-cov-2 infections. the 3dp, which has a more rigid brush-like head compared to the standard flnp, likely collected additional epithelial cells which may increase sensitivity in sample collections as seen by the 3dps rpp gene c(t) values, which were significantly better than standard flnp swabs. the fact that the flnp and 3dps comparison results were the same using three different assay methods (cdc assay, neumodx and roche cobas) illustrates that the 3dp can be used reliably across platforms. at the time of this publication, northwell health moved to the panther system (hologic) for their clinical testing. the 3dp swab was innovated to offset standard flnp shortages not only at our local facilities but with the intention to share the design with any site throughout the world where 3d printer capabilities may be established. as 3d-printing allows for design alterations to be rapidly put into production, several iterations in swab design have been created and readily available by partner institutions. for example, the current swab being produced by northwell contains a brush diameter that is 10% smaller compared to the usf swab. furthermore, several other groups have utilized 3dp swabs in place of flnp swabs. one group examined 160 designs from 48 different materials from 48 manufacturers, focusing on four designs in a clinical trial. [25] one of their main findings was to balance tip design to maximize surface are while balancing it for patient comfort. 3d printing technologies used in that clinical trial are not as readily available as the form 2/form 3b printer utilized in this study. a c c e p t e d m a n u s c r i p t additionally, material costs of an individual 3dp swab ranges from $0.25-$0.46 depending on whether an institution has an in-house printing team and sterile processing unit with the necessary equipment and materials. comparatively, commercial flnp swabs cost approximately $1.00 per unit. regarding the effort required to print and process 3dp swabs, sites were able to shift staff with available extra capacity due to decreased workloads caused by a decrease in surgical caseloads. this further illustrates the role the 3dp swab can play in filling the supply gap that is cost efficient and rapidly deployable. the initial cost of a formlabs system is approximately $3,600 (form 2) to $6,000 (form 3b). this includes washing and curing stations for post processing. each surgical guide resin cartridge is $249. print times range from 11-15 hours per batch. our clinical trial showed that 3dp swabs were an efficient and reliable alternative to standard flnp swabs. the data supports the feasibility of medical teams across the us to deploy 3dp swabs at local sites allowing for rapid local production and distribution that can utilize the same production designs and protocols, facilitating supply maintenance and data comparison. flnp swabs are also routinely used to collect nasopharyngeal samples testing for other respiratory viruses including influenza and rsv. our preliminary results suggest that 3dp may be used for diagnosis of other respiratory viruses, but our clinical study was conducted when the incidence of these infections was low in our respective geographic regions, so future studies will need to be conducted to evaluate whether 3dp swabs can be used for all indications that flnp swabs are used. if so, it is possible that 3dp swabs may be deployed in under resourced or remote settings to enable local manufacturing of swabs. some study limitations include sample size and lack of pediatric testing. as this study was developed in response to a critical shortage of testing swabs, our hospitals were unable to provide large flnp swab samples to be used for clinical trial. once we were able to a c c e p t e d m a n u s c r i p t demonstrate the validity of the clinical results, the data was presented to the hospitals' advisory committees and were accepted as standard of care swabs due to the crisis. additionally, this trial focused on adults. while a pediatric version of the swab was created, the current clinical trial only included one subadult. as a result of this study, all three sites and others participating in our clinical trial have moved forward using 3dp swabs as the alternate to the standard of care swab in patient testing when commercial swabs and kits are not available. as demand for swabs increases, validated designs, such as the one described here, can be designed on multiple printing platforms, assisting healthcare facilities worldwide with diagnosis testing for sars-cov-2. in addition, combining in-house utm production [17, 18] and 3dp swabs may provide an effective, cost-efficient, and fast alternative to standard flnp and viral transport media kits used for covid-19 testing and with the potential to alleviate a major supply chain hurdles, while increasing testing capabilities. a c c e p t e d m a n u s c r i p t notes statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(2019-ncov) an interactive web-based dashboard to track covid-19 in real time report of the who-china joint mission on coronavirus disease aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 covid-19-navigating the uncharted virological assessment of hospitalized patients with covid-2019 asymptomatic transmission, the achilles' heel of current strategies to control covid-19 research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases united states food and drug administration. coronavirus (covid-19) update: fda issues emergency use authorization for potential covid-19 treatment | fda feasibility of controlling covid-19 outbreaks by isolation of cases and contacts. the lancet global health united states centers for disease control and prevention. interim guidance: healthcare professionals 2019-ncov | cdc critical supply shortages-the need for ventilators and personal protective equipment during the covid-19 pandemic overview of testing for sars-cov-2 | cdc covid-19 and the role of 3d printing in medicine a 3d-printed nasopharyngeal swab for covid-19 diagnostic testing. 3d printing in united states centers for disease control and prevention. standard operating procedure format and template -viral-transport-medium essential genome of pseudomonas aeruginosa in cystic fibrosis sputum molecular systems. neumodx sars-cov-2 assay -neumodx adverse effects of nasopharyngeal swabs: three-dimensional printed versus commercial swabs the measurement of observer agreement for categorical data open development and clinical validation of multiple 3d-printed nasopharyngeal collection swabs: rapid resolution of a critical covid-19 testing bottleneck the authors would first like to thank all of the sites who participated in this trial. the authors would also like to thank the following individuals and institutions for their roles in this project. usf health/ tampa no funding was received to perform this study. drs. decker, ford, goldstein, hazelton, kim, sinnott, and teng hold the provisional patent for 3d-printed swab for diagnostic testing.dr. kim serves on the editorial board of the sanford guide to antimicrobial therapy and is site director for covid-19 clinical trials funded by regeneron, romark and abbott.dr. wilson reports grants and personal fees from gilead pharmaceuticals, pfizer, janssen, and portola outside of the submitted work.all other authors have no potential conflicts to disclose. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-007068-vcfs41eb authors: moradi, tony; bennett, nicholas; shemanski, shelby; kennedy, kevin; schlachter, andrew; boyd, sarah title: use of procalcitonin and a respiratory polymerase chain reaction panel to reduce antibiotic use via an electronic medical record alert date: 2019-10-22 journal: clin infect dis doi: 10.1093/cid/ciz1042 sha: doc_id: 7068 cord_uid: vcfs41eb background: respiratory tract infections are often viral and but are frequently treated with antibiotics, providing a significant opportunity for antibiotic de-escalation in patients. we sought to determine whether an automated electronic medical record best practice alert (bpa) based on procalcitonin and respiratory polymerase chain reaction (pcr) results could help reduce inappropriate antibiotic use in patients with likely viral respiratory illness. methods: this multisite, pre–post, quasi-experimental study included patients 18 years and older with a procalcitonin level <0.25 ng/ml and a virus identified on respiratory pcr within 48 hours of each other, and 1 or more systemic antibiotics ordered. in the study group, a bpa alerted providers of the diagnostic results suggesting viral infection and prompted them to reassess the need for antibiotics. the primary outcome measured was total antibiotic-days of therapy. results: the bpa reduced inpatient antibiotic-days of therapy by a mean of 2.2 days compared with patients who met criteria but did not have the alert fire (8.0 vs 5.8 days, respectively, p < .001). the bpa also reduced the percentage of patients prescribed antibiotics on discharge (20% vs 47.8%, p < .001), whereas there was no difference in need for antibiotic escalation after initial discontinuation (7.6% vs 4.3%, p = .198). conclusions: the automated antimicrobial stewardship bpa effectively reduced antibiotic use and discharge prescribing rates when diagnostics suggested viral respiratory tract infection, without a higher rate for reinitiation of antibiotics after discontinuation. lower respiratory tract infections (lrtis) are a leading cause of hospitalizations in the united states [1, 2] . acute differentiation of viral and bacterial causes of these infections presents a challenge, though viral pneumonia may be more common than bacterial pneumonia [3] . difficulty with differentiation of viral vs bacterial presentations often leads to use of empiric antibiotics, thereby risking unnecessary antibiotic exposure. often no pathogen is isolated and the cause of the infection remains unknown [3] . these factors can lead to prolonged continuation of antibiotics to cover potential bacterial pathogens that may not be the source. reducing inappropriate antibiotic use in this setting could decrease drug-resistant organisms, adverse drug events, and healthcare costs. rapid diagnostic tests and biomarkers are available to assist in diagnosis of lrtis. procalcitonin (pct) is a biomarker that can assist in differentiating bacterial vs nonbacterial causes of lrtis, with elevated levels in acute bacterial infections. despite its association with reduction in days of antibiotic therapy, pct use is not ubiquitous in the united states [4] . however, it is a safe and effective predictor of bacterial infections, particularly respiratory tract infections [4] [5] [6] [7] [8] [9] [10] . despite pct sensitivity ranging up to 88%, its use must be clinically correlated with other findings, such as physical examination, history, laboratory tests, and diagnostic imaging. use of multiplex respiratory polymerase chain reaction (pcr) assays has also been increasing. these tests allow laboratories to quickly detect a wide array of respiratory viruses and select bacteria [11] . while individual test use, particularly pct, has been well studied, impact on antibiotic therapy varies significantly [12] [13] [14] [15] [16] [17] [18] . in a study with stewardship intervention for patients on broad-spectrum antibiotics with a respiratory pcr positive for viruses, time to antibiotic de-escalation was not significantly affected [19] . only a small number of studies have examined pct and pcr in tandem. branche and colleagues examined pct use vs usual care in a randomized trial and found no difference in rates of antibiotic use at 48 hours or less, but subgroup analysis noted a trend toward improvement when pct and pcr were suggestive of viral illness. there was also a reduction in patients prescribed antibiotics on discharge (20% vs 45%, p = .002) [20] . the results showed promise regarding influencing prescribing and suggested need for further study. a recent report found that pct plus respiratory pcr results can influence antibiotic duration in viral lrtis, especially with active antimicrobial stewardship input [21] . conclusively, the available literature suggests that leveraging pct and respiratory pcr test results, when suggestive of viral illness, appears to be a viable option to minimize antibiotic exposure. the aim of our study was to determine if antibiotic use could be reduced by deploying an automated antimicrobial stewardship provider alert that prompted antibiotic de-escalation if 3 criteria were met: pct <0.25 ng/ml, virus detected on respiratory pcr, and active use of systemic antibiotics. while the electronic medical record (emr) has been used in various manners for antibiotic stewardship [22, 23] , we are unaware of its use to automate stewardship recommendations for viral respiratory infections. this was a quasi-experimental multisite study at 5 hospitals (4 community, 1 academic) within saint luke's health system, kansas city, missouri. the study received investigational review board waiver approval. patients were included if they had both a positive virus on pcr and a pct value <0.25 ng/ml within 48 hours of each other, and at least 1 active systemic antibiotic. an automated, emr best practice alert (bpa) for these patients was implemented in december 2017 ( figure 1 ) in epic (verona, wisconsin; www.epic.com). the alert fired upon any provider opening an emr when criteria were met. it contained the message "antimicrobial stewardship alert: your patient has a positive viral pcr + negative procalcitonin + one or more antibiotics ordered. these results suggest viral infection-please reassess necessity of antibiotics as indicated. " it contained the pcr and pct results and listed active antibiotics. three options to proceed were available: "acknowledge"; "does not meet criteria"; and "not making antimicrobial decisions. " the first 2 suppressed the alert permanently, and the last allowed it to continue firing each time the emr was opened, until 1 of the other 2 options were selected. electronic time stamps allowed assessment of provider responses and alert firing time(s). the prospective bpa group included all patients on which the alert fired from 15 december 2017 to 28 february 2018. the retrospective comparator group included patients who met the alert firing criteria from 1 december 2015 to 30 march 2016. patients were excluded if they were <18 years old or if antibiotics were also being used for concomitant, nonrespiratory indications. these were identified based on indications included with antibiotic orders (which are required for all antimicrobial orders at our institution), as well as manual records review. the primary endpoint was inpatient antibiotic-days of therapy, defined as each individual antibiotic given on any day. this was calculated by adding together the total number of days the patient received each individual antibiotic. secondary endpoints were discontinuation of antibiotics within 24 hours of initiation, days of antibiotics after alert firing, reinitiation of antibiotics after discontinuation, clostridioides difficile infection, discharge prescription rate, and days of antibiotics prescribed on discharge. the alert firing endpoint for the retrospective group was defined as the time point when all the information (pct, pcr, and antibiotic ordered) was available to providers. reinitiation of antibiotics after discontinuation was defined as any new antibiotic order for a respiratory indication after all antibiotics had been stopped for any significant period (eg, 1 day or more). serum pct levels were measured by vidas brahms (biomérieux, durham, north carolina). the clinical detection range is 0.05-200 ng/ml. our internal guidance for pct in lrtis strongly discourages antibiotic use if the pct value is <0.1 ng/ml and discourages use if it is ≤0.25 ng/ml, consistent with us food and drug administration labeling for pct testing in lrtis [24] . respiratory pcr samples were tested using the filmarray respiratory panel (biofire diagnostics, salt lake city, utah), which detects 17 common respiratory viruses and 3 atypical bacteria. continuous variables are shown as mean ± standard deviation and were analyzed using student t test, and categorical or nominal variables are shown as number (%) and were compared using χ 2 or fisher exact test, as appropriate. a multivariable linear regression model was developed to assess the independent association between our prospective (bpa) group and days of therapy. we adjusted for the following variables based on clinical judgement: age; ventilator-days; charlson comorbidity index; respiratory viral illness; community-acquired pneumonia; healthcare-associated pneumonia, hospital-acquired pneumonia, or ventilator-associated pneumonia; chronic obstructive pulmonary disease; upper respiratory infection; rhinovirus; adenovirus; human metapneumovirus; influenza a; influenza b; respiratory syncytial virus (rsv); and intensive care unit (icu) length of stay. two-tailed statistical tests were utilized, with a significance level set at p < .05. statistical analysis was completed using sas 9.4 software (sas institute, cary, north carolina). two hundred twenty-six patients were included in the prospective (bpa) group and 161 in the retrospective group. there were no significant differences in age, sex, or race among the groups ( table 1 ). the bpa group had a significantly higher mean charlson comorbidity index score (4.8 vs 4.0, p < .001) and a lower mean icu length of stay (5.0 vs 6.9 days, p = .043). viral detection rates on pcr varied between the groups (table 1) . influenza a and b were more common in the bpa group (27.4% vs 11.8%, p < .001 and 8.8% vs 1.9%, p = .004, respectively). rsv was also more common in the bpa group (26.1% vs 15.5%, p = .012), whereas fewer bpa group patients had rhinovirus (5.3% vs 22.4%, p < .001). the primary endpoint of antibiotic-days of therapy was significantly reduced in the bpa group by a mean of 2.2 days (5.8 vs 8.0 days, p < .001). several secondary endpoints were also improved in the bpa group including mean days of therapy after bpa firing (4.5 vs 6.3, p < .001), more patients having antibiotics discontinued within 24 hours of initiation (37.8% vs 18.6%, p < .001), and fewer patients discharged on antibiotics (20.0% vs 47.8%, p < .001). there was no difference in rates of antibiotic reinitiation after discontinuation (7.6% vs 4.3%, p = .198) or c. difficile infection (0.4% vs 1.9%, p = .174). results can be found in table 2 . after adjusting for possible confounding variables, we showed that bpa is associated with 1.48 fewer days of therapy (p = .0002). antibiotic resistance continues to be a major threat to our healthcare community. despite the advent of more expansive rapid diagnostic tests and biomarkers, our efforts are still insufficient to outpace resistance development. this has been supported by national and global efforts to raise awareness to the significance of this issue. to our knowledge, this is the first study to implement an automated clinician antimicrobial stewardship intervention by leveraging emr-driven data for likely viral lrtis, defined by negative pct and positive viral respiratory pcr results. our intervention was highly effective in reducing antibiotic prescribing, with the bpa decreasing antibiotic-days of therapy by 2.2 days. even after adjusting for select variables of interest, the bpa was still associated with a significant reduction in days of therapy. there is some provider concern that early (inappropriate) cessation of antibiotics may pose a risk of failure, but there was no increased need for reinitiation of antibiotics in the bpa group after they were initially stopped. the alert led to a 19.2% higher rate of antibiotic discontinuation within 24 hours of the alert firing. the primary aim of the intervention is to quickly identify patients who no longer need antibiotics for lrtis. however, some providers still feel compelled to continue therapy. without active oversight of the patient discharge process, antibiotics can often be prescribed without regard for appropriateness or duration. the bpa group had a 27.8% reduction in rate of discharge prescriptions and notably decreased postdischarge antibiotics duration by 1.5 days, showing an impact beyond just initial de-escalation of therapy. of note, this decrease in postdischarge duration of therapy is not accounted for in the primary outcome of inpatient-days of therapy, possibly extending the benefit of the alert beyond the inpatient stay. previous studies have shown inpatient viral respiratory infection management to have vast potential for targeted intervention, as providers often continue antibiotic therapy even when there is a low likelihood of bacterial involvement. a study by timbrook and colleagues found low rates of antibiotic discontinuation in patients with positive viral respiratory pcr, negative pct, or both, which were suggestive of viral etiology. because this study did not include a direct intervention, the authors concluded that clinician intervention was likely needed to affect antibiotic prescribing in this subset of patients [17] . branche and colleagues implemented a 1:1 randomized feasibility study in similar patients using respiratory pcr and pct testing. in contrast to the previous study, they employed an intervention to inform providers of likely viral infections, though no difference was found in antibiotic use. however, they did detect a reduction in antibiotic discharge prescriptions by 25%, which is in line with our findings of nearly 28% [20] . they note that they may have encountered a spillover effect, in which their intervention with the study group indirectly influenced the practices of providers in the control group. our study was able to avoid this as it was carried out in 2 distinct time periods with no overlap. importantly, the branche study emphasized the need for provider intervention to leverage diagnostic testing output [20] . a study by file and colleagues evaluated respiratory pcr results coupled with pcr and/or active antimicrobial stewardship intervention. stewardship input, as compared to availability of pcr plus pct alone, yielded the most significant reduction in antibiotic use, though this required contact with providers [21] . our findings suggest that similar efforts can be achieved without direct stewardship input, which allows shifting of efforts to other high-risk patients. benefits of respiratory pcr testing include its rapid turnaround time and inclusion of the most common respiratory viral pathogens. however, concerns for bacterial coinfection limit provider willingness to quickly de-escalate antibiotics based solely on pcr results. this concern is not unfounded, as coinfection rates may be as high as 40% [16] . by coupling temporally related pct values (within 48 hours) to viral pcr results, we were able to suggest to providers a subset of patients who were unlikely to have bacterial coinfection. the targeted stewardship alert enhanced the use of rapid diagnostic tests in determining infectious source. the ability of the bpa to affect provider decision making on antibiotic prescribing played a large part in our study as there was no directed follow-up to bpa results or responses. providers were willing to stop antibiotics in many cases, with fewer antibiotic-days of therapy and a 37.8% rate of antibiotic discontinuation within 24 hours of the alert firing. while providers may not be willing to immediately discontinue antibiotics in some cases, they may still do so earlier than if they had not been prompted with the initial emr alert. a question of what factors caused providers to continue antibiotics is raised. in their follow-up analysis of their randomized trial, branche and colleagues found that while several factors were mentioned by providers as reasons for deviation from their pct de-escalation protocol including illness severity, fever, abnormal complete blood count, and others, only diagnosis of pneumonia was significantly associated with nonadherence [25] . an important distinction in this case is that viruses are able to cause radiographic changes [26] [27] [28] . the emr has untapped potential to enhance antimicrobial stewardship functions by extracting meaningful data points. leveraging effective alerts allows stewardship principles to be active all times of the day, meaning patients admitted or evaluated during off-hours or at sites with less antimicrobial stewardship presence still receive the same interventions. while we do not suggest that alerts should replace staffing as many factors contribute to appropriate evaluation of therapy optimization, alerts have been shown to increase appropriate antimicrobial selection [22] . our study highlights the value of minimally invasive stewardship by allowing the emr to assist in identifying patient subsets and affecting antibiotic use. our study did have limitations. first, our evaluation of data from a single health system may not be representative of prescribing of other institutions. second, the retrospective design did not allow for the most minimally biased comparison between the groups. while our study did attempt to minimize confounding variables, there are always potential unidentified effects. one such effect may have been differences in influenza seasons. the 2017-2018 influenza season was more severe than the 2015-2016 season, as evidenced by centers for disease control and prevention influenza data [29] . our regression analysis still supported a significant reduction in days of therapy with the bpa. we began collecting the prospective data immediately after the alert was launched in december 2017, not allowing for an adaptation period to lapse, which may have skewed the true effect of the bpa. another limitation was nonconsistent timing of the bpa regarding days of therapy. it is possible that earlier firing of bpa led toward earlier discontinuation of antibiotics. however, a temporal relationship cannot be established based on our data alone. a future study might examine how the timing relates to the outcome to further determine bpa effect on antibiotic prescribing. other limitations to the study included lack of stratification by provider and lack of follow-up on influence of bacterial culture result with therapy duration. it is possible certain providers were inherently more open or resistant to the alert intent, which may lend itself to more targeted feedback in the future when providers decline a suggestion. other factors beyond pcr and pct results may have influenced treatment decisions. for example, imaging changes can affect prescriber habits, though imaging alone cannot differentiate bacterial vs viral illness. another consideration is that our alert does not fire when only respiratory pcr or pct is suggestive of viral infection, nor does the alert include standalone pcr tests such as influenza or influenza/rsv combination tests. finally, an issue with the bpa is that it fires for all providers. if one inadvertently selects "acknowledge" or "does not meet criteria, " the alert stops firing. we did not track unintentional alert suppression but realize that it could have affected success rate of the alert. there are several future directions for research using similar approach. first, we did not include any nonneonatal pediatric inpatients as we do not currently provide care for this population in our health system. second, we did not characterize the cost benefit to implementing a targeted bpa intervention on care received. finally, there may be a relationship between timing of the bpa and antibiotic exposure, an area that may prove to be related but was not fully answered in this study. in conclusion, our study showed a significant reduction in antibiotic exposure for patients with likely viral respiratory illness. it also proves that well-constructed emr provider alerts that integrate pcr, pct, and antibiotic data can target patients in whom antibiotic therapy can be rapidly narrowed, without need for direct antimicrobial stewardship oversight. this minimally invasive stewardship practice can easily be replicated by other institutions and represents a step forward in the fight against antibiotic misuse. potential conflicts of interest. s. b. was given travel reimbursement and an honorarium from kurin for 1 day of travel to the centers for disease control and prevention for vendor day to express her personal views on the importance of blood culture contamination on antimicrobial stewardship and clinical impacts. all other authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults healthcare cost and utilization project statistical brief 162 cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. adults procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections serum procalcitonin and c-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis diagnostic and prognostic accuracy of clinical and laboratory parameters in community-acquired pneumonia the diagnostic accuracy of procalcitonin for bacteraemia: a systematic review and meta-analysis systematic review and meta-analysis of procalcitonin-guidance versus usual care for antimicrobial management in critically ill patients: focus on subgroups based on antibiotic initiation, cessation, or mixed strategies procalcitoninguided use of antibiotics for lower 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parainfluenza virus in adults influenza surveillance report key: cord-007073-soov8q3q authors: wang, claire y t; ware, robert s; lambert, stephen b; mhango, lebogang p; tozer, sarah; day, rebecca; grimwood, keith; bialasiewicz, seweryn title: parechovirus a infections in healthy australian children during the first 2 years of life: a community-based longitudinal birth cohort study date: 2019-08-13 journal: clin infect dis doi: 10.1093/cid/ciz761 sha: doc_id: 7073 cord_uid: soov8q3q background: hospital-based studies identify parechovirus (pev), primarily pev-a3, as an important cause of severe infections in young children. however, few community-based studies have been published and the true pev infection burden is unknown. we investigated pev epidemiology in healthy children participating in a community-based, longitudinal birth cohort study. methods: australian children (n = 158) enrolled in the observational research in childhood infectious diseases (orchid) study were followed from birth until their second birthday. weekly stool and nasal swabs and daily symptom diaries were collected. swabs were tested for pev by reverse-transcription polymerase chain reaction and genotypes determined by subgenomic sequencing. incidence rate, infection characteristics, clinical associations, and virus codetections were investigated. results: pev was detected in 1423 of 11 124 (12.8%) and 17 of 8100 (0.2%) stool and nasal swabs, respectively. major genotypes among the 306 infection episodes identified were pev-a1 (47.9%), pev-a6 (20.1%), and pev-a3 (18.3%). the incidence rate was 144 episodes (95% confidence interval, 128–160) per 100 child-years. first infections appeared at a median age of 8 (interquartile range, 6.0–11.7) months. annual seasonal peaks changing from pev-a1 to pev-a3 were observed. infection was positively associated with age ≥6 months, summer season, nonexclusive breastfeeding at age <3 months, and formal childcare attendance before age 12 months. sole pev infections were either asymptomatic (38.4%) or mild (32.7%), while codetection with other viruses in stool swabs was common (64.4%). conclusions: in contrast with hospital-based studies, this study showed that diverse and dynamically changing pev genotypes circulate in the community causing mild or subclinical infections in children. parechovirus can cause severe illnesses in children. however, studies focus mainly on hospitalized populations. true disease burden in the community remains largely unknown. from our community-based cohort, we found diverse parechovirus genotypes in the community, causing mild or subclinical infections in children. . these pev-a3 outbreaks occur biennially, arising in odd and even years in the southern and northern hemispheres, respectively. furthermore, a recent australian study reported that almost 20% of infants hospitalized with a pev-a3 infection had impaired neurodevelopment 12 months later [9] . much of what is known of pev epidemiology, including pev-a3 outbreaks, relies upon hospital-based studies, and uncertainty exists over the actual disease burden within the community [3, 6] . seroprevalence studies from europe and japan suggest that infection is common and occurs early in life with pev-a1 antibodies present in 25%-30% of infants by age 1 year, increasing to 70%-90% by age 2-5 years, with high antibody levels maintained in older children and throughout adulthood [10, 11] . recently, pev-a3 seroepidemiology in australia, the netherlands, and the united states was shown to be similar [12] . the overall prevalence of neutralizing antibodies to pev-a3 increased from nearly 33% in children aged 1-2 years, to 65% in those aged 5-9 years, peaking at 78% in adults aged 20-29 years, and then declining to 42% in older age groups [12] . the lower pev-a3 seroprevalence in older adults may indicate waning childhood immunity or the relatively recent emergence and circulation of pev-a3 globally. beyond seroprevalence, few studies describe the epidemiology and clinical characteristics of pev infections in the community [13] [14] [15] [16] [17] [18] . these have included secondary analyses of monthly stool samples collected from 3 months until 1-3 years of age in participants of 3 case-control studies from norway and finland. these studies were designed originally to determine the incidence of type 1 diabetes in genetically at-risk children [13] [14] [15] . by age 12 months, 43% of 108 norwegian children recruited between 2001 and 2006 had at least 1 pev detection, which increased to 86% by 2 years of age [13] . a similar pattern of pev infection, but with lower incidence rates, was observed in 200 finnish children enrolled between 1996 and 2007 where by age 12 months 22% had pev detected in their stools on at least 1 occasion, which increased to 48% approaching their second birthday [15] . in these longitudinal cohort studies, pev-a1 was the most commonly observed genotype (76%-93%) with pev-a3 and pev-a6 detected only occasionally [13] [14] [15] . limited symptom and epidemiological risk data were reported by the 2 norwegian studies, both of which found no association between pev detection and respiratory or gastrointestinal symptoms [13, 14] . other studies assessed upper airway samples for various viruses, including pev, in healthy older children and adults, but either lacked sufficient numbers or clinical or sociodemographic data to determine risk factors for pev infections or only sampled infrequently [16] [17] [18] . longitudinal, community-based studies employing sensitive molecular diagnostic assays with regular and frequent sampling, irrespective of illness, are best suited to explore the true nature and disease burden of pev infections in young children. we therefore aimed to describe the epidemiology of pev in the first 2 years of life by the means of an unselected communitybased birth cohort whose recruitment coincided with the first reported outbreak of severe pev-a3 disease in australia [5, 19] , and to investigate the risk factors and symptoms associated with acquiring pev in these young children. the observational research in childhood infectious diseases (orchid) project (clinicaltrials.gov identifier nct01304914) is a community-based, longitudinal, birth cohort study of acute respiratory infections (aris) and acute gastroenteritis (age) in unselected, healthy australian children in their first 2 years of life [20] [21] [22] [23] . recruitment was progressive over 2 years; participants needed to be healthy, born at term (36-42 weeks), and without congenital or underlying chronic disorders. the children's health queensland, royal brisbane and women's hospital, and the university of queensland human research ethics committees approved the study. at enrollment, baseline sociodemographic and health data were collected by parental interview. parents maintained daily symptom diaries related to aris and age and a separate illness impact diary healthcare visits for these episodes. diaries were returned monthly by mail. telephone interviews were conducted to collect data on feeding and childcare attendance every 3 months. parents collected from their child weekly anterior nasal swabs and diaper stool swabs from birth until age 2 years. swabs were mailed to the laboratory where they were processed and stored at -80°c (supplementary methods). table 1 lists definitions for pev episodes and ari and age symptoms [22, 23] . nucleic acid was extracted from the swabs using previously described protocols implemented with quality control [22, 23] (supplementary methods). pev in extracts were detected using a previously published reverse-transcription polymerase chain reaction (rt-pcr) assay (supplementary table 1 ) [24] . stool swabs were screened for 8 additional enteric viruses (supplementary table 2 ) and nasal swabs for 17 respiratory viruses [22] using published assays [20] . a selection of specimens positive for pev within the same episode ( figure 1 and supplementary methods) were genotyped using published methods targeting the vp3/1 and vp1 regions (supplementary methods) [25] [26] [27] . one representative sequence per pev episode was selected for the phylogenetic analyses. incidence rates with 95% confidence intervals (cis) were assessed using poisson regression, including the natural logarithm [22] . ari symptoms presence of nasal congestion/discharge, dry or wet-sounding cough, wheezing, shortness of breath, or doctor-diagnosed otitis media or pneumonia [22] age symptoms ≥3 loose stools recorded during a 24-h period [23] symptomatic episode ari, age, or fever only symptoms present within 7 d before or after the first positive pev detection [22, 23] . abbreviations: age, acute gastroenteritis; ari, acute respiratory infection; pev, parechovirus a. of the number of swabs returned as an offset. each swab returned was defined as representing 1 week of study time. the time to first detection was calculated using life tables. infants were censored at either the date of the last swab submitted if the next swab was not returned for >42 days, or at 730 days, whichever came first. the association between potential risk factors and pev detection was examined using mixed-effects logistic regression with the child entered as a random effect to account for repeated measurements. risk factors considered were age (categorized as 0-<3 months, 3-<6 months, 6-<12 months, 12-24 months), sex, exclusive breastfeeding, older siblings in the household, childcare attendance (none/informal/formal), season of pev detection, and season of birth. given the strong association between breastfeeding and childcare with age, both breastfeeding-by-age and childcare-by-age interaction terms were included in the models. all variables (except sex) were analyzed as time-varying variables. univariable and multivariable analyses were conducted. in multivariable analyses, all variables were included in the regression models. a linear regression model was used to investigate the association between symptoms and pev cycle threshold (ct) values, with the latter being inversely proportional to the amplified target nucleic acid in the sample, representing a semiquantitative estimate of viral load. ct values from the first pev detection in each episode were analyzed. data were analyzed using stata version 15.1 software (statacorp, college station, texas). of 165 infants enrolled, 7 were excluded: 1 due to preterm birth (born <36 weeks), and 6 due to failure to provide any swabs. the remaining 158 children (75 male) provided 11 208 stool and 11 192 nasal swabs, of which 11 124 and 8100, respectively, met the inclusion criteria ( figure 1 ). symptom diaries were submitted for 154 children, constituting 88 811 childdays of observation. cohort characteristics are summarized in supplementary table 3 . pevs were detected in 1423 (12.8%) stool and 17 (0.2%) nasal weekly swabs ( figure 1 ). based on stool swab results, 306 distinct pev episodes were identified during the study, including 16 (5.2%) episodes that coincided with the 17 positive nasal swab detections. of the 295 of 306 (96.4%) episodes meeting genotyping inclusion criteria (figure 1 ), 284 (96.2%) were typed successfully (supplementary table 4 figure 3) [28, 29] . the overall pev incidence rate was 144 (95%ci, 128-161) episodes per 100 child-years. incidence rates for the 3 major types (pev-a types 1, 3, and 6) were 64 (95% ci, 54-75), 24 (95% ci, [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] , and 27 (95% ci, 21-35) episodes per 100 childyears, respectively. after the first 3 months of life, the incidence of new pev infections rose steadily until age 8 months before plateauing and then gradually declining in the second year (supplementary figure 4) . the maximum episode number detected was 6 in 2 children (supplementary results and supplementary figure 5 ). the mean individual episode duration was 4.8 (standard deviation, 3.5) weeks with a maximum shedding duration of 16 weeks. in the 26 occurrences with different pev genotype detections from sequential swabs, the maximum duration of combined genotype shedding was 23 weeks (supplementary figure 1) . the first pev infection appeared at a median of 8.0 months of age (interquartile range [iqr], 6.0-11.7; supplementary table 5 ) with the earliest detection at day 5 of life in a stool swab. by 12 months, 76.4% had experienced at least 1 pev infection, increasing to 94.9% by their second birthday. first infections by pev-a1 occurred typically much earlier than for pev-a3 and pev-a6 ( figure 3 ). pevs were detected throughout each year, but peaked in summer and early autumn (december through march; supplementary figure 6 ). seasonality of the 3 main genotypes varied in magnitude between years, with dominant genotypes alternating or sharing seasonal peaks ( figure 4 ). of particular note was pev-a3, whose magnitude rose sharply and became the dominant genotype during the spring/summer months of 2013-2014 ( figure 4) . characteristics independently associated with pev infections included increasing age (particularly >6 months), summer season, not exclusively breastfeeding at a younger age (0-3 months), and attending formal childcare at a younger age (<12 months) (supplementary table 6 ). sex, season of birth, and presence of older siblings in household were not associated with pev infections. of 306 discrete pev episodes (figure 1 ), 138 (45.1%) were asymptomatic, 120 had ari symptoms (39.2%), 7 had age (2.3%), 17 had both (5.6%), 6 had an undifferentiated febrile illness (2.0%), and 18 lacked symptom data (5.9%). when pev was the sole detected virus in stools, 38.4% of episodes were asymptomatic (table 2) , whereas if respiratory viruses in nasal swabs were also absent, the proportion without symptoms increased to 65.9% (table 3) . similar findings were observed when pev-a3 episodes were considered alone (supplementary table 7 ). of the 96 of 150 symptomatic episodes with recorded burden information, 63 (65.6%) received a primary care consultation, but none needed hospitalization. overall, no association was observed between pev viral load and symptomatic episodes (symptomatic vs asymptomatic mean ct difference, orc hid -s1 13e 04 orc hid-s10 4e0 2 orc hid-s130 e03 pev-a 3-kx5 79848 orchi d-s93e 04 orchid-s99e02 orchid-s109e 03 orchid-s160e03 orchid-s117e02 79 orchid-s147e01 orchid-s1 31e02 orchid -s91e03 pev-a 3-ky55 6675 orch id-s9 8e01 orc hid-s157 e02 orc hid -s11 1e0 4 or ch id-s 100 e03 or ch id-s14 4e0 3 or ch id-s1 46e 02 or ch id-s1 48 e0 table 8) . overall, in 197 (64.4%) pev-associated episodes, at least 1 other virus was detected in stool swabs collected within 7 days of the onset of the pev infection (table 2) . indeed, shedding of non-pev viruses was also relatively common in the stools of orchid subjects (supplementary table 9 ). in 137 pevassociated ari episodes, 96 (70.1%) had at least 1 respiratory virus detected in nasal swabs during the 7-day window ( figure 1 and table 3 ); these were primarily rhinoviruses, which were present in 76 of 137 (55.5%) symptomatic ari episodes. the orchid study provides high-resolution representation of pev infection in the first 2 years of life in healthy australian children. we observed frequent pev detections in stools, but not in nasal swabs, from these young children. diverse pev genotypes circulated in the community, with distinct seasonal peaks and genotype transitioning during summer and early autumn. prolonged pev shedding was from sequential infections with different genotypes rather than single infections, suggesting limited heterotypic protection. a sharp increase in pev-a3 detections in cohort children coincided with a pev-a3-related sepsis outbreak in australian infants where both sets of strains clustered phylogenetically [19] . however, infections in cohort children were either community-managed or asymptomatic in nature with symptom association confounded by high viral codetection rates. primary pev infection occurred early in orchid subjects with >75% affected by their first birthday, compared with 22% [15] and 43% [13] of children with pev detections by the same age in 2 other community-based studies. however, neither study recruited before 3 months of age, and both sampled less frequently (monthly) and could sequence fewer (30%-73%) positive samples, meaning short or sequential episodes may have been missed [13, 15] . by age 24 months, 94.9% of orchid children had at least 1 pev infection, agreeing with the norwegian (86%), but not finnish (48%) studies [10, 13, 15] , and aligning with seroprevalence surveys [10, 11, 30] . in orchid, primary pev-a1 detections occurred earlier than pev-a3, agreeing with seroprevalence findings [10, 11] , but conflicting with rt-pcr-based studies [11, 31] . however, the rt-pcr studies relied upon clinical samples, which may bias the pev detection ages as pev-a3 is associated primarily with disease before age 3 months [2, 3, 5, 7, 11] . unlike stools, pev was detected rarely in nasal swabs (0.2%) and was always associated with a pev-positive stool of the same genotype, supporting fecal-oral as one transmission route. previous community-based studies reported higher pev detection rates (4%-9%) in respiratory samples from asymptomatic subjects [16, 18] , possibly resulting from different sampling techniques (gargle and nasopharyngeal swabs). in contrast, infants hospitalized in brisbane with pev-a3-associated sepsis had high and comparable positive nasopharyngeal swab (86.2%) and stool (88.9%) detection rates [19] . given the asymptomatic or mild nature of cases in our study, this observed discrepancy with hospitalized infant pev detection rates is likely a reflection of more severe systemic disease. across the study, pev-a3 was detected infrequently until the 2013-2014 spring/summer when it predominated. this coincided with the outbreak of pev-a3-associated sepsis in australian infants [5, 19] , and aligns with the predicted timing of a genomic recombination event leading to a more virulent phenotype [28] . nevertheless, in our cohort pev-a3 was not associated with severe symptoms, even when detected during the outbreak period or within the high-risk first 3 months of life. case-control studies [32] and the sharp increase in pev infections after age 3 months in our cohort suggest maternal antibody protection against pev-related disease early in life. thus, emergence of a novel recombinant pev-a3 strain and declining pev-a3 neutralizing antibodies in women of childbearing age at a population level may contribute to the biennial outbreaks in australian infants [12, 32] . pev infection risk increased during summer, in agreement with some [2, 3, 7, 11, 15] , but not all studies as late autumn peaks are also reported [6, 11, 13, 15] . presence of older household siblings was not associated with pev or pev-a3 infections within orchid, a finding contrasting with hospital-based studies focusing mainly on pev-a3 cases [33, 34] . it however, agrees with the norwegian community-based study where a sibling age gap >2 years increased the likelihood of infection, possibly originating from similar-aged peers attending kindergarten [13] . almost half the sole pev episodes within orchid were asymptomatic, while virus codetection was common in the remaining symptomatic episodes, primarily from rhinovirusassociated aris, but also other enteric viruses. however, we may have underestimated the etiologic role of pev in aris as rhinoviruses themselves have also been shown to be detected frequently in asymptomatic cases from the same cohort [22] . nevertheless, these results highlight the overall mild nature of pev infections within the community with none of the participants hospitalized, including infants infected with pev-a3. the strength of our study lies in the systematic, highfrequency sample and symptom data collection from an unselected community-based birth cohort, a combination not previously reported in pev epidemiology studies. weekly samples allowed us to observe infections <4 weeks' duration (45.4% of all episodes) that would not be captured by sampling less frequently [13, 15] . in the finnish study, 8 (10.3%) children had secondary episodes, with only 1 genotyped [15] . this contrasts with our findings where 90.7% of subsequent infections were typed successfully allowing multiple samples within the same month being available for sequencing and potentially allowing identification of sequential infections. however, several limitations should be considered. first, the orchid study was conducted in an urban, subtropical setting and most enrolled families were socioeconomically advantaged with higher rates of early age childcare attendance [22] . while the findings are valid, they may not be entirely generalizable to other populations. second, the study relied upon parents diligently recording symptoms and collecting samples. although completion rates were excellent for such a demanding study, not all diaries and swabs were returned. finally, the 2 genotyping regions used in this study are highly conserved within the genome and may not reflect the true genetic diversity shown by the pev-a3 recombinant variant associated with sepsis [28] . the sharp increase in orchid pev-a3 cases observed during the first pev-a3 outbreak [19] suggests this recombinant variant was circulating within the community. in summary, orchid extends previous limited seroprevalence and community-based surveys by confirming that pev infections from multiple genotypes are common during the first 2 years of life, with virus shedding primarily through the fecal route. prolonged pev shedding is from sequential infections with different genotypes rather than a single virus episode. annual outbreaks occurred in the summer/autumn months with the severe disease-associated pev-a3 succeeding pev-a1 as the predominant strain in the 2013-2014 season, but it and other pev infections were typically asymptomatic or associated with mild ari or age symptoms. despite participants being predominantly from advantaged families, which may limit the extrapolation of findings to lower socioeconomic settings, these observations illustrate the value of unselected, high-resolution longitudinal community-based cohort studies helping to identify the overall epidemiology, risk factors, and clinical features associated with infection rather than focusing exclusively upon the few cases with severe disease. of total episodes and episodes per enrolled child each month of all parechovirus a (pev-a) genotypes as a function of enrolled subjects human parechovirus in respiratory specimens from children in kansas city, missouri pediatric parechovirus infections strategies to improve detection and management of human parechovirus infection in young infants specific association of human parechovirus type 3 with 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materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-273956-mruywa71 authors: mathers, amy j title: the practical challenges of making clinical use of the quantitative value for sars-cov-2 viral load across several dynamics date: 2020-07-10 journal: clin infect dis doi: 10.1093/cid/ciaa958 sha: doc_id: 273956 cord_uid: mruywa71 nan a c c e p t e d m a n u s c r i p t we are now approaching the half year mark since the first case of covid-19 was identified and we have already learned a great deal about viral dynamics but obviously continue to need to learn more. as the numbers of patients infected with sars-cov-2 continues to increase in the united states so does access diagnostic molecular testing especially for hospitalized patients. most of the molecular diagnostic platforms provide a polymerase chain reaction (pcr) crossing time (ct) indicative of the amount of viral rna in the original sample. most labs are not currently releasing the ct value into the clinical result. however, based on the findings of ct value tightly correlating with mortality and need for mechanical ventilation when patients present for hospital admission with covid-19 infection this may need to change. from the very beginning of the pandemic for those of us on the laboratory side seeing both the amount of viral rna detected and progression of illness has been striking. here satlin et al demonstrate the prognostic value of the amount of sars-cov-2 rna from a nasopharyngeal swab at the time of hospital admission for those who were sick enough require hospitalization. this data clearly provides valuable means to assess the potential severity of illness to triage at the time of admission. although similar smaller data sets have hinted at this finding it is shown clearly in a large number of patients even though the analysis is retrospective [1, 2] . the ct could be valuable data in determining prognosis and the need for intensive care unit support. predicting location of admission is especially important in the setting of the pandemic where we are frequently trying to allocate resources for optimizing medical care while trying to decrease the number of bed movements in a patient with a contagious respiratory illness. however, there are several hurdles and nuances which need to be addressed to deploy ct value as a meaningful clinical metric. laboratory results are static and capture a point in time whereas the sars-cov-2 replication throughout illness is dynamic [2] . there are a variety of contexts where clinical microbiology laboratories have been asked to provide sars-cov-2 diagnostic testing (e.g. asymptomatic screening, outpatient symptomatic, clearance after infection). however, interpreting a low ct value at the time of hospital admission can a c c e p t e d m a n u s c r i p t only be applied for prognosis of patients in this context while learning about the natural history of a novel infection. as the authors state they were not testing patients whom they were planning to discharge from the ed and thus it is important to note that the findings only apply to the hospitalized cohort. others have established that early in symptom onset the ct value in the nasopharynx is the lowest 1-4 days after symptoms develop [1, 3] . in the cohort presenting for hospital admission there were more days since symptom onset between the low, medium and high viral load groups. presumably viral counts would have been initially higher for all patients had they presented earlier. but we do not know if there would be the same prognostic value this early in disease and there is reason to believe that it would not be equivalent with several descriptions of high initial viral loads even in mild illness [1] . we are learning that the replication which is high early in infection the nasopharynx that later in disease replication likely occurring lower in the lungs which then may be a sign of patients who cannot control the virus well into infection [2] . the highest viral loads are seen early in infection when patients are more likely to be outpatient are not evaluated in this cohort [1] . lastly, there may also be clinical value in using the ct value late in disease to determine the level of contagion after symptom resolution. obviously this will be a completely different setting but it has been demonstrated that late in the disease low ct value correlates with recovery of viable virus but this makes another potential clinical value for reporting the ct [4] . in addition to the viral dynamics of infection there are also several variables which may impact the amount of rna detected in the sample. because of the shortages in diagnostics many laboratories are using more than one platform for sars-cov-2 molecular detection. different diagnostic platforms have varying sensitivity and will likely have differences in the ct values and methods. this likely means a laboratory would need to perform a ct value equivalency study to use the release the result as clinicians typically assume that results from the same lab are roughly equivalent. another factor potentially influencing ct values has been variability of specimen collection. in the setting of a shortages of flocked nasopharyngeal swabs and viral transport media many hospitals substituting flocked swabs for wrapped or 3-d printed swabs and use variable media in place of viral transport media. a c c e p t e d m a n u s c r i p t the use of differing collection tools will likely also result in variable rna recovery. shortages in personnel, swabs and the needed personal protective equipment for safe collection have also prompted health systems to explore anatomic site alternatives to nasopharyngeal collection (e.g. throat, nasal, saliva) which will alter the test sensitivity and ct value [4] . because many of these factors are driven by the dynamics of shortages it may be challenging for a lab to consistently report the ct value and have it with equivalent meaning across all of these variables within a single result. even though there are many logistic challenges in comparing the ct value from one platform, specimen type and disease state as demonstrated in this manuscript there is value in understanding viral burden. our job now as infectious disease clinicians and laboratorians will be to work together to come up with reporting parameters across the various contexts for using the ct value to maximum effect. laboratorians will need to develop a standard equivalency across their own diagnostic platforms and specimen types to validate the numbers for accuracy and reproducibility. more critical will be for clinicians to interpret the reported ct value of the in the context of the patient who is having the test. if a laboratory can only release the ct value on inpatients then maybe the ct value will be interpreted as the data supports. it is not clear that a ct<25 at day one of symptom onset has the same mortality prediction that the data in satlin et al does at day seven (median for patients in the high viral load group in this study). as most laboratory reports are not context aware this may mean that additional information for interpretation of the ct value may need to be amended to the report so that we can use this valuable data to understand how it can help us care for patients infected with sars-cov-2. we need to continue to work together to develop all the tools we possibly can to understand the disease progression, transmission risk and recovery with a rapidly spreading novel infectious disease. temporal dynamics in viral shedding and transmissibility of covid-19 virological assessment of hospitalized patients with covid-2019 variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure swabs collected by patients or health care workers for sars-cov-2 testing a c c e p t e d m a n u s c r i p t key: cord-254183-98o0dssj authors: waggoner, jesse j.; soda, elizabeth a.; deresinski, stan title: rare and emerging viral infections in transplant recipients date: 2013-10-15 journal: clin infect dis doi: 10.1093/cid/cit456 sha: doc_id: 254183 cord_uid: 98o0dssj emerging viral pathogens include newly discovered viruses as well as previously known viruses that are either increasing, or threatening to increase in incidence. while often first identified in the general population, they may affect transplant recipients, in whom their manifestations may be atypical or more severe. enhanced molecular methods have increased the rate of viral discovery but have not overcome the problem of demonstrating pathogenicity. at the same time, improved clinical diagnostic methods have increased the detection of reemerging viruses in immunocompromised patients. in this review, we first discuss viral diagnostics and the developing field of viral discovery and then focus on rare and emerging viruses in the transplant population: human t-cell leukemia virus type 1; hepatitis e virus; bocavirus; ki and wu polyomaviruses; coronaviruses hku1 and nl63; influenza, h1n1; measles; dengue; rabies; and lymphocytic choriomeningitis virus. detection and reporting of such rare pathogens in transplant recipients is critical to patient care and improving our understanding of posttransplant infections. emerging infectious diseases are caused by pathogens that are newly recognized or whose incidence has either increased in the preceding 2 decades or threatens to increase. viral diseases account for a large proportion of such infections. in the context of transplant recipients, important emerging viruses can be considered to be 1 of 3 types: (1) novel viruses; (2) known viruses increasing in incidence in the general population and, potentially, in transplant recipients; and (3) previously known viruses that cause disease of increased severity in the immunocompromised host. in this review, we begin by discussing viral diagnostics and the evolving field of viral discovery, which has increased the speed of virus identification but has created new challenges. our focus then shifts to specific emerging and reemerging viral pathogens in the transplant community. viruses described in case series or multiple case reports are listed in table 1 . viruses described only in single case reports are listed in table 2 . the potential risks of viral transmission as the result of xenotransplantation will not be addressed [1] . viral discovery has typically relied on the ability to detect new viruses in cell culture. although clinical virology laboratories affiliated with transplant centers routinely perform viral culture, many pathogens do not grow well or do not grow at all, and viral detection using culture is further limited by the number of cell lines a laboratory can realistically maintain. pathogen detection in the clinical laboratory is also limited by the available tests, which often target conserved sequences (polymerase chain reaction [pcr] or real-time [rt]-pcr) or specific antigens or antibodies to detect known viruses. multiplex testing for clinical syndromes, particularly for respiratory-tract infections, allows for a less biased approach to viral diagnosis but still faces limitations in identifying emerging pathogens [2] . in rare situations, an unusual virus may be detected by testing for known pathogens, as in the case of a woman who presented with usutu viremia, which gave a low-positive result by west nile virus rt-pcr [3] . a number of more rapid molecular methods are now being employed in viral discovery, categorized as sequence dependent (such as the pan-viral microarray) or sequence-independent techniques [4] . the pan-viral microarray is an array spotted with oligonucleotide sequences representing known viral pathogens. novel viruses can be identified if sufficient similarity exists between sequences in the new virus and those on the array. amplicons can then be recovered from the array, then cloned and sequenced [5] . this technology was used in the identification of sars coronavirus from a cultured patient isolate [4] . pcr based on conserved sequences generally has limited applicability in viral diagnostics, as viruses do not contain highly conserved sequences analogous to 16s ribosomal rna sequences utilized in bacterial identification [6] . the sequence-independent amplification and sequencing of viral nucleic acids in biological samples has been termed viral metagenomics [4, 7] . sequence-independent approaches include subtractive hybridization or representation difference analysis, sequence-independent single-primer amplification, and rolling circle amplification. these techniques have been used to identify agents such as human herpes virus 8, torque teno viruses (ttv), hepatitis e virus (hev), norwalk virus, parvovirus 4, and human bocavirus (hbov) [4, 7] . viral metagenomics has been aided by the development of a number of new sequencing platforms. termed next-generation sequencing (ngs, or deepsequencing), such technologies allow for the rapid and parallel generation of 10 6 to over 10 9 sequences per run. most current technologies rely on nonspecific amplification of viral dna or rna from samples treated to remove host nucleic acids. amplification is followed by sequencing by synthesis using different technologies to detect base incorporation [6] [7] [8] . ngs has been utilized to identify novel viruses in patient samples and in studies of fevers of unknown origin [9, 10] . ngs has a great ability to detect both known and previously unknown (divergent) viruses, but mere detection does not demonstrate causation. for many of these viruses, classical koch's postulates cannot be applied, and as demonstrated with ttv and hbov, establishing a causative role for these agents can be difficult [6, 11, 12] . mokili and colleagues [6] proposed "metagenomic koch's postulates," but whether they are sufficient remains moot. at this time, ngs is largely a tool for research purposes. sequencing reactions take a good deal of time to set up and perform [8] . these runs generate massive amounts of data that must be filtered prior to analysis using various alignment programs designed to handle the large numbers of short reads [8, 9] . finally, results must be interpreted carefully. contaminants from the laboratory and even from commercial reagents are often identified (eg, xenotropic murine leukemia virus-related virus), and confirming the presence of a virus identified with small numbers of reads may not be possible [9, 13] . human t-cell leukemia virus type 1 human t-cell leukemia virus type 1 (htlv-1) seroprevalence rates range from 3% to 30% in endemic areas, to <1% in western countries [14] . chronic htlv-1 infection is associated with adult t-cell leukemia (atl) and htlv-1-associated myelopathy (ham) in 5% or fewer of those infected, but there is concern that immunosuppression in htlv-1-positive transplant recipients may trigger progression to these complications [15] [16] [17] . yoshizumi et al [17] identified 26 htlv-1-positive, living donor liver transplant recipients. atl developed in 4 patients at 181-1315 days post transplantation; all 4 patients died, including 3 from atl. overall survival rates did not differ between htlv-1-positive recipients and 305 htlv-1-negative liver transplant recipients from the same institution [17] . case reports of atl following renal transplantation in htlv-1positive patients have been documented, though in case series of renal transplant recipients (totaling 46 patients with 5-17 years of follow-up), no cases of atl or ham developed [18] [19] [20] [21] . ham has been reported in 1 htlv-1 d+/r+ living-related liver transplant recipients [22] . atl responds poorly to conventional chemotherapy, with the highest median survival rates reported in clinical trials being approximately 13 months [23] . as a consequence, hematopoietic stem cell transplantation (hsct) has been evaluated for the treatment of atl in htlv-1-positive patients. (hsct will be used to describe the transplantation of multipotent stem cells from bone marrow, peripheral, or cord blood.) the largest study of hsct for atl involved the retrospective analysis of 386 patients with atl who had undergone an allogeneic hsct at 3 centers in japan [23] . their 3-year survival rate was 33%; atl recurred in 41% of patients who survived to 30 days post transplant. those who received transplants from a related htlv-1 seropositive donor had a higher risk of disease-associated mortality relative to those whose related donor was htlv-1 negative. hsct recipients in complete remission at the time of transplantation had a higher rate of survival compared to patients not in complete remission (51% vs 26%) [23] . the transmission of htlv-1 through transplantation or transfusion has been documented. in spain, 3 htlv-1-negative recipients of organs from a single htlv-1-positive donor (1 liver and 2 kidney transplants) developed ham within 2 years of transplantation [24] . two case reports document the occurrence of ham in a heart transplant recipient and an hsct recipient who acquired htlv-1 through blood transfusions [16, 25] . in low-prevalence areas, however, universal donor screening with enzyme-linked immunosorbent assay followed by western blotting resulted in many false positives, and the practice is no longer recommended by the united states organ procurement and transplantation network [14] . hev is a common cause of acute liver disease in the developing world, primarily from fecal-oral spread through contaminated drinking water. infections in developed nations are well described, but typically result from the consumption of undercooked pork products. in immunocompetent hosts, hev acute infection is self-limited with rare progression to fulminant liver failure. however, in the immunosuppressed host, chronic infection marked by persistent viremia and abnormal liver function with eventual progression to cirrhosis can occur [26] [27] [28] . cases have been described in recipients of a variety of transplants, including kidney, liver, heart, and lung [27, 28] . in a multicenter review of 85 cases of acute hev infection, 65.9% of the solid organ transplant (sot) recipients developed chronic hepatitis of whom 14.3% developed cirrhosis. the use of tacrolimus compared with cyclosporine a was an independent predictor of chronic infection [28] . rare cases of encephalitis and polyradiculopathy with hev rna detection in the cerebrospinal fluid (csf) have also been described [26] . the majority of hev infections following sot result from de novo infections and are unlikely to represent virus reactivation [26, 29] . rare instances of transmission through blood transfusion or the donated graft have been reported [26] . determining the overall incidence of hev-related disease following transplantation is hampered by the available diagnostic tests, none of which are food and drug administration (fda) approved. commercial serological assays have variable test characteristics, and tests for hev rna detection in serum or stool samples are not routinely available [26] . reports of hev infection following hsct are limited. while an individual case of hev reactivation following hsct has been reported, a review of 32 anti-hev immunoglobin gpositive patients prior to hsct showed no evidence of disease reactivation [30] . treatment of prolonged hev viremia often involves reducing immunosuppression. pegylated interferon administration has been shown to induce a sustained virologic response in a limited group of patients [26] . both approaches to viral control may increase the risk of graft rejection. ribavirin monotherapy has induced sustained virologic responses without the risk of rejection and may represent the first-line agent for treatment [26] . there are 5 clinically relevant non-sars human coronaviruses (hcov): oc43; 229e; hku1 and nl63, both identified in the last decade; and the middle east respiratory syndrome hcov (mers-cov), identified in 2012. a prospective study found that 41% of hcov infections were asymptomatic and none of 22 infected allogeneic hsct recipients developed lower-respiratorytract infection, although prolonged viral excretion is frequent [30] . nonetheless, there have been reports of fatal hcov infection following hsct. there is evidence among sot recipients that hcov can cause severe lower-respiratory-tract infections and increase the risk of graft rejection [31, 32] . hcov-hku1 and nl63 do not appear to be more virulent than the previously discovered hcov-oc43 and 229e; however, the recently identified mers-cov has been associated with severe pneumonia and a high mortality rate. cases have not yet involved immunocompromised hosts. efforts to identify novel respiratory pathogens have led to the discovery of hbov and ki and wu polyomaviruses (kipyv and wupyv) [12, [33] [34] [35] . while these viruses have been detected in patients with respiratory symptoms, evidence to support a causative role for these agents in severe disease is lacking [12, 33] . studies evaluating hbov as a respiratory pathogen in immunocompromised adults have detected the virus infrequently and have not documented an effect on patient outcomes [2, 12] . the establishment of hbov as a respiratory pathogen has also been complicated by high rates of copathogen detection and hbov detection in asymptomatic patients [2, 12] . viral dissemination in transplant recipients occurs, with hbov detected in blood and stool. however, patients often had hbov detected after weeks of hospitalization, and other pathogens were also detected during these episodes [36, 37] . some commercial platforms for multiplex detection of respiratory pathogens include hbov. no specific antiviral treatment is available [2] . kipyv and wupyv have been detected in nasopharyngeal and bronchoalveolar lavage samples from sot and hsct recipients [33] [34] [35] . detection of these viruses has been associated with sputum production and wheezing following hsct [34] . however, similar to hbov, these viruses are often codetected with other pathogens, and they have not been associated with severe respiratory tract disease or mortality [34, 35] . immunocompromised hosts are more susceptible to complications of influenza; however, it is not clear that emerging strains will necessarily cause more severe disease. one case series of 237 sot patients with h1n1 influenza showed that 16% required icu admission and 4% died [38] . a series among hsct recipients showed similar findings [39] . in a comparison of outcomes in kidney transplants and immunocompetent patients with h1n1, there were no differences in morbidity or mortality [40] . to date, no cases of h5n1 or h7n9 influenza have been reported in transplant recipients. the recent rise in measles incidence brings it into consideration here. the most significant manifestation may be subacute measles encephalitis (sme), though severe cases of pneumonia have been documented [2] . sme has developed in renal transplant recipients and a single hsct recipient. patients may present with a measlescompatible illness, which improves. they develop altered mental status and seizures 2-4 weeks later; fever is uncommon. the first imaging changes are seen by magnetic resonance imaging, and diagnosis is confirmed by immunoglobin m (igm) seroconversion or rt-pcr. the clinical course is one of deteriorating mental status and treatment-refractory seizures [2] . four of 6 transplant cases of sme have died. the 2 survivors both had significant neurological deficits [41] . the incidence of measles in transplant recipients, as well as the proportion with severe disease, is unclear. two series identified 2 cases of interstitial pneumonia (1 fatal) among 24 hsct recipients diagnosed with measles, though methodological limitations existed in both studies [2] . dengue virus (denv) is the most common vector-borne viral disease worldwide and has been detected in an increasing number of countries over the last 40 years. in 2 case series involving 33 renal transplant recipients, only a single case of severe dengue developed, with no fatalities or loss of graft function [42, 43] . severe cases of dengue, including 4 deaths, have been reported in renal transplant recipients along with fatal cases in a liver transplant recipient and an hsct recipient [2] . in patients who died, disease typically developed within the first month post transplant. human-to-human transmission of denv as a result of sot or hsct has been postulated, and transfusion-related denv infections have been reported [44] . fda-approved diagnostics include tests for igm detection and a centers for disease control and prevention-developed rt-pcr; management consists of supportive care. seventeen cases of rabies have been reported in transplant recipients, and to date, all have been transmitted through the transplanted tissue or organ [2, 45] . nine cases followed corneal transplantation, including 8 deaths [2] . the sole survivor, reported in 1981, began postexposure prophylaxis (pep) on postoperative day 1 [46] . two clusters (texas, 2004, and germany, 2005) , totaling 7 rabies cases, have occurred following sot [47, 48] . these cases followed the transplantation of liver, lung, kidney, kidney-pancreas, and iliac artery grafts. patients typically developed encephalitis between 30 and 60 days post transplant, and all symptomatic patients died [48] . patients in germany received pep and antiviral treatment, though not until postoperative day 45 [47] . the liver recipient in this cluster had been previously vaccinated and never developed disease [2] . both donors were later determined to have rabies exposures (bat and dog bites, respectively) [47, 48] . a recent report (maryland) documented a fatal case of rabies developing a year after kidney transplant. transmission of raccoon-variant rabies through the donated graft was confirmed. three other graft recipients from the same donor are alive, though full details are not available [45] . the management of rabies focuses on prevention with vaccination in high-risk patients or pep. transplant recipients who receive pep can mount adequate responses (antibody titers of 0.5 international units/ml), though titers are lower than in immunocompetent patients [2] . based on the experience of the german liver transplant recipient, rabies vaccination may remain effective even after transplantation. cases of lymphocytic choriomeningitis virus (lcmv) transmitted through organ transplantation (4 clusters, including 14 cases and 11 deaths) document the ability of this pathogen to cause severe disease in the immunocompromised host [10, [49] [50] [51] . another cluster involved the transmission of a related arenavirus in australia, with similar outcomes (1 liver and 2 kidney recipients; 3 deaths) [10] . as with rabies infections post transplant, all cases resulted from transmission through organ transplantation [10, [49] [50] [51] . at this time, cases have not been described in the hsct population. the 4 case clusters of lcmv infection occurred in the united states and involved kidney, liver, and lung transplants [49] [50] [51] . symptoms developed between 2 and 23 days post transplant and included fever, abdominal pain, nausea, diarrhea, and altered mental status. patients often developed a peri-incisional rash and tenderness. csf findings included elevated protein (often marked), normal to low glucose, and a mild pleocytosis [49] [50] [51] [52] . three patients survived lcmv infection following sot, 2 kidney recipients and a liver recipient. ribavirin has been employed in some cases, though the benefit remains unclear [2] . three corneal transplant recipients were potentially exposed to lcmv, though none of them developed symptoms or seroconverted [2] . contact investigation revealed exposure to rodents or positive testing for lcmv in 3 donors [49] [50] [51] . investigation into the fourth donor revealed no exposure, and all testing performed on remaining tissues was negative [50] . it has been advised that immunocompromised patients avoid contact with rodents, including pets, though this was not the mode of lcmv acquisition in these outbreaks [53] . for the majority of viral infections discussed here, data are insufficient to determine the true incidence of disease in transplant recipients. measles, mumps, and yellow fever are vaccinepreventable illnesses, though these vaccines are live-attenuated and not recommended following transplantation. also, antibody response to vaccines is less than in immunocompetent patients. donor-transmitted rabies carries a dire prognosis, and though limited data exist, the use of pep in transplant recipients appears safe. given their apparent rarity, screening for many of these diseases in organ donors is not recommended. the examples of htlv-1 (discussed earlier) and lcmv are illustrative of some of the difficulties involved with donor screening. in the outbreak investigations for lcmv, only 1 of 4 donors had detectable antibodies. indeed, rt-pcr from multiple samples failed to detect lcmv from 1 donor, and yielded a positive result in only a single lymph node in another [49] [50] [51] . it seems prudent to obtain a comprehensive history of potential organ donors, though it remains unclear how certain findings, such as rodent ownership, should affect one's donor status. reporting rare infections in transplant recipients will help to identify agents for which more research is needed and screening may be warranted. however, it is likely that these infections are underdiagnosed as symptoms may be attributed to more common, and potentially coincident, posttransplant infections. xenotransplantation-associated infectious risk: a who consultation rare and emerging viral infection in the transplant population usutu virus infection in a patient who underwent orthotropic liver transplantation viral 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virus type 1 in living donor liver transplantation prognosis of htlv-1 positive renal transplant recipients in iran prognosis of htlv-i-positive renal transplant recipients renal transplantation in patients with human t-cell lymphotropic virus type 1 long-term results in human tcell leukemia virus type 1-positive renal transplant recipients human t-cell leukemia virus type i-associated myelopathy following living-donor liver transplantation transplantation of allogeneic hematopoietic stem cells for adult t-cell leukemia: a nationwide retrospective study posttransplantation htlv-1 myelopathy in three recipients from a single donor htlv-i-associated myelopathy following allogeneic bone marrow transplantation hepatitis e virus: what transplant physicians should know hepatitis e virus and chronic hepatitis in organ-transplant recipients factors associated with chronic hepatitis in patients with hepatitis e virus infection who have received solid organ transplants hepatitis e virus infection without reactivation in solid-organ transplant recipients low risk of hepatitis e virus reactivation after haematopoietic stem cell transplantation a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant human polyomaviruses in disease and cancer wu and ki polyomaviruses in respiratory samples from allogeneic hematopoietic cell transplant recipients no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease persistence of human bocavirus dna in immunocompromised children disseminated bocavirus infection after stem cell transplant outcomes from pandemic influenza a h1n1 infection in recipients of solid-organ transplants: a multicentre cohort study outcome of pandemic h1n1 infections in hematopoietic stem cell transplant recipients morbimortality of pandemic influenza a h1n1 infection in kidney transplant recipients requiring hospitalization: a comparative analysis with nonimmunocompromised patients measles-associated encephalopathy in children with renal transplants dengue in renal transplant patients: a retrospective analysis dengue has a benign presentation in renal transplant patients: a case series non vector-borne transmission modes of dengue centers for disease control and prevention. cdc confirms rabies death in organ transplant recipient prevention of inter-human rabies transmission after corneal graft management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus transmission of rabies virus from an organ donor to four transplant recipients lymphocytic choriomeningitis virus transmitted through solid organ transplantation -massachusetts transmission of lymphocytic choriomeningitis virus by organ transplantation solid organ transplant-associated lymphocytic choriomeningitis, united states lymphocytic choriomeningitis virus infection in organ transplant recipients -massachusetts update: interim guidance for minimizing risk for human lymphocytic choriomeningitis virus infection associated with pet rodents acknowledgments. the authors thank dr benjamin a. pinsky for his comments on this manuscript. we also thank dr jose g. montoya for his support during the preparation of this review.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-266150-wox7pnkr authors: torres, juan pablo; piñera, cecilia; de la maza, verónica; lagomarcino, anne j; simian, daniela; torres, bárbara; urquidi, cinthya; valenzuela, maría teresa; o’ryan, miguel title: sars-cov-2 antibody prevalence in blood in a large school community subject to a covid-19 outbreak: a cross-sectional study date: 2020-07-10 journal: clin infect dis doi: 10.1093/cid/ciaa955 sha: doc_id: 266150 cord_uid: wox7pnkr background: a sars-cov-2 outbreak affecting 52 people from a large school community in santiago, chile was identified (march 12), nine days after the first country case. we assessed the magnitude of the outbreak and the role students and staff played using a self-administered antibody detection test and survey. methods: the school was closed on march 13, and the entire community was placed under quarantine. we implemented a home-delivery, self-administered, igg/igm antibody test and survey to a classroom stratified sample of students and all staff from may 4-19. we aimed to determine overall seroprevalence rates by age group, reported symptoms, contact exposure and to explore dynamics of transmission. results: antibody positivity rates were 9.9% (95%ci: 8.2-11.8) for 1,009 students and 16.6% (95%ci: 12.1-21.9) for 235 staff. among students, positivity was associated with younger age (p=0.01), lower grade level (p=0.05), prior rt-pcr positivity (p=0.03), and history of contact with a confirmed case (p<0.001). among staff, positivity was higher in teachers (p=0.01) and in those previously rt-pcr positive (p<0.001). excluding rt-pcr positive individuals, antibody positivity was associated with fever in adults and children (p=0.02; p=0.002), abdominal pain in children (p=0.001), and chest pain in adults (p=0.02). within antibody positive individuals, 40% of students and 18% of staff reported no symptoms (p=0.01). conclusions: teachers were more affected during the outbreak and younger children were at higher infection risk, likely because index case(s) were teachers and/or parents from preschool. self-administered antibody testing, supervised remotely, proved to be a suitable and rapid tool. our study provides useful information for school re-openings. as of may 30, 2020, reported covid-19 cases had reached 5,817,385 globally, including 362,705 deaths [1] , with the majority of infections occurring in adults 18-64 years of age (74%) [2] . children under 18 years of age have made up a relatively small proportion of all cases (3% in the us) [2] , and children under 5 years of age account for less than 1% of reported cases [3] . however, early studies from china suggested that as the epidemic progressed in a large community, children became infected at higher rates [4] . further information from europe and the americas also seem to indicate that children could play a minor role in the infection process, at least in terms of detected cases and transmission [5, 6] . nce the true percentage of the childhood population infected with sars-cov-2 is not known, it is unclear the role that children play in transmission of the virus. however, they may interact with a greater number of contacts, as compared to adults, under normal non-social distancing conditions [7] . additionally, they may have higher viral loads compared to adults [8] , suggesting that they may still be transmitters of disease, similar to other respiratory viruses [9] . the possibility of children transmitting infection, especially to adult populations, led many countries worldwide to implement school closures as an important component of sars-cov-2 transmission mitigation policies [10] ; the impact of such measures is still being explored [11] [12] [13] [14] . the reference method for screening and diagnosing covid-19 infection is rt-pcr, nevertheless detection of antibodies against sars-cov-2 (igg, igm and iga) play a complementary role, with particular importance in providing epidemiological information [15] . seroprevalence has been extensively explored in covid-19 patients confirmed by rt-pcr, as recently reviewed [16] . a few studies have assessed seroprevalence, primarily in asymptomatic individuals using different methods, with rates among healthcare workers who had direct contact with covid-19 patients ranging from 1.6-44% [17, 18] and in the general population 2.8-5% [19] [20] [21] . covid-19 was first detected in chile on march 3, 2020; over the following two weeks, cases increased slowly, largely via travelers arriving from asia and europe [22] . the first communities to report an increase in detected cases were in eastern santiago, and the first covid-19 outbreak to be reported in chile was in a private school in this area. the school outbreak began on march 12, nine days after the index covid-19 case was detected in the country. the school year began on march 4, and during the first week of school parent-teacher meetings were held nearly every evening (figure 1 ). on march 13, when there were 58 confirmed cases nationwide, two cases were reported within the school community, one teacher and another staff member. that same day, the chilean ministry of health declared quarantine for the entire school community. in the next few days, confirmed symptomatic cases (rt-pcr) continued to appear, primarily amongst staff and parents, and to a lesser extent within the student population. at the time of the study, pcr testing was available with medical referral when patients were symptomatic or had contact with a known case. as of april 6, 52 m a n u s c r i p t 5 members of the school community had been confirmed positive for sars-cov-2 and there had been one associated death; cases were distributed as follows: 7 (13%) students, 18 (35%) staff, and 27 (52%) parents. the index case was a staff member who worked with the entire preschool and elementary school staff, and was present at all of the parent-teacher meetings for prekinder through 4 th grade. the school outbreak provided a unique opportunity to determine the role of children as potential asymptomatic transmitters of sars-cov-2. while children are believed to play a minor role in detected symptomatic cases, if a large number of students were asymptomatically infected, this could be determined after the fact by testing for anti-sars-cov-2 antibodies. thus, we designed a contactfree field study using finger-prick chromatographic-based igm/igg antibodies tests, in order to evaluate a large number of students and school staff, 8-10 weeks after the start of the outbreak. our primary aim was to determine the overall sars-cov-2 antibody prevalence in blood during the outbreak and characterize antibody prevalence among students by grade level, as well as in school staff. secondary aims were to retrospectively identify symptoms, confirmed covid-19 cases, and contact history within the school community in order to correlate with antibody positivity and explore possible transmission dynamics within the school community. the school is co-educational, located in the community of vitacura (population 85,000; 2017 census), in the metropolitan region (population 7.1 million). the school has 2,616 students in 14 grade levels. there are 318 staff members, of which 195 are teachers. classrooms in the preschool have two head teachers and 25-27 students; in the rest of the school classes are 36-38 students, and there is one teacher in 3 rd -12 th grades. the academic researchers presented the study proposal to the school authorities in mid-april, followed by submission to the ethical committee for research in human subjects of the faculty of medicine, universidad de chile, which was approved on april 30. all school staff and a randomized selection of students, evenly distributed by classroom, were invited to participate in the study. participants were sent an email describing the study and asking them to provide their address if they were interested in participating. in the case of students, parents and/or legal guardians were initially contacted, and if they declined, a random replacement was selected. after indicating an intention to participate, a package containing a flyer with simple step-by-step instructions (supplement) and the antibody testing supplies (test card, lancet, a cryovial with diluent, 2 capillary tubes and an alcohol pad) were distributed to each household using a georeferenced-home delivery service (uber flash). the flyer included a link/qr code that directed participants to a web m a n u s c r i p t 6 page where study data were collected using redcap electronic data capture tools hosted at the medical school of the universidad de chile. [23] using the redcap platform, participants could read and digitally sign the informed consent, and in the case of children 8 years and older, assent. once these forms were signed, a copy was emailed to participants for their records and they were directed to a secure survey that i) asked basic demographic questions, ii) requested information on any previous rt-pcr test for sars-cov-2 and potential contact with any covid-19 positive cases, and iii) asked about symptoms experienced since the outbreak (date and duration in days of each symptom). next, a detailed video (supplement), prepared by the research team, demonstrated how to perform the antibody test. participants were asked to attach a photo of the test after 15 minutes had elapsed and self-report the appearance of the three lines, g (igg), m (igm), and c (test control). after completion, participants were told they would receive results as interpreted by the research team, within the next few days. all tests were to be performed within one hour of opening the sealed aluminum foil bag. the novel coronavirus (2019-ncov) igg/igm test kit (colloidal gold) from genrui biotech inc, china, was used according to the manufacturer's specifications. for details on training staff in interpreting photos of the test cards see the supplement. in a first screening phase, the study nurse and/or technician viewed the photo provided by the participant along with the participant's self-report as to the visibility of the three bands, and determined whether the tests were igg+, igm+, igg & igm+, negative, invalid, or indeterminate. in this screening phase, all self-reported positive responses, in addition to any visual presence according to the study nurse and/or technician, no matter how faint, were to be considered. all samples considered positive, including those where parents/participants and study staff did not agree, and indeterminate samples (doubt of line presence), along with a random selection of negative samples, were then individually reviewed by a three-physician panel to determine their final positivity/negativity (details in supplement). data was analyzed in stata version 15 [24] . statistical differences for categorical variables were calculated using pearson's chi-squared test or fisher's exact test; continuous variables were tested using the mann-whitney u test or kruskal-wallis test. sample size calculations and details on model adjustment are in the supplement. m a n u s c r i p t 7 parents of 1,224 children were contacted by email; 1,105 manifested interest in participating and were sent a study package. a total of 1,029 child/parents signed consent and participated in the study, of whom 20 (1.9%) were excluded from analysis due to invalid or indeterminate results. this resulted in 1,009 students for inclusion in the analysis, representing 38% of the entire student body (supplementary figure 1a) . all staff (n=318) were contacted by email, and a total of 272 responded and were sent packages; 240 completed the online consent process, of which 235 were included in the final analysis, representing 74% of the entire school staff (supplementary figure 1b) . overall antibody positivity rates were 9.9% (95%ci: 8.6-11.5) for students and 16.6% (95%ci: 12.1-21.9) for school staff ( table 1 ). high school students had a lower positivity rate compared to younger levels. there were no differences in positivity between women and men in either students or staff, or between teachers and support staff. among students, antibody positive children were younger, had a higher pcr positivity rate (in those who underwent pcr testing during the outbreak), and were more likely to self-report contact with one or more confirmed cases, as compared to seronegative children ( table 2 ). in staff, the only variable that differed by antibody positivity was a previous history of pcr positive results; most individuals reported contact with one or more covid-19 cases and there was no difference by antibody positivity (74 vs. 54%). overall 490/1,009 (49%) students and 171/235 (73%) staff declared the presence of one or more symptoms since the start of the outbreak (figure 2) . in children, abdominal pain and fever were significantly more common among antibody positive (21% and 17%) compared to antibody negative children (9% and 6%, respectively). after removing children that were pcr positive, these differences remain significant (22% and 14% compared to 9% and 5%). among school staff (figure 2) , antibody positive individuals reported significantly higher frequency of weakness (54% vs. 30%), abdominal pain (21% vs. 7%), fever (36% vs. 7%), myalgia (46% vs. 24%), dyspnea (26% vs. 6%), chest pain (33% vs. 8%), and hyposmia (31% vs. 5%). after removing pcr positive individuals, these differences remain significant only for fever and chest pain (14% vs. 6% and 25% vs. 7%, respectively). when comparing symptoms between staff and students, a significantly higher frequency of headache, weakness, fever, myalgias, dyspnea, chest pain, sore throat, and hyposmia was observed in staff. within antibody positive individuals, 40% (95%ci: 30-50%) of students and 18% (95%ci: 8-34%) of staff reported no symptoms (p=0.01). m a n u s c r i p t 8 regarding the timing of onset of reported symptoms, in the staff the median date was significantly earlier in antibody positive versus negative participants (p=0.02). there was greater dispersion in the timing of symptom onset in seronegative participants (march-may), while in antibody positive subjects symptoms predominantly occurred during march (closer to the school outbreak). in students, the median date of symptom onset was also significantly earlier in antibody positive children (p=0.04). the median duration from self-reported symptom onset to sampling tended toward fewer days for an igm positive result (44 days for students and 51 for staff), compared to an igm/igg positive result (51 and 60 days) or an igg positive result (58 and 56 days) (students p=0.06, staff p=0.17). for seropositive individuals, symptoms occurred earlier in adults compared to students (median: march 15 vs. march 21; p=0.03). antibody positive students were distributed across all grade levels at the school (figure 3 ), but were significantly less common in high school students. there was a significantly greater number of teachers that were antibody positive and/or with a history of positive rt-pcr results in the preschool compared to the other levels. the median percent of antibody positive students per classroom was 8.3% (iqr 1.6-14.3%). in 7 classrooms, over 25% of students were positive for antibodies, of which four had a primary teacher who was antibody positive and/or rt-pcr positive. antibody positive children had an average of 1.8 contacts with a confirmed rt-pcr covid-19 case, while antibody negative children had 1.4 contacts (p=0.01). we found that the greater the number of contacts, the greater the probability that the child was antibody positive (or: 1.4; p=0.05; bivariate logistic model; response variable: positive/negative test result; explanatory variable: number of covid-19 positive contacts). the most common covid-19 contact in antibody positive (versus negative) children was their teacher (21% vs. 12%; p= 0.022), a household relative (11% vs. 2%; p<0.001), a classmate (9 % vs. 4%; p= 0.03), followed by a home caregiver (4% vs. 0.1%; p<0.001). sources with the greatest likelihood of possible contagion in students were: a home caregiver (or: 27.9), a household relative (or: 5.4), a classmate (or: 3.2), and teacher (or: 2.2). sars-cov-2 antibody prevalence was 10% in students and 17% in staff from a large school in santiago, chile, 8 to 10 weeks after a covid-19 outbreak affecting mostly staff and parents, and to a lesser extent students. these prevalence rates are higher than reports from community settings, where rates reported to date are about 5% [19] [20] [21] . however, due the fact that not everyone who develops covid-19 develops an immune response [26] , true infection rates may have been even higher. consistent with the detection of pcr confirmed cases during the outbreak, more adults than students were antibody positive. among students, antibody positivity was higher in younger children, pre-high m a n u s c r i p t 9 school, and among teaching staff as compared to non-teaching staff. increased positivity in younger children was likely due to the fact that the index case was a member of the preschool community. factors associated with antibody positivity in children were sars-cov-2 pcr positivity during the outbreak and a history of contact with an infected case; in staff, only pcr positivity was significantly associated with antibody positivity. overall, pcr testing and contact history was significantly higher in staff compared to students, which in addition to the higher antibody positivity observed in this study, support the more significant role of adults within the outbreak, in proportion to the overall population. nineteen participants had prior positive pcr results, of which 14 (73.7%) were also antibody positive. this outbreak has particular epidemiological features that may differ from other current or future school outbreaks. the index case(s) was(were) incoming travelers, teachers, parents and possibly students. initial cases in non-traveling members of the community were mostly related to parentteacher meetings, especially among adults related to the preschool. it is possible that a future outbreak, with a different index case(s), may result in a different age distribution of infection within the school. the duration of the virus's circulation period within the school lasted a maximum of 10 days from the beginning of the school year to school closure (if an infected individual circulated on opening day). the virus most likely continued to circulate in the households of infected individuals, and a thorough investigation of intra-familiar outbreaks was not performed. additional external exposures both during and following the outbreak period, while possible, seem less significant, as a majority of families in the area were under relatively strict stay-at-home orders imposed on march 20. our findings, if replicated in other schools, have several potential implications for the prevention of future covid-19 outbreaks in schools. in this outbreak, school closure occurred within two days of detection of the first two cases and within a few days of the start of the school year. nevertheless, newly detected cases occurred mostly after school closure, with a declining trend in school staff and an increase in parents and students. we hypothesize that adults, mainly through adult-to-adult contact seem to have been most affected during this outbreak. students on the other hand, were most likely to be infected by adults, either their teacher or parent, who most likely were infected during parentteacher meetings. thus, reopening schools, under scenarios in which community transmission levels may be as low as during the pre-epidemic phase, should focus on avoiding new cases among teachers. assuring household detection of cases in adults will also be important. general recommendations for reopening schools are focused on maximizing person-to-person distancing as much as possible given the reality of a school setting: reducing both the number of students and hours within classrooms, limiting group activities, rapid identification and isolation of symptomatic cases, robust parent-school reporting of cases and action plans in case of outbreaks, among others. our study, does not contradict these recommendations, but may lead to prioritize actions, by especially focusing on reducing adult-to-adult transmission. certainly, school related outbreak studies from other settings and situations, especially after the first wave, are required to further increase our knowledge of transmission dynamics within m a n u s c r i p t 10 schools, in order to pinpoint recommendations which may end up being most effective. it is likely that current infection awareness will allow early identification of future school outbreaks and implementation of school measures within a short period, which should rapidly curtail a potential outbreak. antibody detection tests have limitations. although manufacturers claim sensitivities and specificity of 72-100% and 98.7-100% respectively [25] , how these values relate to the true infection status of individuals in a community setting is not fully clear. according to the manufacturer, any visible band at the igg and/or igm level indicates a positive test. nevertheless, visibility of the band is subject to individual interpretation, as evaluated in our pilot testing. bands can range from clearly visible to faintly visible. to deal with this issue we defined criteria for positivity, with four levels of visibility, based on a medical panel's review of photos of the test card. overall there was agreement between parents and the medical panel in 931/1,009 student samples (92%) and 213/235 (91%) staff samples. discrepancies largely occurred in samples deemed negative by parents, but positive by the panel. antibody positivity levels would have been different based exclusively on parent/staff ascertainment: 65/1,009 (6.4%) and 31/235 (13%), respectively. a second test to confirm results in samples with discordant interpretations between the panel and participants would have been desirable (e.g. serum antibody test by elisa); however this was not possible due to a citywide quarantine, which is still in place to date. importantly, the self-applied tests were successful in terms of self-application with only 3.7% determined to be invalid and 0.5% indeterminate (1.9% were invalid/indeterminate after the option to perform a second test). while it would have been ideal to test parents as well, due to funding limitations this was not possible. symptoms were retrospectively reported with the known risk of recall bias. importantly, we asked participants to report symptoms prior to viewing the instructional sampling video, thus it is unlikely that test results influenced self-report of symptoms. the fact that both antibody negative students and staff reported symptoms at later dates leads us to conclude that their symptoms were due to other causes. self-application of the antibody test, delivered using a home delivery system, in combination with the use of electronic data capture via the participant's cell phone in a secure web platform proved to be an effective, contact-free epidemiological tool in the current pandemic setting, where limiting person-toperson contact is a relevant public health measure. such methodology could be considered in other settings worldwide, including remote areas. m a n u s c r i p t 11 in this school-based covid-19 outbreak in chile, affecting nearly 50 people among school and household members, antibody positivity rates based on a self-administered test were 10% and 17% among students and staff, respectively. adults, mainly through adult-to-adult contact seem to have been most affected during this outbreak. self-administered, remotely supervised antibody testing allowed us to determine the magnitude of infection and characterize outbreak features. m a n u s c r i p t 12 m a n u s c r i p t m a n u s c r i p t m a n u s c r i p t 17 world health organization. coronavirus disease (covid-19) situation report-132 covid-19): cases in the the novel coronavirus pneumonia emergency response epidemiology t. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study coronavirus disease 2019 in children -united states spread of sars-cov-2 in the icelandic population changes in contact patterns shape the dynamics of the covid-19 outbreak in china an analysis of sars-cov-2 viral load by patient age journal of clinical virology : the official publication of the pan american society for united nations educational saco. covid-19 educational disruption and response school closure and management practices during coronavirus outbreaks including covid-19: a rapid systematic review impact of school closures for covid-19 on the us healthcare workforce and net mortality: a modelling study school opening delay effect on transmission dynamics of coronavirus disease 2019 in korea: based on mathematical modeling and simulation study impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study connecting clusters of covid-19: an epidemiological and serological investigation antibody tests in detecting sars-cov-2 infection: a meta-analysis sars-cov-2-specific antibody detection in healthcare workers in germany with direct contact to covid-19 patients asymptomatic seroconversion of immunoglobulins to sars-cov-2 in a pediatric dialysis unit covid-19 antibody seroprevalence seroprevalence of sars-cov-2-specific antibodies among adults informe de situación covid-19 n16 research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support statacorp. stata statistical software: release 15. college station, tx: statacorp llc diagnostic performance of covid-19 serology assays clinical and immunological assessment of asymptomatic sars-cov-2 infections we thank the school rector david halm, c.s.c. and vicerector macarena lópez for their enthusiastic support for the study and the entire school community for participation in such difficult times. we thank dr. sergio george for his support in the writing of the manuscript, uber for the support with free rides for package delivery, cowork latam for granting us office space, and nicole huerta for her revision of photos of test cards. the authors have no conflicts of interest to declare. the ethics committee of the medical school of the universidad de chile approved of all aspects of this study. key: cord-284782-51mbq7qb authors: huang, jing; liu, fangkun; teng, ziwei; chen, jindong; zhao, jingping; wang, xiaoping; wu, renrong title: care for the psychological status of frontline medical staff fighting against covid-19 date: 2020-04-03 journal: clin infect dis doi: 10.1093/cid/ciaa385 sha: doc_id: 284782 cord_uid: 51mbq7qb nan according to preliminary estimates, there were more than 80,000 medical workers in 131 covid-19 designated hospitals, more than 170,000 medical workers in hubei province fighting against covid-19. besides, a total of 6, 097 medical workers from different provinces in china were in hubei province to assist in the covid-19 by jan 30, 2020 2 . more medical teams were then sent out to the hardest-hit areas in hubei province, including rescue and medical teams from different provinces, the army medical teams, specialized doctors and nurses' teams, and emergency medical teams from different hospitals. for example, the emergency medical team from the second xiangya hospital is able to set up field hospitals and ambulances to help and serve the with the increasing spreading of the virus worldwide, covid-19 outbreak is now becoming a pandemic. while there are many challenges and struggles with covid-19, we know that we are engaged and prepared for the fight. with the huge efforts of mental health care for medical professionals, patients and others affected by the outbreak, we hope this can bring courage, confidence, and willpower to these fighters, and the resulting the end the outbreak comes soon. none of the authors has any potential conflicts of interest to disclose. timely mental health care for the 2019 novel coronavirus outbreak is urgently needed the immediate psychological and occupational impact of the 2003 sars outbreak in a teaching hospital protecting health care workers during the covid-19 coronavirus outbreak -lessons from taiwan's sars response severe acute respiratory syndrome (sars) in hong kong in 2003: stress and psychological impact among frontline healthcare workers key: cord-261270-jkm9c5yv authors: annweiler, cédric; sacco, guillaume; salles, nathalie; aquino, jean-pierre; gautier, jennifer; berrut, gilles; guérin, olivier; gavazzi, gaetan title: national french survey of covid-19 symptoms in people aged 70 and over date: 2020-06-18 journal: clin infect dis doi: 10.1093/cid/ciaa792 sha: doc_id: 261270 cord_uid: jkm9c5yv the objective of this national french survey was to determine the covid-19 semiology in seniors(n=353; mean,84.7±7.0y). 57.8% of patients exhibited ≤3symptoms, including thermal dysregulation(83.6%), cough(58.9%), asthenia(52.7%), polypnea(39.9%), gastrointestinal signs(24.4%). patients≥80y exhibited falls(p=0.002) and asthenia(p=0.002). patients with neurocognitive disorders exhibited delirium(p<0.001) and altered consciousness(p=0.001). clinical peculiarities of covid-19 were reported in seniors. since december 2019, the covid-19 caused by sars-cov-2 is spreading worldwide from china, affecting millions of people. although older adults do not appear more prone than younger ones to be infected, they are more at risk of developing severe and lethal forms of covid-19 [1] [2] [3] . the core question is thus to properly discuss the diagnosis of covid-19 in older patients. it is commonly admitted that the semiology of older adults differs from that encountered in younger ones. changes in the clinical expression of the diseases and difficulties in interpreting the clinical signs in older patients could blur the diagnosis process. if these peculiarities were also retrieved with covid-19, it could be the cause of delayed diagnosis among older patients, responsible for delayed care and isolation measures with subsequent higher risk of virus propagation. the objective of this national french survey was to describe and identify the symptoms most frequently encountered in people aged 70 and over diagnosed with covid-19. this cross-sectional study was conducted by the french society of geriatrics and gerontology (sfgg). an online standardized questionnaire was sent by email to all sfgg members and widely communicated through the professional networks in geriatrics and infectious diseases. physicians were asked to report, between 22 march and 5 april 2020, their last 10 patients aged ≥70years with confirmed sars-cov-2 infection (as defined as a positive rt-pcr test result). those who had treated less than 10 diagnosed patients were asked to submit a questionnaire for each of them. the study was conducted in accordance with the ethical standards set forth in the helsinki declaration (1983) , was declared to the national commission for information a c c e p t e d m a n u s c r i p t 5 technology and civil liberties (ar20-0031v1), and was registered on clinicaltrials.gov under number nct04343781. the following characteristics were collected for each patient: demographic (age, gender, place of life, place of care, most recent disability score according to the iso-resource group) [4] , medical history (major neurocognitive disorders [mnd], hypertension, diabetes mellitus, asthma or chronic obstructive pulmonary disease (copd), cardiomyopathy, severe chronic renal failure defined as creatinine clearance under 30ml/min, solid or hematological cancer). the following symptoms observed within the first 72 hours of sars-cov-2 infection (i.e., 72h from suspicion, possibly before diagnostic confirmation by rt-pcr test) were collected for each patient using yes/no questions: general signs (sudden deterioration of general condition, temperature, blood pressure), respiratory signs (cough, polypnea), ear nose and throat (ent) signs (rhinorrhea, odynophagia, otalgia, conjunctivitis, dysgeusia or ageusia, anosmia), gastrointestinal signs (diarrhea, nausea or vomiting) and geriatric syndromes (falls, hypo or overactive delirium, altered consciousness). changes in complete blood count (leukopenia, lymphopenia, thrombocytopenia) were also collected, with details when available. qualitative variables were described using numbers and percentages, and quantitative variables using means and standard deviations. comparisons between participants aged ≥80years and <80years, and between those with and without mnd, were performed using chi² test for qualitative variables (or exact fisher test where appropriate), and student t test for quantitative variables (or mann-whitney u test where appropriate). univariate logistic regressions were conducted to determine the association of each covid-19 sign with age≥80years and history of mnd. finally, the profiles of covid-a c c e p t e d m a n u s c r i p t 6 using a multiple correspondence analysis (mca). two-sided p-values<0.05 were considered significant. analyses were performed with sas® (sas institute inc.; v9.4) and r (r core team 2020; v3.6.3) using the factominer and factoshiny packages. older patients' characteristics are presented in table 1 finally, the mca results distinguished between two profiles of older patients. the first profile matched with patients under age 80 without mnd, who exhibited more frequent hyperthermia and cough during the first 72h of the infection, but no fall, altered a c c e p t e d m a n u s c r i p t 7 consciousness or hypoactive delirium. in contrast, the second profile matched with patients aged 80 and over with mnd; the latter exhibiting more frequently no specific symptoms, and most often an absence of hyperthermia, polypnea, cough and dysgeusiaageusia. this national french survey shows that older adults with covid-19 exhibit a paucisymptomatic clinical picture with less than 3 signs during the first 72h of the infection, generally combining general and respiratory signs (e.g. hyperthermia and cough) with peculiarities that should alert the clinician (e.g. sudden deterioration of general condition, diarrhea, lymphopenia, and/or geriatric syndromes including falls and delirium). various clinical profiles were highlighted across older adults, especially among the oldest-old ≥80years and those with chronic diseases such as mnd. our survey provides the first description of the covid-19 signs in older, and even oldest-old, adults with comorbidities [1] [2] [3] . compared to previous meta-analyses in younger adults [5] [6] [7] , we found that older adults with covid-19 often exhibit thermal dysregulation, which however results less often in hyperthermia (56% here versus 82% [5] to 91% [6] in younger adults) and more often in subfebrile temperatures or alternations of hyperthermia and hypothermia (not described thus far to our knowledge). the prevalence of cough was similar (59% here versus 61% [5] to 72% [7] in younger adults). in contrast, the sudden deterioration of general condition, mostly illustrated by marked asthenia, was particularly frequent in older adults (53% here versus 36% [5] to 51% [6] in younger adults). also, older adults exhibited more often dyspnea (40% here versus 26% [5] to 30% [6] in younger adults) and gastrointestinal signs (24.4% here a c c e p t e d m a n u s c r i p t 8 with mostly diarrhea (21.8%) versus 10% in younger adults [5, 8] ). this should encourage clinicians to integrate the gastrointestinal signs into the diagnostic reasoning for sars-cov-2 infection in older adults. older adults had less often anosmia (2% here versus 86% in younger adults [9]) and dysgeusia-ageusia (7% here versus 89% in younger adults [9] ). the latter prevalence should however be cautiously interpreted due to olfactory and gustatory dysfunctions with advancing age [10]. finally, we found a higher proportion of lymphopenia in older adults compared to the general population (75% here versus 55% [3] ). the lymphopenia was more significant than that usually observed in the normal aging population (750/mm 3 versus 1432/mm 3 in the literature in conclusion, this national french survey revealed that the clinical picture of older adults with covid-19 includes both general and respiratory signs like in younger adults (e.g. hyperthermia and cough), but also more peculiar features such as marked asthenia, diarrhea, lymphopenia and geriatric syndromes. we also reported various clinical profiles across older adults, notably in those aged 80 years and over and those with a history of mnd who appeared particularly pauci-or asymptomatic during the first 72h of the infection. these findings should be integrated into the clinical reasoning in geriatric medicine, and encourage the systematization of diagnostic tests for sarsa c c e p t e d m a n u s c r i p t -ca has full access to all of the data in the study, takes responsibility for the data, the analyses and interpretation and has the right to publish any and all data, separate and apart from the attitudes of the sponsors. all authors have read and approved the manuscript. -study concept and design: ca, gs, ns, gb, og and gg. the study was conducted in accordance with the ethical standards set forth in the a c c e p t e d m a n u s c r i p t 13 helsinki declaration (1983). the study protocol was declared to the national commission for information technology and civil liberties (cnil) under the number ar20-0031v1, and was registered on clinicaltrials.gov under number nct04343781. patient level data are freely available from the corresponding author at cedric.annweiler@chu-angers.fr. there is no personal identification risk within this anonymized raw data, which is available after notification and authorization of the competent authorities. symptoms of covid-19 among older adults: systematic review of biomedical literature coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up clinical characteristics and outcomes of older patients with coronavirus disease 2019 (covid-19) in wuhan, china (2019): a single-centered, retrospective study novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis clinical characteristics of hospitalized patients with sars-cov-2 infection: a single arm meta-analysis epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms leukocytes°/°mm 3 (n=17), mean ± sd 2 394 ± 881 2613±1174 copd: chronic obstructive pulmonary disease; ent: ear nose and throat; gir: iso resource group; n: total number of patient included in the study; n: number of patients according to the considered group; sd: standard deviation; y: years; *: comparisons based on chi2 test or exact fisher test for qualitative variables, and student t test or mann-whitney u test for quantitative variables, as appropriate; †: 27 missing data; ‡: 2 missing data; §: 22 missing data; ¶: 20 missing data; |: 18 missing data the authors wish to thank all participants and services for their cooperation; melinda all authors state that they have no conflicts of interest with this paper. the authors have no relevant personal financial interest in this manuscript.a c c e p t e d m a n u s c r i p t key: cord-280954-wd89nka9 authors: ackerson, bradley; tseng, hung fu; sy, lina s; solano, zendi; slezak, jeff; luo, yi; fischetti, christine a; shinde, vivek title: severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults date: 2019-07-15 journal: clin infect dis doi: 10.1093/cid/ciy991 sha: doc_id: 280954 cord_uid: wd89nka9 background: respiratory syncytial virus (rsv) is an important cause of serious respiratory illness in older adults. comparison of rsv and influenza infection in hospitalized older adults may increase awareness of adult rsv disease burden. methods: hospitalized adults aged ≥60 years who tested positive for rsv or influenza between 1 january 2011 and 30 june 2015 were identified from kaiser permanente southern california electronic medical records. baseline characteristics, comorbidities, utilization, and outcomes were compared. results: the study included 645 rsvand 1878 influenza-infected hospitalized adults. patients with rsv were older than those with influenza (mean, 78.5 vs 77.4 years; p = .035) and more likely to have congestive heart failure (35.3% vs 24.5%; p < .001) and chronic obstructive pulmonary disease (copd) (29.8% vs 24.3%; p = .006) at baseline. in adjusted analyses, rsv infection was associated with greater odds of length of stay ≥7 days (odds ratio [or] = 1.5; 95% confidence interval [ci], 1.2–1.8; p < .001); pneumonia (or = 2.7; 95% ci, 2.2–3.2; p < .001); intensive care unit admission (or = 1.3; 95% ci, 1.0–1.7; p = .023); exacerbation of copd (or = 1.7; 95% ci, 1.3–2.4; p = .001); and greater mortality within 1 year of admission (or = 1.3; 95% ci, 1.0–1.6; p = .019). conclusions: rsv infection may result in greater morbidity and mortality among older hospitalized adults than influenza. increased recognition of adult rsv disease burden will be important in the evaluation and use of new rsv vaccines and antivirals. respiratory syncytial virus (rsv) initially was found to be a common cause of severe respiratory illness in young children [1, 2] . while mild disease in healthy young adults has also been described [3] , the potential for rsv to cause severe disease in adults was not recognized until reports of outbreaks among older adults residing in long-term care facilities emerged [4, 5] . subsequently, rsv was shown to cause serious illness among healthy, community-dwelling older adults. a landmark study of high-risk and community-dwelling adults aged ≥65 years and high-risk adults in the united states detected rsv infection in 3%-7% of healthy, community-dwelling older adults, 4%-10% of high-risk adults, and 16% of adults hospitalized with cardiopulmonary infections [6] . furthermore, rsv infection was estimated to result in approximately 177 000 hospitalizations, 10 000 to 14 000 deaths, and more than $1 billion dollars in healthcare costs per year in the united states, mostly among older adults [6, 7] . nonetheless, rsv remains clinically underrecognized by most healthcare providers as a cause of severe respiratory disease in adults [7] [8] [9] . a current comparison of the clinical characteristics and burden of rsv illness with those of influenza, a well-recognized cause of severe morbidity and mortality in older adults [10] , may increase provider awareness of the potential of rsv infection to cause severe lower respiratory tract disease in older adults. appreciation of the potential for rsv infection to cause serious illness similar to that caused by influenza will likely trigger more testing for rsv in addition to testing for influenza and direct therapy to the identified etiology in patients as newer agents become available. in addition, recent data on the epidemiology and clinical outcomes of older adults hospitalized with rsv are critical to the evaluation of more than 50 rsv vaccines, monoclonal antibodies, and antivirals currently under development prior to their introduction [11] . while a number of important studies have described the burden and clinical characteristics of rsv and influenza disease in hospitalized older adults, most of these studies were conducted at least a decade ago [6, [12] [13] [14] [15] . furthermore, longterm mortality has not been analyzed previously. therefore, we evaluated a large cohort of adults aged ≥60 years hospitalized with rsv or influenza a/b infection during 5 consecutive seasons. demographics, prehospitalization characteristics, hospital utilization, and clinical outcomes, including long-term mortality, were assessed and compared between adults with rsv infection and those with influenza virus infection. this observational, retrospective, cohort study was conducted at kaiser permanente southern california (kpsc), an integrated healthcare system that provides comprehensive prepaid health services for 4.4 million members cared for at 15 large, community-based hospitals that are owned and operated by kpsc. the demographic makeup of the kpsc membership closely mirrors the racially and sociodemographically diverse southern california population and the california census population; more than 99% are community dwelling [16] . as kpsc is a prepaid healthcare system, there is a very strong motivation for members to use services internally. furthermore, reimbursement by kpsc for outside care requires that claims be submitted with documentation of the care provided; claims are subsequently entered into the administrative data systems. thus, capture of care delivered to kpsc members by electronic data is very comprehensive. the study population consisted of hospitalized kpsc members aged ≥60 years at the time of admission who had a positive rsv or influenza a/b laboratory test (multiplex polymerase chain reaction [pcr] [filmarray, luminex xtag], influenza a/b/ h1n1 [cepheid xpert flu], or viral culture) that was ordered between 1 january 2011 and 30 june 2015 during a hospitalization encounter or an emergency department encounter that resulted in hospitalization at 1 of 15 kpsc hospitals. all samples were submitted to a single, centralized kpsc laboratory for processing. participants were required to have continuous kpsc membership for at least 6 months (allowing a 31-day gap) prior to the hospital admission date to allow for ascertainment of baseline characteristics. if there were patients with multiple hospitalizations who met the above criteria (eg, had a positive rsv or influenza a/b test on 2 separate hospitalization encounters), the first rsv or first influenza hospitalization was kept for the rsv or influenza cohort, respectively. for the purpose of statistical comparison, independent samples of rsv and influenza cohorts were defined. twenty-five individuals with both a positive rsv test and a positive influenza test during different hospitalizations during the study period were kept in the rsv cohort and removed from the influenza cohort. nineteen individuals coinfected with rsv and influenza during the same hospitalization were excluded from the comparison. the kpsc institutional review board reviewed and approved the study. electronic medical records were used to obtain information for the study population, including membership history, baseline characteristics, demographics, comorbidities, treatments, utilization, and outcomes. each kpsc health plan member has a unique medical record number that is used as an identifier to retrieve and link all variables from different databases. outcomes of hospitalization included length of stay, occurrence of complications, use of respiratory support, intensive care unit (icu) admission, readmission, use of vasopressors, mortality, exacerbation of chronic conditions, discharge location, and healthcare utilization after discharge. patient demographics, prehospital course, comorbid conditions, and in-hospital utilization were compared between patients hospitalized with rsv and those hospitalized with influenza a/b using a χ 2 test, fisher exact test, t test, or wilcoxon ranksum test, as appropriate. categorical factors were presented using frequencies and percentages. continuous variables were categorized based on distribution and described using mean, standard deviation, and range, in addition to frequencies and percentages. multivariable logistic regression was used to estimate the odds ratio (or) and 95% confidence interval (ci) associated with hospitalization outcomes between those infected with rsv and those infected with influenza after adjustment for potential differences between cohorts, including age, sex, race/ethnicity, history of influenza and pneumococcal vaccination, prior chronic obstructive pulmonary disease (copd)/chronic bronchitis/emphysema, charlson comorbidity index [17] , history of healthcare utilization, and recent use of antivirals, antibiotics, or steroids. one-year survival after admission was estimated using the kaplan-meier method, and survival rates between hospitalized persons infected with rsv or influenza were compared using the log-rank test. the analyses were performed using sas enterprise guide 5.1 (sas institute inc, cary, nc). among patients hospitalized with rsv (n = 645) during the study period, rsv infection was detected by multiplex pcr in 91.8% and by culture in 8.2% of patients. among patients hospitalized with influenza (n = 1878) during the study period, influenza infection was detected by multiplex pcr in 82.6%, by influenza a/b/h1n1 pcr in 0.05%, and by culture in 17.4% of patients. the distribution of demographic characteristics, medical history, and comorbidities of patients hospitalized with rsv (n = 645) or with influenza (n = 1878) is presented in table 1 . patients hospitalized with rsv infection were slightly older (mean age, 78.5 vs 77.4 years; p = .035), with a higher proportion of those aged ≥85 years compared to those hospitalized with influenza infection. there was a higher proportion of females in the rsv cohort than in the influenza cohort (60.5% vs 50.3% female; p < .001). race/ethnicity distributions were similar between the rsv and the influenza cohorts. a higher proportion of rsv patients than influenza patients had received influenza vaccine in the 1 year prior to admission (83.1% vs 73.1%; p < .001). smoking status and body mass index were similar between cohorts. the prevalence of several comorbidities was significantly different between patients hospitalized with rsv and those with influenza, including congestive heart failure (35.3% vs 24.5%; p < .001), diabetes (38.9% vs 44.6%; p = .012), copd/chronic bronchitis/emphysema (29.8% vs 24.3%; p = .006), asthma (26.0% vs 18.6%; p < .001), any solid cancer (11.2% vs 8.0%; p = .016), and leukemia (1.9% vs 0.9%; p = .050). use of any antiviral therapy, including oseltamivir, within 14 days prior to admission was not common but was more prevalent in the rsv cohort than the influenza cohort (4.3% vs 2.7%; p = .034 for any antiviral medication and 2.3% vs 1.1%; p = .026 for oseltamivir; data not shown). the in-hospital characteristics of the rsv and influenza cohorts are described in table 2 . the mean time from admission to the first positive test result for either rsv or influenza was similar between the 2 cohorts (2.5 vs 2.6 days; p = .570), and approximately 98% of patients in either cohort did not have test results available until some time after the hospital admission date. among the rsv and influenza cohorts, respectively, use of antivirals (47.1% vs 78.6%; p < .001), 99% of which was oseltamivir (data not shown); antibiotics (94.1% vs 88.9%; p < .001); and any inhalation, injection, intravenous, or oral steroid (64.5% vs 47.9%; p < .001) during the hospitalization period was significantly different. in the adjusted analyses, the odds of all outcomes were similar or significantly higher among participants hospitalized with rsv compared to those hospitalized with influenza ( figure 1 ). hospital utilization was greater among those infected with rsv than with influenza as measured by length of stay ≥7 days among the entire population (or = 1.4; 95% ci, 1.2-1.7), length of stay ≥7 days among survivors (or = 1.5; 95% ci, 1.2-1.8), and icu admission (or = 1.3; 95% ci, 1.0-1.7). in addition, respiratory complications during hospitalization were more common among rsv-infected older adults including pneumonia diagnosis (or = 2.7; 95% ci, 2.2-3.2); highest measured respiratory rate (rr; measured by breaths per minute; rr ≥26 vs ≤22 reference group [or = 1.5; 95% ci, 1.2-1.9]); lowest oxygen saturation (≤84% vs ≥93% reference group [or = 1.6; 95% ci, 1.2-2.1]); greatest level of oxygen supplementation (≥16 l/min vs ≤5 l/min reference group [or = 1.5; 95% ci, 1.0-2.2]); exacerbation of copd, chronic bronchitis, or emphysema (or = 1.7; 95% ci, 1.3-2.4); and exacerbation of asthma (or = 1.5; 95% ci, 1.1-1.9). more older adults infected with rsv than with influenza required home health service after discharge (or = 1.3; 95% ci, 1.0-1.6). while mortality during hospitalization (or = 1.1; 95% ci, 0.8-1.7) and within 6 months of admission (or = 1.2; 95% ci, 0.9-1.5) for patients with rsv did not differ significantly from those with influenza, long-term mortality within 1 year of admission was significantly greater in the rsv cohort compared to the influenza cohort (or = 1.3, 95% ci, 1.0-1.6). the 1-year survival rate after admission was significantly lower (figure 2 , 74.2% vs 81.2%; p < .001) in older adults hospitalized with rsv compared to those hospitalized with influenza. this retrospective cohort study that spans 5 consecutive winters demonstrates that rsv is an important cause of serious and life-threatening respiratory illness that results in morbidity and mortality among older adults that were comparable or more severe than those caused by influenza. to our knowledge, this study describes the largest and most recent cohort of older adults hospitalized with rsv or influenza reported to date. by utilizing patient electronic health records, we were able to obtain important information, including demographics and comorbidities prior to hospitalization and short-and long-term healthcare utilization and hospitalization outcomes. consistent with earlier reports, we found that adults hospitalized with rsv were slightly older and had greater frequency of baseline comorbidities, particularly cardiopulmonary disease, than those hospitalized with influenza virus infection [6, 12, 14] . however, after adjustment for comorbidities, we found that hospitalization outcomes among older adults hospitalized with rsv were either comparable to or significantly worse than they were among those hospitalized with influenza. while previous studies found similar hospital length of stay among older adults hospitalized with rsv or with influenza [6, 14] , we found that longer hospitalizations (length of stay ≥7 days) occurred more often among the rsv cohort than they did in the influenza cohort. this may reflect increased use of antiviral therapies in recent years that ameliorate influenza but not rsv disease [18] or differences in clinical recovery associated with a more pronounced rsv-associated severe lower respiratory tract syndrome. we also found that pulmonary complications and findings, including pneumonia, tachypnea, hypoxia, and greater need for oxygen supplementation, and exacerbation of asthma and of copd, chronic bronchitis or emphysema were significantly greater among the rsv cohort after adjustment for potential confounders. these findings are similar to those of lee et al. who reported that hospitalized adults with rsv in hong kong had a greater frequency of lower respiratory complications than those with influenza [14] . while increased baseline cardiopulmonary disease in the rsv cohort likely increased their risk of pulmonary complications, including exacerbation of chronic respiratory disease and asthma, these differences were observed after adjustment for comorbidities, including baseline cardiopulmonary disease. discharge to hospice and skilled nursing facilities was similar in the rsv and influenza cohorts, but a greater proportion of the rsv cohort required home health services after discharge. finally, similar to previous findings [6, 14] , short-term mortality for patients infected with rsv was similar to that of patients infected with influenza. however, longer-term survival among the rsv cohort, which has not been reported previously, was significantly worse than that of the influenza cohort. the impact of rsv on long-term survival may be a reflection of the potential for rsv to drive chronic inflammation associated with persistent, sometimes progressive, pulmonary disease, although the mechanism for this is not clear [19] [20] [21] [22] . in addition to confirming the severe impact of annual influenza epidemics, our findings also underscore the substantial morbidity, mortality, and healthcare utilization associated with rsv infection in the expanding population of older adults and the need for rsv vaccines and antivirals. interestingly, influenza vaccine uptake within 1 year of admission was higher among the rsv cohort than the influenza cohort while receipt of pneumococcal vaccine was similar. this may reflect, in part, the effectiveness of influenza vaccine in preventing influenza infections and is unlikely due to differential vaccine acceptance. on the other hand, the increased use of oseltamivir within 14 days prior to hospitalization among older adults hospitalized with rsv compared to those hospitalized with influenza may reflect presumed influenza infection and delay in diagnosis of rsv infection. conversely, decreased in-hospital use of antiviral therapy among the rsv cohort compared to the influenza cohort likely reflects the discontinuation of oseltamivir after identification of rsv and initiation of oseltamivir after identification of influenza. the greater use of antibiotics and systemic and inhaled steroids in the rsv cohort during their hospitalizations may mirror the higher frequency of pneumonia and exacerbation of chronic lung disease and asthma among older hospitalized adults with rsv infection than those with influenza infection. whereas rsv has long been recognized as a cause of serious illness among young children, its role as a significant pathogen among older adult patients appears to be underrecognized by many adult providers [7] [8] [9] . this is understandable since in many settings, respiratory pathogen testing is limited to influenza virus testing, so that noninfluenza pathogens, including rsv, have not been recognized as possible etiologies of serious respiratory illness similar to that caused by influenza. in addition, even in settings in which multiplex pathogen testing is used, local circulation of influenza virus in the community and clinical suspicion for influenza may drive testing patterns among hospitalized older adults and, thus, may underestimate the relative frequency of other viral respiratory pathogens, including rsv, which may have either nonoverlapping or broader periods of circulation in the same community. therefore, the number of hospitalizations with rsv or with influenza detected in this study may not reflect the relative incidences of each pathogen over the study period since rsv infections that occur during intervals when influenza virus is less prevalent are less likely to be detected. increased provider recognition of rsv as an important cause of severe influenza-like illness among older adults and other high-risk populations will become increasingly important in order to increase vaccine uptake among high-risk populations after rsv vaccines are developed. in addition, recognition and early identification of rsv as a potential cause of illness that is sometimes indistinguishable from influenza [23, 24] will be important in the timely implementation of diagnostic testing, adjustment of antiviral medication administration, including early discontinuation of medications targeted to influenza, and initiation of medications effective against rsv as they become available in order to optimize effectiveness and to minimize cost and complications. the strengths of this study include detailed clinical and health outcomes data extracted from a large patient sample drawn from a racially and socioeconomically diverse population receiving care at multiple medical centers over 5 seasons. in addition, the widespread inpatient use of a sensitive multiplex pcr to detect rsv and influenza allowed for the possible detection of a wide spectrum of both diseases [6] . moreover, since these test results were not immediately available, initial medical management decisions, including hospital admission and treatment decisions, were made based on severity of clinical symptoms rather than virologic diagnosis, minimizing bias resulting from differential admission or management plans for different infections. rsv is a cause of serious illness among hospitalized older adults with morbidity and mortality that may be even more substantial than that caused by influenza, with possibly greater impact on long-term survival. the projected continued increase in the proportion of older adults in the population [25] underscores the growing unmet medical and public health need to develop vaccines and therapeutics directed against rsv. increased appreciation among adult providers of the frequency and severity of rsv disease among adults will be important to the uptake of rsv vaccines and antivirals after they are introduced. recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization respiratory syncytial virus and parainfluenza virus natural reinfection of adults by respiratory syncytial virus. possible relation to mild upper respiratory disease an outbreak of respiratory syncytial virus infection in an old people's home an outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly respiratory syncytial virus infection in elderly and high-risk adults mortality associated with influenza and respiratory syncytial virus in the united states rsv in adult ed patients: do emergency providers consider rsv as an admission diagnosis? respiratory syncytial virus infection in older adults: an under-recognized problem excess pneumonia and influenza associated hospitalization during influenza epidemics in the united states, 1970-78 identifying gaps in respiratory syncytial virus disease epidemiology in the united states prior to the introduction of vaccines rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults vaccine safety datalink adult working group. influenza-and rsv-associated hospitalizations among adults high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections respiratory syncytial virus and influenza a infections in the hospitalized elderly sociodemographic characteristics of members of a large, integrated health care system: comparison with us census bureau data a new method of classifying prognostic comorbidity in longitudinal studies: development and validation oseltamivir prescribing in pharmacy-benefits database detection of respiratory syncytial virus in adults with chronic obstructive pulmonary disease respiratory syncytial virus-induced pulmonary disease and exacerbation of allergic asthma antiviral immune responses and lung inflammation after respiratory syncytial virus infection respiratory syncytial virus, airway inflammation, and fev1 decline in patients with chronic obstructive pulmonary disease respiratory syncytial virus infection in adults is clinical recognition of respiratory syncytial virus infection in hospitalized elderly and high-risk adults possible? the nation's older population is still growing, census bureau reports acknowledgments. the authors acknowledge songyue chen for his contribution to programming. the authors thank the patients of kaiser permanente for helping us improve care through the use of information collected via our electronic health record systems. key: cord-007050-ibmr5bev authors: xu, wei; lu, lu; shen, bo; li, jun; xu, jianqing; jiang, shibo title: serological investigation of subclinical influenza a(h7h9) infection among healthcare and non–healthcare workers in zhejiang province, china date: 2013-09-15 journal: clin infect dis doi: 10.1093/cid/cit396 sha: doc_id: 7050 cord_uid: ibmr5bev nan infection with avian influenza a(h7n9) virus, including 37 deaths [1] . zhejiang province is the most affected area, where 46 cases were reported. poultry workers are at the highest risk for a(h7n9) infection in china [2] [3] [4] . however, the retrospective serologic study provided no evidence for a(h7n9) infection in poultry workers before november 2012 in eastern china [5] . another high-risk population is the healthcare workers who may have close contacts with patients with symptomatic or asymptomatic a(h7n9) infection. a number of healthcare workers were infected, through contacts with patients, by middle east respiratory syndrome coronavirus (mers-cov), a novel human coronavirus causing outbreaks of severe acute respiratory syndrome (sars)-like illness in middle east and europe, although there is no confirmed evidence of efficient human-to-human transmission of mers-cov, like sars-cov [6] . here, we investigated the possible subclinical or asymptomatic infection with a(h7n9) virus among the healthcare workers in zhejiang province during the outbreak of a(h7n9) in 2013 by detecting the prevalence of anti-h7n9 antibodies in the leftover serum samples from 126 healthy healthcare workers and 615 healthy non-healthcare workers (as controls) in the cities of taizhou, hangzhou, and shaoxing in zhejiang province, who underwent regular physical examinations between 1 april 2013 and 1 may 2013. a hemagglutination inhibition (hi) assay using guinea pig red cells and pseudoviruses expressing hemagglutinin of a representative a(h7n9) strain (a/hangzhou/ 1/2013) was conducted for determining the hi titers of the serum samples, as previously described [7] . we found that none of the serum samples from the healthcare and nonhealthcare workers contained h7-specific antibodies with hi titers of ≥20, whereas the hi titer of the serum sample from a patient with laboratory-confirmed a (h7n9) infection was ≥40. hi titers of 10 were detected in 3 of 123 serum samples (2.4%) from healthcare workers and 9 of 585 serum samples (1.5%) from the nonhealthcare workers. however, there was no significant difference between these 2 groups (table 1 ). no significant difference was observed between the male and female groups and among different age groups in who had hi titer ≥10 ( table 1 ). the results suggest that none of our tested healthcare and non-healthcare workers in zhejiang province may have subclinical or asymptomatic a(h7n9) infection, because none of the serum samples contains h7-specific antibodies with hi titer ≥20. these results also implied that a(h7n9) might not be able to cause asymptomatic or subclinical infection. to the best of our knowledge, no case of asymptomatic a(h7n9) infections have been reported so far. this also suggests that people may be unable to acquire h7n9-specific immunity through asymptomatic or subclinical a(h7n9) infection, which may explain why people lack population immunity against a (h7n9) viruses. however, more extensive serological investigation on the asymptomatic or subclinical a(h7n9) infection among different high-risk groups at different regions should be performed. a serum sample with a hemagglutination inhibition (hi) titer of ≥40 from a patient with influenza a(h7n9) infection admitted to the shanghai public health clinical center (shaphc) was included as a control in the hi assay. the study was reviewed and proved by the ethics committee of shaphc, and written informed consent was provided to the participants. human infection with avian influenza a(h7n9) virus in china: update human infection with a novel avian-origin influenza a (h7n9) virus origin and diversity of novel h7n9 avian influenza viruses causing human infection human infections with the emerging avian influenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome serologic study for influenza a (h7n9) among high-risk groups in china novel coronavirus infection-update (middle east respiratory syndrome-coronavirus) receptor specificity and erythrocyte binding preferences of avian influenza viruses isolated from india contributed equally to this work. correspondence: shibo jiang, md, phd, shanghai medical college, fudan university, 130 dong an road on behalf of the infectious diseases society of america. all rights reserved. for permissions key: cord-266808-wyuodzyt authors: nagler, arielle r; goldberg, eric r; aguero-rosenfeld, maria e; cangiarella, joan; kalkut, gary; monahan, carolyn rooke; cerfolio, robert j title: early results from sars-cov-2 pcr testing of healthcare workers at an academic medical center in new york city date: 2020-06-28 journal: clin infect dis doi: 10.1093/cid/ciaa867 sha: doc_id: 266808 cord_uid: wyuodzyt covid-19 rt-pcr employee-testing was implemented across nyu langone. over eight-weeks, 14,764 employees were tested: 33% of symptomatic employees, 8% of asymptomatic employees reporting covid-19 exposure, 3% of employees returning to work were positive. positivity rates declined over time possibly reflecting the importance of community transmission and efficacy of ppe. a c c e p t e d m a n u s c r i p t a novel coronavirus, sars-cov-2, was identified in late 2019 in wuhan, china, and rapidly became a pandemic. new york quickly became the american epicenter. [1] hcw are at the frontline in the fight against covid-19 and are at increased risk for infection. in an early review of 139 hospitalized covid-19 patients in china, 41% had nosocomial covid-19. [2] as a group at high-risk for exposure, widespread testing for hcw has been proposed in the literature. [3] although specifically designed for symptomatic testing, rt-pcr can be used to detect covid-19 infection in both symptomatic and asymptomatic individuals. identifying both asymptomatic and symptomatic hcw can promote a safe environment within the healthcare system as asymptomatic carriers can transmit covid-19. [4, 5] nyulh is an academic medical center encompassing four hospital campuses in manhattan, brooklyn, and long island and over 250 ambulatory sites, with approximately 43,000 employees. at the center of the hardest hit region in the united states, from march 14 to may 18, 5,767 covid-19 patients were admitted to nyulh. herein we describe the widespread employee covid-19 testing program that was initiated at nyulh to promote a safe and informed environment for employees and patients. on march 25, 2020, nyulh began a dedicated, on-demand covid diagnostic program for employees. over the following eight weeks three groups were tested: 1) symptomatic staff with fever or respiratory illness; 2) asymptomatic employees with self-reported exposure to covid-19 which included any degree or duration of contact with an individual who had a documented covid-19 infection in the work place or in the community; and 3) all employees who were returning to work in services that had been suspended during the epidemic's peak. this study was exempt from irb review. a c c e p t e d m a n u s c r i p t employees meeting criteria for testing contacted a call center which screened employees for testing eligibility. the request for testing was forwarded through epic® to a pool of physicians and nurse practitioners who ordered the covid-19 rt-pcr. using mychart, the nyulh patient portal, the employee then scheduled the test at one of nyulh's testing centers. nyulh developed three testing centers in former conference rooms, building testing cubicles for specimen collection that were under negative pressure. samples were obtained by nurses in ppe (n95 respirators, face shields, and impermeable gowns). nasopharyngeal samples were obtained by inserting a swab into the anterior nares until reaching the nasopharynx and rotating the swab for several seconds. samples were placed in tubes containing viral transport medium and were submitted to the hospital laboratory. detection of sars-cov-2 rna using real time rt-pcr was performed on the roche fully automated cobas 6800 system under emergency use authorization. results were reported as detected (positive) or not detected (negative). the sars-cov-2 results interfaced into epic® and were automatically released to employees through mychart. simultaneously, employee rt-pcr results returned to the ordering-provider pool. positivity rates amongst all groups of employees being tested including symptomatic employees, asymptomatic employees with self-reported exposure to covid-19, and employees being screened for returning work declined over time ( figure 1 ). overall, for symptomatic employees, the positivity rate was 33%, but the positivity rate declined from 51% in the first week of testing (march 25-march 31) to 3% in the most recent week of testing starting may 13 th . the overall positivity rate of asymptomatic employees with self-reported exposures was 8%, dropping from 12% in the first week of testing (april 1-april 7) to 0% during the most recent week of testing starting may 13th. all asymptomatic staff who were returning to work in re-opening services were tested starting on april 20th had a mean positivity rate of 3% (figure 1 ). in the first week of testing 5% of these employees were positive, but in the most recent week of testing 0% were positive. a c c e p t e d m a n u s c r i p t hcw are at increased risk for exposure to persons with covid-19. nyulh began a dedicated, ondemand covid-19 diagnostic program for employees in late march 2020. over the following eight weeks, nearly 15,000 symptomatic and asymptomatic employees were tested. testing results were used to guide treatment, self-isolation, and to reinforce the rigorous ppe standards used during covid-patient care. this comprehensive testing program helped to maintain and manage the workforce. even amongst the symptomatic employees, over 67%of those tested were covid-19 negative, enabling these workers to go back to work after being fever-free for 72-hours rather than being isolated for a minimum of 7-10 days. similarly, in one british study, 87% of the 1,654 symptomatic hcw tested were negative and were able to return to work more quickly. [6] thus, in addition to providing comfort and reassurance to employees who test negative, broad testing programs help to maintain the workforce. testing of employees and patients is also a critical step to safely re-open essential non-covid clinical services. staff and patients want reassurance that healthcare systems are taking all possible steps to exclude persons with active infection from the healthcare environment. while covid-free environments simply cannot be guaranteed, our goal is to use all current tools, including diagnostic covid-19 testing for patients and staff, daily symptomatic checks, and mandatory use of ppe in all clinical settings, to minimize exposure to covid for staff and patients. while, in the future, we expect that covid antibody testing will be utilized within the healthcare work force; currently antibody testing is not being employed at nyulh as antibody status does not influence or change our policy for patients or staff. widespread community transmission existed in metropolitan new york starting in mid-march. although the nyulh covid-19 patient census peaked in early april, throughout the duration of the nyulh employee testing program, the covid patient census remained consistently greater than 500 and the vast majority of the care delivered at nyulh was direct covid-19 patient care. thus during this testing program, most nyulh employees had continued exposure to covid patients, yet a c c e p t e d m a n u s c r i p t employees still demonstrated sharp declines in covid infection rates. ppe and infection control standards and procedures are unlikely to account for this decline as they remained comprehensive, stringent, and largely unchanged since the onset of the pandemic in early march. notably, however, the reduction in employee covid positive rates is coincident with the decline in new york city cases that began after peaking on april 6 th . [7] the temporal correlation between the reduction in community cases and hcw covid infections despite continued covid workplace exposures may suggest the impact of community transmission on the hcw covid infection rate and the effectiveness of properly donned ppe. the importance of ppe in effectively preventing hcw infections was further evidenced in the new york state testing data released by governor cuomo showing that 12.2% of hcw tested positive for covid antibodies as compared to 19.9% of the approximately 3,000 randomly tested individuals from the general new york city population. [8] additional studies are needed to be better understand the relative impact of community and workplace exposures to covid-19 in hcw. we have shown that widespread testing can be developed and operationalized in a large academic hospital that simultaneously has large patient demands for testing. employee testing is critical for workforce planning since covid-19 positive employees require home-isolation for 10 days. testing combined with conventional infection control measures, supports the culture of safety for patients and workers. importantly, widespread testing of hcw offers valuable information for hospital workflow and workforce amid an epidemic that threatened to overwhelm the healthcare system. public health response to the initiation and spread of pandemic covid-19 in the united states clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in covid-19: the case for health-care worker screening to prevent hospital transmission presumed asymptomatic carrier transmission of covid-19 transmission of 2019-ncov infection from an asymptomatic contact in germany first experience of covid-19 screening of health-care workers in england covid-19 data: cases, hospitilizations, deaths amid ongoing covid-19 pandemic, governor cuomo announces results of state's antibody testing survey at churches in lower-income nyc communities of color show 27 percent of individuals tested positive for covid-19 antibodies a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-283788-04gwxo4b authors: pinninti, swetha; trieu, connie; pati, sunil k; latting, misty; cooper, joshua; seleme, maria c; boppana, sushma; arora, nitin; britt, william j; boppana, suresh b title: comparing nasopharyngeal and mid-turbinate nasal swab testing for the identification of sars-cov-2 date: 2020-06-29 journal: clin infect dis doi: 10.1093/cid/ciaa882 sha: doc_id: 283788 cord_uid: 04gwxo4b testing of paired mid-turbinate (mt) nasal and nasopharyngeal (np) swabs, collected by trained personnel from 40 patients with covid-19 showed more np (76/95, 80%) than mt swabs tested positive (61/95, 64%; p=0.02). among samples collected a week after study enrollment, fewer mt than np samples were positive (45% vs 76%; p=0.001). a c c e p t e d m a n u s c r i p t 3 initial reports of pneumonia caused by a novel coronavirus, severe acute respiratory syndrome coronavirus-2 (sars-cov-2), and the infection referred to as covid-19, were reported from wuhan, china in december 2019 [1, 2] . covid-19 has since been declared a pandemic and has been reported in almost all regions across the globe, contributing to significant mortality, morbidity, and economic losses. use of nasopharyngeal samples for the detection of respiratory viruses is considered the standard of care. however, the centers for disease control and prevention (cdc) [3] and the infectious disease society of america (idsa) [4] currently recommend testing of np, mid-turbinate nasal (mt), anterior nares, oropharyngeal and saliva swabs or washes from upper or lower respiratory tract for sars-cov-2 by reverse transcription (rt)-pcr based on limited data [5] [6] [7] . moreover, mt swab testing is gaining wider acceptance due to the ease of sample collection, ability to self-collect and personal protective equipment may not be needed [8] . however, few studies have directly compared the reliability of np and mt swabs for the detection of sars-cov-2 rna [9] . the objective of this study is to determine whether mt swabs are comparable to np swabs in detecting sars-cov-2 by rt-pcr in patients with confirmed covid-19. subjects and specimens: all hospitalized patients with confirmed covid-19 infection were eligible for participation in the study and 40 hospitalized patients with covid-19 were enrolled in a prospective study between april 5 and may 16, 2020, from whom serial np and mt nasal swabs were collected weekly and analyzed by rt-pcr. both swabs were collected by the same medical provider from both nares. demographic data and clinical characteristics for the study participants were collected from electronic medical records. the research a c c e p t e d m a n u s c r i p t 4 protocol was approved by the institutional review board for human use and an informed consent was obtained from all study participants or their legally authorized representatives. specimen collection and processing: paired mt and np swabs were collected at enrollment and weekly thereafter by trained bedside nursing staff, placed in transport media, and stored at -80 o c until processed. for the current study, paired samples collected at enrollment and a week after hospitalization were compared. rna was extracted using commercial spin column kits (qamp viral rna mini kit, qiagen, inc., valencia, ca), and stored at -80 o c. (table s2 of the supplementary information) . the demographic and clinical characteristics are provided in table s1 (supplementary information) figure 1a ). while most initial np (34/40, 85%) and the corresponding mt swabs (29/40, 73%) (sampling time point 1; fig 1a) were positive (p=0.53), significantly fewer mt swabs were positive (24/29, 82% np vs 13/29, 45% nasal; p=0.001) about a week later (sampling time point 2; fig 1a) . the relationship between the ct value and the rt-pcr results of np and mt swabs is shown in figure 1b 0001; fig 1c) . a modest correlation between np and nasal swabs for sars-cov-2 rna was observed only at ct values ≤30 (r=0.51) ( figure 1c ). rnase p pcr of discordant np and mt samples showed similar amplification plots without significant differences in ct values (table s2 ). our study of paired np and mt swabs from a cohort of hospitalized covid-19 patients demonstrates that while both np and mt swabs are reliable for diagnosis early during hospitalization, np swabs are more reliable later in disease course (figures 1b and 1c ). both the cdc and the idsa currently endorse testing of np or mt samples for sars-cov-2 with limited data. by analyzing serial samples from 40 patients, we examined the reliability of mt swabs in the setting of varying sars-cov-2 viral load levels. our findings suggest that patients whose np swabs are pcr-positive but have a lower viral load as suggested by high ct values (>30), often test negative by mt swab. to date, only two peer reviewed publications have directly compared np and mt swabs in patients with suspected covid-19 [9, 10] . péré et al. tested np and mt swabs from 44 patients and found that np and mt swabs were positive in 37 and 33 patients, respectively. we report similar findings but only for samples obtained earlier during hospitalization. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention a novel coronavirus from patients with pneumonia in china interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease infectious diseases society of america guidelines on the diagnosis of covid-19 detection of sars-cov-2 in different types of clinical specimens evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections sars-cov-2 viral load in upper respiratory specimens of infected patients self-collection: an appropriate alternative during the sars-cov-2 pandemic nasal swab sampling for sars-cov-2: a convenient alternative in time of nasopharyngeal swab shortage swabs collected by patients or health care workers for sars-cov-2 testing comparison of commercially available and laboratory developed assays for viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china acknowledgements: we thank the nursing staff for assistance with sample collection and the study participants and their families. a c c e p t e d m a n u s c r i p t 9 key: cord-287676-qh7zeyyx authors: angoulvant, françois; ouldali, naïm; yang, david dawei; filser, mathilde; gajdos, vincent; rybak, alexis; guedj, romain; soussan-banini, valérie; basmaci, romain; lefevre-utile, alain; brun-ney, dominique; beaujouan, laure; skurnik, david title: covid-19 pandemic: impact caused by school closure and national lockdown on pediatric visits and admissions for viral and non-viral infections, a time series analysis date: 2020-06-03 journal: clin infect dis doi: 10.1093/cid/ciaa710 sha: doc_id: 287676 cord_uid: qh7zeyyx a time series analysis of 871,543 pediatric emergency visits revealed that the covid-19 lockdown and school closure were associated with a significant decrease in infectious diseases disseminated through airborne or fecal-oral transmissions: common cold, gastro-enteritis, bronchiolitis, acute otitis. no change was found for urinary tract infections. m a n u s c r i p t in late december 2019, patients with viral pneumonia due to an unidentified microbial agent were reported in wuhan, hubei province, central china. this disease outbreak, covid-19, then grew substantially and was declared a pandemic by who on march 11, 2020 . [1] in 1995, a major french nationwide strike paralyzed france. for 19 days from november 30, 1995 to december 18, people stayed at home, including children who normally went to day-care centers and a significant decrease of bronchiolitis cases was observed. this decline might have been caused by workplace and school absenteeism, and lower attendance of day-care centers. [2] likewise, during the 2013-2014 measles epidemic, a reduction in contact rate during school vacations was associated with 4900 averted cases in the netherland. [3] after reaching france on january 24, 2020, a major progression of covid-19 from february to march lead to public health interventions. partial lockdown and school closure were initiated early march and a national lockdown was officially started on march 17 th , 2020. [4, 5] no previous public health intervention can be compared to the extent of the lockdown established for the covid-19 epidemic. we hypothesized that this unusual situation in france would be associated with a sharp decrease in pediatric infectious diseases that usually disseminate through social contacts, with schools at their center. being able to prevent these infections, responsible for many pediatric hospitalizations, would be an unwanted direct benefit of the lockdown for children, that seem otherwise significantly more protected than the adult from the sars-cov-2 infection. [6] even more, this could open the road for future guidelines to control future major health issues once the covid-19 pandemic is under control. therefore, the evolution of several major diseases usually correlated with dissemination through contact, such as gastroenteritis, common cold, and acute otitis media, were investigated before and after the start of the national lockdown. urinary tract infections, which are not reported to be correlated with contacts in children, were used as control outcome. we conducted a quasi-experimental interrupted time series analysis based on multicenter prospective french surveillance data for pediatric emergency department (ped) visits and related hospital admissions. the regional centre of observation and action on emergencies e-cerveau, agence régionale de santé, is an official network of emergency departments dedicated to public health that automatically transmit a summary of anonymized data from all their visits to the regional database. the database has been approved by the french data protection authority. these data include discharge diagnosis coded by the physicians in a c c e p t e d m a n u s c r i p t charge of the patient at the end of the peds visits according to icd-10 th revision and hospital admission or discharge. this study covers 6 peds from academic hospitals being part of assistance publique -hôpitaux de paris, located in and around paris, gathering 250,000 annual visits that daily transmitted their from january 1 st , 2017 to april 19 th , 2020. we used the e-cerveau database for this research. data are anonymous. patient informed consent is not required according to current dispositions. groups of diagnosis extracted were: gastroenteritis, common cold, bronchiolitis, acute otitis media, considered as infectious diseases thriving through social contact, and urinary tract infection (table s1). visits were grouped by calendar weeks for each year. the main outcome was the evolution of the number of hospital admissions following the french decision to close schools and start a lockdown for the whole country. [5] the secondary outcomes were the number of peds visits for gastroenteritis, bronchiolitis, common cold, acute otitis media. as recommended to prevent potential confusion, [7, 8] urinary tract infections were analyzed as a control outcome, given that this common pediatric infectious disease is not expected to be impacted by social distancing, although indirect effect such as stress or diet change cannot be excluded. this outcome was already used as a control concerning previous acute respiratory tract infections studies. [9] statistical analysis outcomes were analyzed by quasi-poisson regression, accounting for seasonality, secular trend before and after lockdown, and overdispersion of data. [7, 8, 10, 11] seasonality was taken into account by including harmonic terms (sines and cosines) with 12-months and 6months periods to adjust for the seasonal pattern. [11] the time unit chosen was one week to provide optimal precision to the model. [8] we hypothesized that the intervention would have an immediate impact, meaning after one analysis. analysis of acute gastroenteritis was performed on data from four hospitals combining 81% of the visits during the study period. all statistical tests were two-sided, and we considered a result as "significant" when the p-value was <0.05. all statistical analysis involved using r v3.6.1 (http://www.r-project.org). a total of 871,543 peds visits in the six participating centers from january 1 st , 2017 to april (table s2 , figure 1 and figure s1 ). we found a significant decrease of acute gastro-enteritis, common cold and acute otitis media (table s2, table s3 ) with a sharp decrease over 70% compared to the expected values (figures 1b-d) . decrease of bronchiolitis was also significant (-63.5% 95ci [-101.8 ; -25.9]) (table s2) . consistent both with our hypothesis and the incubation time of these different diseases, a dramatic decrease of overall peds visits (-68.5%) and hospitalization (-44.7%) was observed as soon as one week after the start of the lockdown ( figure s1 ). by contrast, urinary tract in this time series analysis of 871,543 peds visits, the number of peds visits and admission after the lockdown decreased by -68% and -45%, respectively. we found a significant decrease over 70% of acute gastro-enteritis, common cold, bronchiolitis and acute otitis media compared to the expected values. unprecedented public health interventions were ordered to reduce the risk of sars-cov-2 transmission. [4] our data suggest that these measures have also a critical impact on the transmission of numerous infectious diseases, more specifically on viral or viral-induced pediatric diseases. this major achievement may also play a critical role in making more health resources with adults admitted in icu and health care workers available to fight covid-19 pandemic. [12] in paris area, children with acute illness could be seen not only in ped but also in private office (general practitioners or pediatrician), general practitioner house calls, and communities' center. however most of these sites of care do not work 24/7 and rarely perform additional tests especially for the younger children. our study have limitations; we cannot exclude a change in clinical management such as avoidance of ent examination because of covid-19 fear which could have influenced diagnosis coding; we did not collect data regarding severity and so we cannot exclude that reduction in presentations was associated with children presenting later in their illness. while the dramatic decrease in peds could be partially due to transportation limitations and a fear of going to the hospital and increase of telemedicine, the stability in the number of urinary tract infections cases that we used as control outcome and the significant decrease in hospital admissions do not favor this hypothesis. moreover visits to private doctors' offices decrease by 40%. [13] while the role of the children in the dissemination of sars-cov-2 is still being discussed, finding that school closure and national lockdown were linked to a dramatic decrease in pediatric emergency visits concerning gastroenteritis, acute otitis media, bronchiolitis, and other viral diseases could be not only an unexpected benefit for the children but could also raise the question of the impact on the health care system of starting lifting the french national lockdown by reopening the schools. [14] complementary studies using more granular data such as severity could be useful to better understand lockdown's impact on children's health. this nation-level quasi-experiment is unprecedented in the modern era. it provides unique evidence which could be key in the post covid-19 era, to implement new guidelines and new routines in our way of life, and in order to fight past but also potential future infectious diseases threats reaching both children and adults. m a n u s c r i p t impact of lockdown on weekly pediatric emergency department visits and major pediatric infectious diseases, from january 1 st , 2017 to april 19 th , 2020. -a: overall peds visit (n=871,543). the black line shows the observed data. the bold red slope shows the model estimates based on observed data (quasi-poisson regression modeling). the bold blue slope shows the expected values without lockdown in the post-intervention period (quasi-poisson regression modeling). the start of the lockdown is indicated by the vertical black arrow. a c c e p t e d m a n u s c r i p t figure 1 world health organization. who director-general's opening remarks at the media briefing on covid-19 -11 coincidence of public transport strike with bronchiolitis epidemic the reduction of measles transmission during school vacations adaptation of the national plan for the prevention and fight against pandemic influenza to the 2020 covid-19 epidemic in france. disaster medicine and public health preparedness décret n° 2020-260 du 16 mars 2020 portant réglementation des déplacements dans le cadre de la lutte contre la propagation du virus covid-19 mmwr morbidity and mortality weekly report interrupted time series analysis in drug utilization research is increasing: systematic review and recommendations segmented regression analysis of interrupted time series studies in medication use research impact of implementing national guidelines on antibiotic prescriptions for acute respiratory tract infections in pediatric emergency departments: an interrupted time series analysis. clinical infectious diseases : an official publication of the infectious diseases society of america regression based quasiexperimental approach when randomisation is not an option: interrupted time series analysis interrupted time series regression for the evaluation of public health interventions: a tutorial paediatric intensive care society. pics and ics joint position statement 14. french government. french government measures m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-260457-m1jbpo5l authors: allander, tobias; jartti, tuomas; gupta, shawon; niesters, hubert g. m.; lehtinen, pasi; üsterback, riikka; vuorinen, tytti; waris, matti; bjerkner, annelie; tiveljung-lindell, annika; van den hoogen, bernadette g.; hyypiä, timo; ruuskanen, olli title: human bocavirus and acute wheezing in children date: 2007-04-01 journal: clin infect dis doi: 10.1086/512196 sha: doc_id: 260457 cord_uid: m1jbpo5l background. human bocavirus is a newly discovered parvovirus. it has been detected primarily in children with acute lower respiratory tract infection, but its occurrence, clinical profile, and role as a causative agent of respiratory tract disease are not clear. methods. we investigated the presence of human bocavirus by quantitative polymerase chain reaction of nasopharyngeal aspirate specimens and selected serum samples obtained from 259 children (median age, 1.6 years) who had been hospitalized for acute expiratory wheezing. the samples were analyzed for 16 respiratory viruses by polymerase chain reaction, virus culture, antigen detection, and serological assays. results. at least 1 potential etiologic agent was detected in 95% of children, and >1 agent was detected in 34% of children. human bocavirus was detected in 49 children (19%). a large proportion of the cases were mixed infections with other viruses, but human bocavirus was the only virus detected in 12 children (5%). high viral loads of human bocavirus were noted mainly in the absence of other viral agents, suggesting a causative role for acute wheezing. in addition, infections that had uncertain clinical relevance and low viral loads were prevalent. human bocavirus dna was frequently detected in serum specimens obtained from patients with acute wheezing, suggesting systemic infection. conclusions. human bocavirus is prevalent among children with acute wheezing and can cause systemic infection. results suggest a model for bocavirus infection in which high viral loads are potentially associated with respiratory symptoms and low viral loads indicate asymptomatic shedding. therefore, quantitative polymerase chain reaction analysis may be important for additional studies of human bocavirus. acute viral respiratory tract infection is the leading cause of hospitalization for infants and young children in developed countries and is a major cause of death in developing countries [1, 2] . human bocavirus (hbov) is a newly discovered parvovirus that was first identified in sweden from pooled nasopharyngeal aspirate specimens by large-scale molecular virus screening [3] . in the original study, 540 nasopharyngeal aspirate specimens obtained from hospitalized patients were analyzed for hbov, and 3.1% yielded positive results. all hbov-positive samples had been obtained from young children with respiratory distress (mainly acute expiratory wheezing). many of the children had pneumonia, with interstitial infiltrates noted by chest radiography [3] . recent studies have detected hbov in 1.5%-11.3% of investigated respiratory tract samples in north america, europe, asia, and australia, suggesting that the virus has a global distribution [4] [5] [6] [7] [8] [9] [10] . respiratory distress and abnormal chest radiography findings have been frequent findings associated with hbov in these studies. however, the causative role of hbov remains unclear. in fact, hbov was detected concurrently with other potential pathogens in 33%-56% of cases in which it was studied [5, 7, [9] [10] [11] . these findings have raised the question of whether hbov is at all a causative agent of respiratory tract disease. we investigated the prevalence of hbov and the genome hbov load in the respiratory tract and blood specimens obtained from children who had been hospitalized for acute expiratory wheezing-the most common manifestation of lower respiratory tract infection in children-to investigate the association between hbov infection and acute respiratory tract illness. importantly, the samples had earlier been investigated for evidence of different respiratory virus infections, and a potential causative agent had been detected in 88% of cases [12] . in the present, expanded study, we searched for a total of 16 respiratory viruses. as part of a randomized, placebo-controlled clinical trial evaluating the efficacy of systemic corticosteroids for the treatment of acute expiratory wheezing in children, we performed extensive diagnostic evaluations for respiratory virus infection. the study included 293 children who presented to the department of pediatrics, turku university hospital (turku, finland), during the period from september 2000 through may 2002 [12, 13] . acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children aged !3 years. asthma was diagnosed on the basis of the national asthma education and prevention program guidelines [14] . all other episodes of acute expiratory wheezing were considered to be recurrent wheezing. all patients were examined by 1 of the 2 study physicians (t.j. and p.l.) twice daily during hospitalization. signs and symptoms were recorded on a standardized form. of the 293 children who were randomized, 259 children (median age, 1.6 years; range, 3 months to 15 years) who had sufficient sample material available for complete virus diagnostic evaluation (nasopharyngeal aspirate specimens were used for pcr [for 16 viruses], virus culture [for 9 viruses], and antigen detection [for 7 viruses]; acute-and convalescent-phase serum samples were used for serologic testing [for 7 viruses]) were included in the present study. sixty-four asymptomatic children (median age, 4.1 years; range, 5 months to 14 years) who had been admitted to the division of pediatric surgery, turku university hospital, during the period from september 2000 through october 2002 and who did not have respiratory symptoms during the preceding 4 weeks were also investigated for the presence of hbov [15] . the study protocol was approved by the ethics committee of the turku university hospital. at hospital admission, a nasopharyngeal aspirate sample was obtained using a standardized procedure, as described elsewhere [12] . antigen detection and virus culture were performed immediately, and the samples obtained for pcr assays were stored in tubes at ϫ70њc before processing. blood samples were collected at the time of the patient's admission and 2-3 weeks after discharge from the hospital and were stored at ϫ20њc. nasal swab specimens were obtained from the asymptomatic children. nasopharyngeal aspirate specimens could not be obtained from healthy children, because they did not have mucus in the nasopharynx. real-time pcr for hbov. nucleic acids were extracted from 400 ml of the nasopharyngeal aspirate samples using the magattract virus mini m48 kit and the biorobot m48 instrument (qiagen), and samples were eluted in 100 ml of rnasefree water. for serum samples, 100 ml of serum was processed using the qiaamp blood mini kit (qiagen), and dna was eluted in 50 ml. the pcr assay targeted the np-1 gene of hbov. the 20-ml amplification reaction contained 5 ml of sample dna, 10 ml of taqman universal pcr master mix (pe applied biosystems), 0.1 ml of bovine serum albumin (20 mg/ ml), 300 nmol/l each primer (boca-forward, gga aga gac act ggc aga caa; and boca-reverse, ggg tgt tcc tga tga tat gag c), and 150 nmol/l boca probe (fam-ctg cgg ctc ctg ctc ctg tga t-tamra). amplification was performed using a lightcycler 1.2 instrument (roche) with the following instrument settings: 95њc for 10 min, 50 cycles of 95њc for 0 s, and 60њc for 1 min. for standardizing the quantification, a plasmid (npsc3.1) containing the hbov np-1 gene was used in serial dilutions covering a range of 6 logs. the criteria for a positive reaction were a cycle threshold !40 cycles and fluorescence count 10.5. the minimum genome viral load that would allow reproducible quantification was 10 copies per reaction, corresponding to 500 copies/ml for nasopharyngeal aspirate specimens or 1000 copies/ml for serum specimens. the analyses were performed at a diagnostic laboratory, with rigorous measures to prevent contamination. the realtime pcr assay was compared with the previously published single end-point pcr assay targeting the np-1 gene [3] by parallel analysis of 74 samples. fifteen samples yielded positive results with both assays, 5 yielded positive results with realtime pcr only, and the remaining 54 yielded negative results with both assays. virus culture was performed for adenovirus, influenza a and b viruses, parainfluenza virus types 1-3 (piv 1-3), respiratory syncytial virus (rsv), enteroviruses, and rhinoviruses, in accordance with routine diagnostic protocols, in a549, hela, and llc-mk 2 and human foreskin fibroblast cells, as well as for human metapneumovirus in tertiary monkey kidney cells [16] . viral antigens were detected for adenovirus, influenza a and b viruses, piv 1-3, and rsv by time-resolved fluoroimmunoassay [17] . levels of igg antibodies specific for the same viruses (except for piv 2 and human metapneumovirus) were analyzed in paired serum samples, in addition to igm antibodies for enteroviruses. rt-pcr was used for the detection of enteroviruses and rhinoviruses, rsv, coronaviruses 229e and oc43, and human metapneumovirus. these results were reported elsewhere [12] . in addition to hbov diagnostic evaluations, the present study included extended diagnostic evaluations by real-time pcr for influenza a and b viruses, adenovirus, piv 1-4, and coronavirus nl63 (in rotterdam) and coronavirus hku-1 (in stockholm). total nucleic acids for these analyses were isolated using a magnapure lc isolation station (roche). amplification was performed with an abi7700 instrument, using commercially available master mixes (applied biosystems) and standard protocols. the primer sequences for piv 1-4 and coronavirus hku-1 are available from the authors. otherwise, the primer and probe sequences, pcr protocols, and other viral detection methods were described elsewhere [12, 16, [18] [19] [20] [21] . the sequencing of the 12 previously reported, nontypable rhinoviruses and enteroviruses [12] identified them as rhinoviruses. a patient was considered to be positive for the virus if results of at least 1 of the tests used were positive. a potential viral pathogen was identified in 245 children (95%) ( tected in 11% of wheezing patients. the nasal swab samples obtained from the 64 asymptomatic children were hbov negative. hbov was found significantly more frequently among children with acute wheezing than among asymptomatic children (19% vs. 0%; , by fisher's exact test). however, p ! .001 because age distributions and sampling techniques differed between the 2 groups, this comparison should be interpreted with caution. we searched for additional evidence of causality by studying whether detection of hbov was unrelated or inversely correlated to the occurrence of other viruses in children with acute wheezing [3] . hbov was indeed more prevalent among children with symptoms of an otherwise unexplained etiology than among those in which other viruses were detected (46% vs. 16%; ) (table 2) . stratification of the patients on p ! .001 the basis of a nasopharynx hbov load of !10 4 copies/ml versus 110 4 copies/ml revealed that only the patients with a high hbov load had this association (table 2). this suggests that the presence of hbov at high-but not at low-viral loads is associated with previously unexplained acute wheezing. detection of hbov dna in serum specimens. acute-and convalescent-phase serum samples were available in sufficient quantity from 43 of the 49 patients who tested positive for hbov in the nasopharynx. hbov dna was detected in 23 (53%) of the 43 acute-phase serum samples and in 8 (19%) of the 43 convalescent-phase serum specimens ( , by p ! .001 fisher's exact test). detection of hbov in serum was mainly, although not exclusively, associated with a high viral load in the nasopharynx (table 3) . viral loads in serum samples ranged from ! to copies /ml and did not correlate 3 5 1.0 ϫ 10 5.9 ϫ 10 with genome viral loads in the corresponding nasopharyngeal aspirate specimens ( ; data not shown). we also an-r p ϫ0.24 alyzed 45 randomly selected serum samples obtained from the 196 wheezing patients who tested negative for hbov in the nasopharynx but who had demonstrated infection with some other virus. three tested positive for hbov dna (2 had low viral loads, and 1 had a viral load of copies/ml). 5 hbov was the only virus detected in 12 children (table 4). the median age of these children was 1.3 years. eight children received a diagnosis of bronchiolitis, 3 had recurrent wheezing, and 1 had acute exacerbation of asthma. acute otitis media was diagnosed in 5 (42%) of the children, and chest radiographs taken from 9 patients all showed interstitial infiltrates. one child had leukocytosis ( cells/l), and 2 children had 9 16.3 ϫ 10 increased levels of serum c-reactive protein (73 and 78 mg/l). in all other cases, total wbc counts and serum c-reactive protein levels were within normal limits. the reported duration of symptoms varied from 2 days to 120 days. when characteristics of the children positive only for hbov were compared with those of children with other demonstrated virus infections, no clinically significant differences were recorded (table 4). we found hbov in the nasopharynx of 19% of children with acute wheezing illness, and it was the fourth most frequent virus detected after rhinoviruses, enteroviruses, and rsv. these findings show that hbov is a common virus in the community. the prevalence of hbov was higher in our study than in previous reports [3] [4] [5] [6] [7] [8] [9] [10] [11] ; this may have been the result of technical factors and study design rather than true differences in virus prevalence. a large number of the samples had low hbov loads, making evaluation of prevalence highly dependent on assay sensitivity. differences in pcr assay sensitivity may, of course, similarly have affected the relative prevalence estimates for all 16 of the viruses studied. it should also be recognized that nasopharyngeal sampling is not a standardized procedure. finally, the present study included only hospitalized patients with acute wheezing illness, which may be the major manifestation of hbov infection, whereas previous studies have included a broader selection of samples. despite the large number of mixed hbov infections, our findings are still consistent with a potential etiologic role for hbov in respiratory tract disease in young children. in the absence of in vitro virus propagation systems and animal models, final proof of causality will remain difficult to obtain. fredricks and relman [22] proposed molecular diagnostic criteria for causality for use in this situation, and the present study provides data relevant for many of these criteria: 1. hbov was present at the site of the symptoms (i.e., in the respiratory tract). genome viral loads were generally higher in the respiratory tract specimens than in the serum specimens. 2. hbov was more prevalent in wheezing patients than in asymptomatic children. the significance of this observation alone should not be exaggerated. first, the age distribution differed between symptomatic and asymptomatic children. second, sampling techniques were not matched. however, these results are consistent with the recent findings of kesebir et al. [23] , who reported that nasal wash samples obtained from 96 asymptomatic children were negative for hbov. nevertheless, the comparison of symptomatic and asymptomatic individuals remains problematic. the large number of mixed infections observed with hbov may indicate that hbov is reactivatedor its detection enhanced-by other infections. this would result in a higher prevalence among symptomatic patients than among control subjects, even in the absence of an etiologic role. respiratory secretions recovered during acute infection are also inherently very different from those recovered from asymptomatic subjects (e.g., with regard to cell counts). therefore, evidence of a virus-disease association must rely also on criteria other than differences in the prevalence between case patients and control subjects. 3. hbov was more prevalent among patients with previously unexplained wheezing than among patients who tested positive for other viruses. 4. this association was seen only for patients with a high viral load (i.e., there was a dose-response relationship). 5. occurrence of hbov in serum specimens was linked in time with an episode of acute wheezing, because hbov became less prevalent in the same patients after clinical recovery. taken together, these findings suggest an association between high hbov load and acute wheezing of otherwise unknown etiology, but they do not prove a causal relationship. at the same time, shedding of hbov (continuous or secondary to other infections) also appears to be common. this model for hbov biology may prove to be valuable for additional studies of the possible causative role of hbov in respiratory tract disease. applying this model to the present study would suggest that hbov is a potential etiologic agent in 28 patients (11%) with a high viral load, of whom 10 (3.9%) had hbov infection alone. however, some studies have reported that dual viral infections are associated with more-severe disease than are infections with a single agent, so the clinical relevance of hbov infection with a low viral load will also require additional study [24] [25] [26] . detection of hbov dna in serum suggests that hbov is a systemic infection, like most parvovirus infections, including animal bocavirus infection [27, 28] . it is possible that children with high-titer hbov in both the respiratory tract and the blood had a primary hbov infection, and this hypothesis is supported by the finding that serum viral loads generally became low or undetectable after clinical recovery. however, confirmation of a primary infection will have to await the development of an hbov antibody assay. an hbov infection with a low viral load may reflect long-term virus persistence after clinical recovery, as has been reported for parvovirus b19 [29, 30] . the findings of hbov in a few convalescent-phase serum samples and in serum samples obtained from some patients whose respiratory tract specimens tested negative for hbov are also consistent with virus persistence. cases of possibly symptomatic primary hbov infection (in the patients with hbov infection alone) seemed to be particularly common among children aged ∼1 year (i.e., soon after the loss of maternal antibodies). such cases emerged throughout the year (table 4) . these findings, together with the high prevalence rate, suggest that hbov is an endemic virus in the study area. we only studied children with bronchiolitis, recurrent wheezing, and acute asthma. thus, possible other manifestations of hbov infection were not addressed. isolated hbov positivity was associated with acute otitis media in nearly one-half of the subjects, and interstitial infiltrates were seen in all chest radiographs, suggesting that hbov could be a frequent causative agent of acute otitis media and childhood community-acquired pneumonia. hbov-associated acute wheezing did not differ clinically from that induced by rhinoviruses, enteroviruses, or rsv. our results indicate that acute wheezing is almost invariably associated with virus infection-and often with multiple-virus infection. many new respiratory viruses, such as human metapneumovirus and 3 new types of coronaviruses, have been identified over the past few years [3, 16, 18, 31, 32] . our results suggest that 5% of suspected respiratory virus infections are still unaccounted for, but that estimate is, of course, highly uncertain. on one hand, 5% of patients may not experience virus infections or may be infected by a known virus not detected because of assay limitations. on the other hand, the large number of mixed infections that we observed indicates that additional respiratory agents may also be found in other patients in addition to the 5% who still had negative results. in conclusion, we found evidence of virus infection in nearly all children with acute wheezing. hbov was detected in onefifth of patients, and it was the fourth most prevalent virus detected. results suggest that hbov at a high viral load could be an etiologic agent of respiratory tract disease, whereas little support was found for the etiologic role of hbov at a low viral load. quantitative analysis may, therefore, be important for future studies of hbov infection. like other parvoviruses, hbov can cause a systemic infection and may persist after resolution of symptoms. global and regional burden of disease and risk factors, 2001: systematic analysis of population health data bronchiolitis-associated hospitalizations among us children cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection human bocavirus in children detection of human bocavirus in japanese children with lower respiratory tract infections evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus: prevalence and clinical spectrum at a children's hospital the association of newly identified respiratory viruses with lower respiratory tract infections in korean children frequent detection of bocavirus dna in german children with respiratory tract infections frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children evaluation of the efficacy of prednisolone in early wheezing induced by rhinovirus or respiratory syncytial virus expert panel report: guidelines for the diagnosis and management of asthma update on selected topics-2002 persistence of rhinovirus and enterovirus rna after acute respiratory illness in children a newly discovered human pneumovirus isolated from young children with respiratory tract disease viruses and bacteria in the etiology of the common cold a previously undescribed coronavirus associated with respiratory disease in humans pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr diagnosing herpesvirus infections by real-time amplification and rapid culture design and performance testing of quantitative real-time pcr assays for influenza a and b viral load measurement sequence-based identification of microbial pathogens: a reconsideration of koch's postulates human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human metapneumovirus in severe respiratory syncytial virus bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis association of rhinovirus infection with increased disease severity in acute bronchiolitis minute virus of canines (mvc, canine parvovirus type-1): pathogenicity for pups and seroprevalence estimate pathological and virological studies of experimental parvoviral enteritis in calves persistent b19 infection in immunocompetent individuals: implications for transfusion safety slow clearance of human parvovirus b19 viremia following acute infection identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia we thank kaisu kaistinen for traveling with the samples between turku and stockholm.financial support. academy of finland (to t.j.), the pediatric research foundation (to o.r. and t.j.), the torsten and ragnar söderberg foundation, the swedish cancer foundation, nana svartz' fund, and the swedish society for clinical microbiology (to t.a.). t.a. is a fellow of the swedish research council.potential conflicts of interest. t.a. is a coinventor on a patent application related to hbov and assigned to karolinska institutet innovations. all other authors: no conflicts. key: cord-279828-es498qul authors: boulle, andrew; davies, mary-ann; hussey, hannah; ismail, muzzammil; morden, erna; vundle, ziyanda; zweigenthal, virginia; mahomed, hassan; paleker, masudah; pienaar, david; tembo, yamanya; lawrence, charlene; isaacs, washiefa; mathema, hlengani; allen, derick; allie, taryn; bam, jamy-lee; buddiga, kasturi; dane, pierre; heekes, alexa; matlapeng, boitumelo; mutemaringa, themba; muzarabani, luckmore; phelanyane, florence; pienaar, rory; rode, catherine; smith, mariette; tiffin, nicki; zinyakatira, nesbert; cragg, carol; marais, frederick; mudaly, vanessa; voget, jacqueline; davids, jody; roodt, francois; van zyl smit, nellis; vermeulen, alda; adams, kevin; audley, gordon; bateman, kathleen; beckwith, peter; bernon, marc; blom, dirk; boloko, linda; botha, jean; boutall, adam; burmeister, sean; cairncross, lydia; calligaro, gregory; coccia, cecilia; corin, chadwin; daroowala, remy; dave, joel a; de bruyn, elsa; de villiers, martin; deetlefs, mimi; dlamini, sipho; du toit, thomas; endres, wilhelm; europa, tarin; fieggan, graham; figaji, anthony; frankenfeld, petro; gatley, elizabeth; gina, phindile; govender, evashan; grobler, rochelle; gule, manqoba vusumuzi; hanekom, christoff; held, michael; heynes, alana; hlatswayo, sabelo; hodkinson, bridget; holtzhausen, jeanette; hoosain, shakeel; jacobs, ashely; kahn, miriam; kahn, thania; khamajeet, arvin; khan, joubin; khan, riaasat; khwitshana, alicia; knight, lauren; kooverjee, sharita; krogscheepers, rene; jacque kruger, jean; kuhn, suzanne; laubscher, kim; lazarus, john; le roux, jacque; lee jones, scott; levin, dion; maartens, gary; majola, thina; manganyi, rodgers; marais, david; marais, suzaan; maritz, francois; maughan, deborah; mazondwa, simthandile; mbanga, luyanda; mbatani, nomonde; mbena, bulewa; meintjes, graeme; mendelson, marc; möller, ernst; moore, allison; ndebele, babalwa; nortje, marc; ntusi, ntobeko; nyengane, funeka; ofoegbu, chima; papavarnavas, nectarios; peter, jonny; pickard, henri; pluke, kent; raubenheimer, peter j; robertson, gordon; rozmiarek, julius; sayed, a; scriba, matthias; sekhukhune, hennie; singh, prasun; smith, elsabe; soldati, vuyolwethu; stek, cari; van den berg, robert; van der merwe, le roux; venter, pieter; vermooten, barbra; viljoen, gerrit; viranna, santhuri; vogel, jonno; vundla, nokubonga; wasserman, sean; zitha, eddy; lomas-marais, vanessa; lombard, annie; stuve, katrin; viljoen, werner; basson, de vries; le roux, sue; linden-mars, ethel; victor, lizanne; wates, mark; zwanepoel, elbe; ebrahim, nabilah; lahri, sa'ad; mnguni, ayanda; crede, thomas; de man, martin; evans, katya; hendrikse, clint; naude, jonathan; parak, moosa; szymanski, patrick; van koningsbruggen, candice; abrahams, riezaah; allwood, brian; botha, christoffel; henndrik botha, matthys; broadhurst, alistair; claasen, dirkie; daniel, che; dawood, riyaadh; du preez, marie; du toit, nicolene; erasmus, kobie; koegelenberg, coenraad f n; gabriel, shiraaz; hugo, susan; jardine, thabiet; johannes, clint; karamchand, sumanth; lalla, usha; langenegger, eduard; louw, eize; mashigo, boitumelo; mhlana, nonte; mnqwazi, chizama; moodley, ashley; moodley, desiree; moolla, saadiq; mowlana, abdurasiet; nortje, andre; olivier, elzanne; parker, arifa; paulsen, chané; prozesky, hans; rood, jacques; sabela, tholakele; schrueder, neshaad; sithole, nokwanda; sithole, sthembiso; taljaard, jantjie j; titus, gideon; van der merwe, tian; van schalkwyk, marije; vazi, luthando; viljoen, abraham j; yazied chothia, mogamat; naidoo, vanessa; alan wallis, lee; abbass, mumtaz; arendse, juanita; armien, rizqa; bailey, rochelle; bello, muideen; carelse, rachel; forgus, sheron; kalawe, nosi; kariem, saadiq; kotze, mariska; lucas, jonathan; mcclaughlin, juanita; murie, kathleen; najjaar, leilah; petersen, liesel; porter, james; shaw, melanie; stapar, dusica; williams, michelle; aldum, linda; berkowitz, natacha; girran, raakhee; lee, kevin; naidoo, lenny; neumuller, caroline; anderson, kim; begg, kerrin; boerlage, lisa; cornell, morna; de waal, renée; dudley, lilian; english, rené; euvrard, jonathan; groenewald, pam; jacob, nisha; jaspan, heather; kalk, emma; levitt, naomi; malaba, thoko; nyakato, patience; patten, gabriela; schneider, helen; shung king, maylene; tsondai, priscilla; van duuren, james; van schaik, nienke; blumberg, lucille; cohen, cheryl; govender, nelesh; jassat, waasila; kufa, tendesayi; mccarthy, kerrigan; morris, lynn; hsiao, nei-yuan; marais, ruan; ambler, jon; ngwenya, olina; osei-yeboah, richard; johnson, leigh; kassanjee, reshma; tamuhla, tsaone title: risk factors for covid-19 death in a population cohort study from the western cape province, south africa date: 2020-08-29 journal: clin infect dis doi: 10.1093/cid/ciaa1198 sha: doc_id: 279828 cord_uid: es498qul background: risk factors for covid-19 death in sub-saharan africa and the effects of hiv and tuberculosis on covid-19 outcomes are unknown. methods: we conducted a population cohort study using linked data from adults attending public sector health facilities in the western cape, south africa. we used cox-proportional hazards models adjusted for age, sex, location and comorbidities to examine the association between hiv, tuberculosis and covid-19 death from 1 march-9 june 2020 among (i) public sector “active patients” (≥1 visit in the 3 years before march 2020), (ii) laboratory-diagnosed covid-19 cases and (iii) hospitalized covid-19 cases. we calculated the standardized mortality ratio (smr) for covid-19 comparing hiv positive vs. negative adults using modelled population estimates. results: among 3,460,932 patients (16% hiv positive), 22,308 were diagnosed with covid-19, of whom 625 died. covid-19 death was associated with male sex, increasing age, diabetes, hypertension and chronic kidney disease. hiv was associated with covid-19 mortality (adjusted hazard ratio [ahr] 2.14; 95% confidence interval [ci] 1.70-2.70), with similar risks across strata of viral load and immunosuppression. current and previous tuberculosis were associated with covid-19 death (ahr [95%ci] 2.70 [1.81-4.04] and 1.51 [1.18-1.93] respectively). the smr for covid-19 death associated with hiv was 2.39 (95%ci 1.96-2.86); population attributable fraction 8.5% (95%ci 6.1-11.1). conclusion: while our findings may over-estimate hivand tuberculosis-associated covid-19 mortality risks due to residual confounding, both hiv and current tuberculosis were independently associated with increased covid-19 mortality. the associations between age, sex and other comorbidities and covid-19 mortality were similar to other settings. the effects of the intersecting pandemics of hiv, tuberculosis and coronavirus disease-19 in sub-saharan africa are unknown. studies to date suggest no increased risk of adverse outcomes in hiv co-infected patients, but these are small studies from europe and north america, often limited to hospitalized patients, and may not be relevant to sub-saharan africa where people living with hiv (plwh) are younger with different comorbidities, frequently including tuberculosis [1] [2] [3] [4] [5] [6] [7] [8] . plwh may experience more severe covid-19 disease due to hiv-related immune suppression, which may be exacerbated by transient immune deficiency from coronaviruses [9, 10] . in support of this hypothesis a large uk cohort study reported increased risk of covid-19 death with immunosuppressive comorbidity, including plwh [8] . two factors may reduce risk of severe covid-19 in plwh, however: dysfunctional immunity may lessen a virus-induced cytokine storm [11, 12] , and some antiretroviral drugs (tenofovir and some protease inhibitors) have in vitro activity against coronaviruses, with better outcomes reported for plwh receiving tenofovir disoproxil fumarate (tdf) vs. other antiretrovirals [12, 13] . tuberculosis may exacerbate covid-19 with impaired immune responses and increased angiotensin converting enzyme 2 receptor expression in respiratory epithelial cells, while covid-19 pneumonia may enhance tuberculosis progression [14] [15] [16] [17] . it is important to establish if hiv and tuberculosis increase risk of covid-19 death so that patients with these conditions can be provided with augmented prevention and potential therapeutic interventions. we used linked data from adults attending public sector health facilities in the western cape province, south africa, to identify factors associated with covid-19 death. 9 we conducted a cohort study using de-identified data from the western cape provincial health data centre (wcphdc) of public sector patients aged ≥20 years with documented sex and not known to have died before march 1, 2020 (before the first diagnosed covid-19 case in south africa, and several weeks before the first documented covid-19 death) and included all follow up through june 9, 2020. the outcome was covid-19 associated death. our main analysis examined risk of covid-19 death in the general population, so all patients were included irrespective of sars-cov-2 testing. the study was approved by the university of cape town and stellenbosch university health research ethics committees and the western cape province department of health. individual informed consent requirement was waived for this secondary analysis of de-identified data. the western cape has nearly 7 million inhabitants, of whom ~520,000 are plwh with >90% of them dependent on public sector health services. the wcphdc has been described in detail [18] . briefly, wcphdc consolidates administrative, laboratory, and pharmacy data from routine electronic clinical information systems used in all public sector health facilities with linkage through a unique identifier. multiple data sources are triangulated to enumerate health conditions such as diabetes mellitus ("diabetes"), hypertension, tuberculosis and hiv, with high or moderate certainty evidence assigned for each inferred condition (supplementary table 1 ). high certainty evidence of hiv comprises a positive hiv diagnostic test and/or hiv-rna test and/or triple antiretroviral therapy (art) and/or registration in the hiv disease management system; moderate certainty is assigned for those with only a cd4 count measure and/or two antiretroviral drugs prescribed (previously used for vertical hiv transmission prevention) and/or icd-10 diagnosis code of hiv. hiv testing coverage is high, as >90% of plwh know their hiv diagnosis [19] . high certainty evidence of tuberculosis comprised laboratory evidence of mycobacterium tuberculosis infection (any anatomical site, using xpert rif/mtb, microscopy, culture) and/or registration on the electronic tuberculosis registers and/or combination tuberculosis treatment and/or admission to a tuberculosis hospital. comorbidities were based on high or moderate certainty evidence but restricted to high certainty evidence in sensitivity analyses. the virologic, immunologic and art status of plwh on march 1, 2020 was categorized, based on most recent measures, as "confirmed virologically suppressed on art" (hiv-rna<1000 copies/ml in last 15 months and art dispensed in last 6 months), "likely virologically suppressed on art" (hiv-rna <1000 copies/ml 15-24 months previously or hiv-rna <1000 copies/ml >24 months previously if art dispensed in last 6 months), "viraemic or immunosuppressed" (hiv-rna >1000 copies/ml in last 15 months or cd4 count <200 cells/µl within 18 months before march 2020) or "unknown". until january 2020, adult first-line art was tdf+emtricitabine/lamivudine+efavirenz, with abacavir replacing tdf for patients with kidney disease; zidovudine+emtricitabine/lamivudine+protease inhibitor was used for second-line art for most patients. dolutegravir was introduced in first and second-line since january 2020. diabetic control was categorized according to glycosylated haemoglobin (hba1c) measurement within the last 2 years as <7% (controlled); 7-8.9% (poorly controlled), ≥9% (uncontrolled). all covid-19 diagnoses were based on a positive sars-cov-2 pcr test. testing was available for all patients with covid-19 symptoms until june 1, 2020; thereafter public sector laboratory testing was restricted to patients requiring admission or aged >55 years or 11 with comorbidities, due to temporary limited testing capacity. hospital admissions and all deaths in sars-cov-2 positive cases are recorded and reviewed daily. we used cox-proportional hazards models adjusted for age, sex and other comorbidities to examine the association between hiv, tuberculosis and covid-19 death among (i) all public sector patients with ≥1 health visit in the 3 years before march 1, 2020 (considered "active patients"), (ii) laboratory-diagnosed covid-19 cases and (iii) hospitalized covid-19 cases. we adjusted for location within cape town vs. rest of the province and subdistrict of residence within cape town to account for geographical variation in infection rates and as a proxy for socio-economic status. patients were censored on date of death if deceased without a covid-19 diagnosis, or on june 9, 2020, whichever was earliest. database closure was 7 days later to allow for death reporting delays. for the analysis of covid-19 death in laboratory-diagnosed cases we included cases diagnosed before june 1, 2020 when testing was available for all patients with covid-19 symptoms, but included all patients diagnosed by june 9, 2020 in sensitivity analysis. the proportional-hazard assumption was assessed with schoenfeld residuals [20] . all analyses were conducted using stata 15.1. we also calculated the standardized mortality ratio (smr) of the actual number of covid-19 deaths in plwh vs. the number that would be expected if plwh had the same risk of covid-19 death as hiv-negative people of the same age and sex. we used data on the age, sex and hiv status of all covid-19 deaths (public and private sector) and the thembisa western cape hiv model to estimate the western cape population size and hiv prevalence, by age and sex, in 2020. [21] we calculated 95% confidence intervals (ci) for the smr using 12 since individual socio-economic status and some comorbidities are not recorded in wcphdc, we calculated e-values to determine the minimum strength of association that an unmeasured confounder (e.g. raised body mass index [bmi] or socio-economic status) would need to have with hiv/ tuberculosis and covid-19 death to fully account for any association between hiv/tuberculosis and covid-19 death [22] . we conducted quantitative bias analysis to assess the impact of potential confounding by obesity on an association between hiv and covid-19 death. among 3,460,932 "active patients" aged ≥20 years on march 1, 2020, 22,308 were diagnosed with covid-19, of whom 625 (2.8%) died (table 1) . among covid-19 cases, 69% were diagnosed and 67% of deaths occurred before the change in testing criteria (june 1). the proportion of men was lower among covid-19 cases vs. non-cases (31% vs. 42%), likely due to initial cases being among essential workers in retail and manufacturing sectors employing predominantly women. the proportion of women peaked (76%) in week 5 of the epidemic, declining thereafter. diabetes and hypertension were common in all patients, with higher prevalence among covid-19 cases than non-cases ( and previous tuberculosis 14% vs. 8%). 13 although the proportion of plwh was similar among surviving and deceased covid-19 cases, a greater proportion of covid-19 deaths were in patients aged <50 years in those with vs. without hiv (39% vs 13%) ( table 2) . a substantial proportion of covid-19 deceased plwh had diabetes (50%) and hypertension (42%), however these conditions were more common in deceased people without hiv (62% for each condition). current and previous tuberculosis were more frequent in plwh irrespective of covid-19 with 14% and 37% of covid-19 deceased cases with hiv having current and/or previous tuberculosis respectively. among all public sector patients, the probability of covid-19 death by 100 days since figure 1) . to assess whether the association between hiv or tuberculosis and covid-19 mortality could be due to residual unmeasured confounding e.g. by socio-economic status, or unrecorded comorbidities, we calculated the e-value for an unmeasured confounder. for among all laboratory diagnosed covid-19 cases, there were 135 deaths among an estimated ~520,000 plwh in the province (260 deaths/million) and 786 deaths among 6.36 million people without hiv (124 deaths/million). the smr for covid-19 mortality in plwh, relative to hiv-negative people was 2.39 (95% ci 1.96-2.86) and the attributable fraction of public and private sector covid-19 deaths due to hiv was 8.5% (95% ci 6.1-11.1). among nearly 3.5 million adults (16% plwh) in south africa we found an approximately two-fold association of covid-19 death with hiv, irrespective of viraemia or immunosuppression prior to the covid-19 episode, and a similar association between covid-19 death and current tuberculosis. among plwh on art, receiving tdf was associated with lower covid-19 mortality compared to other antiretrovirals. while the hivand tuberculosis-associated increased risk of covid-19 death may be over-estimated if there is residual confounding due to socio-economic status or unrecorded comorbidities, our results, supported by sensitivity analyses, demonstrate that plwh and persons with tuberculosis are at increased risk of severe covid-19. nonetheless, despite a high burden of advanced hiv in the province, the attributable fraction of all deaths ascribed to hiv was <10%. while most case series of hiv and sars-cov-2 co-infection have not shown poor outcomes in plwh, [1-3, 5-7] some cohorts of hospitalized plwh with covid-have reported substantial morbidity and mortality including among patients with suppressed viral load on art [23, 24] london have not shown differences in mortality risk [25] [26] [27] , however, the absence of increased mortality risk in hospitalized patients with comorbidities may be explained by selection bias; risk factors for covid-19 death may be attenuated by restricting to the subset of hospitalized patients already at high mortality risk [28] . it is therefore expected that in our analysis the increased risk of death associated with all comorbidities was progressively attenuated when restricting to cases (people with sufficiently severe symptoms to be tested) and hospitalized patients. similar to our findings, several studies have reported a high prevalence of comorbidities among plwh with severe covid-19 [3, 6, 7] . the high prevalence of comorbidities in deceased plwh suggests that the effect of hiv may at least partly be due to an increased risk of comorbidities at younger ages [2, 7] , including those not recorded in wcphdc such as cardiovascular disease. persistent immune dysfunction may also be important in severe covid-19 despite viral suppression; the hazard ratio point estimates for association with covid-19 death were greater in immunosuppressed or viraemic plwh, although the numbers of these patients with covid-19 were small with wide cis. further, cd4 <200 cells/µl during admission was associated with covid-19 death. while this may partly be due to the well-described lymphopenia in severe covid-19 which is prognostic of poor outcomes, about half of patients with low cd4 during admission were either new hiv diagnoses or had previous immunosuppression, viraemia or no recent art [10] . among covid-19 cases in plwh on art, receipt of tdf (vs. other therapies) was associated with reduced covid-19 mortality. however, this association is likely to be over-estimated; in south africa only patients on second-line art or with poor renal function would not be on tdf, and both of these factors may themselves increase mortality. nonetheless, the association remained when adjusting for kidney disease, viral suppression and art duration, and concurs with results from a recently published cohort of plwh on art from spain [13] . we found both current and previous tuberculosis to be associated with covid-19 death, but since current tuberculosis itself causes death, in the absence of autopsy evidence it is difficult to disentangle the effects of covid-19 vs tuberculosis disease on mortality per se [17] . in our study, the overall high prevalence of diabetes in people with and without hiv, high proportion with poor glycaemic control and very elevated risks for covid-19 death for diabetics compared to those reported from other countries are concerning [8] . diabetes is often diagnosed late and/or untreated or poorly controlled in resource-limited settings, and the resulting microvascular disease even in people with good current diabetic control may increase covid-19 mortality [29] . to our knowledge this is the largest report on sars-cov-2 from africa, the largest report on table 1 : characteristics of (i) western cape "active patients" aged ≥20 years in public sector (public sector health care visit in last 3 years before march 1, 2020) according to covid-19 outcome (ii) covid-19 cases in "active patients" and (iii) hospitalized covid-19 cases in "active patients". reference category is hiv negative; restricted to hiv-negative patients and 199 of 601 plwh with cd4 measurement at time of covid-19 diagnosis or admission; adjusted for all other variables listed in this table in a model that included the listed categories of cd4 count instead of the binary variable hiv positive vs negative; the effect o f the other variables on mortality was similar to those presented here. hr hazard ratio; ci confidence interval; hba1c glycosylated haemoglobin; vl viral load; art antiretroviral therapy; mo months; yr years; plwh people living with hiv figure 1 : comparison of adjusted hazard ratios (hr) and 95% confidence intervals (ci) for associations with covid-19 death from cox-proportional hazards models among (i) all public sector patients ≥20 years with a public sector health visit in the previous 3 years (n=3,460,932) (ii) all adult covid-19 cases diagnosed before june 1, 2020 (n=15,203) and (iii) all hospitalized covid-19 cases (n=2, 978). covid-19 in patients with hiv: clinical case series clinical features and outcomes of hiv patients with coronavirus disease 2019 covid-19 in people living with human immunodeficiency virus: a case series of 33 patients covid-19 in patients with hiv a case series of five people living with hiv hospitalized with covid-19 in clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort opensafely: factors associated with covid-19 death in 17 million patients dysregulation of immune response in patients with covid-19 in wuhan, china lymphopenia predicts disease severity of covid-19: a descriptive and predictive study could hiv infection alter the clinical course of sars-cov-2 infection? when less is better why aren't people living with hiv at higher risk for developing severe coronavirus disease 2019 (covid-19)? incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study tuberculosis and type 2 diabetes mellitus: an inflammatory danger signal in the time of covid-19 tuberculosis, covid-19 and migrants: preliminary analysis of deaths occurring in 69 patients from two cohorts active tuberculosis, sequelae and covid-19 co-infection: first cohort of 49 cases active or latent tuberculosis increases susceptibility to covid-19 and disease severity data centre profile: the provincial health data centre of the western cape province, south africa available at: www.thembisa.org 22. vanderweele tj, ding p. sensitivity analysis in observational research: introducing the e-value hospitalized patients with covid-19 and hiv: a case series clinical features and outcome of hiv/sars-cov-2 co-infected patients in the bronx outcomes among hiv-positive patients hospitalized with covid-19 covid-19 and people with hiv infection: outcomes for hospitalized patients comparative outcomes in hospital admissions with covid-19 in people living with hiv and people living without hiv: a retrospective study diabetes mellitus in zambia and the western cape province of south africa: prevalence, risk factors, diagnosis and management reference category for hazard ratio is hiv negative; only included in adjusted analysis; adjusted for all other variables listed in this table in a model that included the listed categories of hiv viral load (vl), antiretroviral therapy (art) and immunosuppression instead of the binary variable hiv positive vs negative; the effect of the other variables on mortality was similar to those presented here. hr hazard ratio; ci confidence interval; hba1c glycosylated haemoglobin; vl viral load; art antiretroviral therapy all public-sector sars-cov-2 cases diagnosed before 1 june2020 key: cord-285403-h8ahn8fw authors: zhang, liangsheng; shen, fu-ming; chen, fei; lin, zhenguo title: origin and evolution of the 2019 novel coronavirus date: 2020-02-03 journal: clin infect dis doi: 10.1093/cid/ciaa112 sha: doc_id: 285403 cord_uid: h8ahn8fw nan to the editor-the 2019 novel coronavirus disease (2019-ncov or covid-19) recently reported from wuhan (china), which has cases in thailand, japan, south korea, and the united states, has been confirmed as a new coronavirus [1] . the 2019-ncov has infected several hundred humans and has caused many fatal cases. determining the origin and evolution of 2019-ncov is important for the surveillance, drug discovery, and prevention of the epidemic. with more and more reported pathogenic 2019-ncov isolates, it is necessary to reexamine their origin and diversification patterns. based on our phylogenomic analysis of the recently released genomic data of 2019-ncov, we showed that the 2019-ncov is most closely related to 2 severe acute respiratory syndrome (sars)-like cov sequences that were isolated in bats during 2015 to 2017 [2] , suggesting that the bats' cov and the human 2019-ncov share a recent common ancestor ( figure 1a) . therefore, the 2019-ncov can be considered as a sars-like virus and named sars-cov-2. the 2 bat viruses were collected in zhoushan, zhejiang province, china, from 2015 to 2017 [2] . there is speculation that the 2019-ncov may have originated near zhoushan or elsewhere. the new coronavirus was first isolated from stallholders who worked at the south china seafood market in wuhan. this market also sells wild animals or mammals, which were likely intermediate hosts of 2019-ncov, which originated from bat hosts ( figure 1b ). it has been speculated that the intermediate hosts (wild mammals) may have been sold to the seafood market in wuhan. the 2019-ncovs have long branches (0.09) for the 2 isolated in the phylogenomic tree ( figure 1a) , indicating that the 2019-ncovs likely share bat hosts. similarly, the 2003 sars-covs (human sars-covs) had short branches (0.03) for the bat hosts ( figure 1a) . this indicates that there should be more bat viruses closer to 2019-ncov. according to their phylogenetic relationships, the 27 isolates of 2019-ncov examined in this study can be divided into at least 6 genotypes (i-vi; figure 1c ). the 27 isolates were mainly obtained from 4 different places-thailand and the 3 chinese cities of wuhan, zhejiang, and guangdong-and all of them were present in people who visited or had contact with people from wuhan. the genotypes vi, v, and iv (guangdong and shenzhen) are located at the basal branch in the phylogenetic tree of 2019-ncov, indicating that those patients infected by these genotypes of cov were among the earliest groups to be infected. there were 3 genotypes present in samples from guangdong province, indicating that the 6 strains were infected from different places in wuhan. there were 2 genotypes found in the zhejiang province, suggesting that the 2 strains were infected from different places in wuhan. the 2 strains detected in nonthaburi, thailand, are from the same genotype and perhaps originated from the same place in wuhan. the sequence diversification between the 24 strains of 2019-ncov are small, and it is difficult to separate them in the phylogenomic tree. compared with the rapid reassortment and mutation of avian influenza (h7n9) [3, 4] , the degree of diversification of 2019-ncov is much smaller. but the 27 isolates can be divided into 6 genotypes, indicating that the 2019-ncov has mutated in different patients. the magnitude of this variation is worthy of attention in the future, and it is necessary to be vigilant for any noticeable, rapid mutations. as of today, the intermediate host of 2019-ncov has not been determined ( figure 1b) . considering that intermediate hosts are generally mammals [5] , they are likely the living mammals sold in the south china seafood market. therefore, strengthening the monitoring of wild mammals is an urgent measure needed to prevent similar viruses from infecting humans in the future. more than 1000 confirmed cases have been reported in china. the number of provinces and cities in china, as well as in other countries, with confirmed cases is steadily increasing. it is necessary to further strengthen monitoring to ensure that these virus strains will not cause diseases like the global outbreak of 2003 sars. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats rapid reassortment of internal genes in avian influenza a(h7n9) virus substitution rates of the internal genes in the novel avian h7n9 influenza virus origin and evolution of pathogenic coronaviruses the authors thank the creators of the originating and submitting laboratories of the nucleotide sequences from betacov2019-2020 of the global initiative on sharing all influenza data's epiflu database (23 january 2020; 18 isolates).potential conflicts of interest. key: cord-287923-ev93r09i authors: apisarnthanarak, anucha; mundy, linda m; tantawichien, terapong; leelarasamee, amorn title: infection prevention and control in asia: current evidence and future milestones date: 2017-05-15 journal: clin infect dis doi: 10.1093/cid/cix071 sha: doc_id: 287923 cord_uid: ev93r09i nan the asia-pacific region is a geographic source for emerging infectious diseases, including multidrug-resistant (mdr) organisms (mdros) and pathogens with pandemic potential. risks for emerging infectious diseases in this geographic region are complex and are presumed to include ecological, socioeconomic, and technological processes favorable to microbial transmission dynamics. in resource-limited settings, relative to resource-adequate settings, there continues to be a paucity of data in support of infection prevention and control, and patient safety interventions to ensure that regional, if not national, healthcare systems work effectively to improve infection prevention and control interventions. in addition, several viral pandemics and annual influenza strains have originated in the asia-pacific region, which, together, has global implications for population health. the prevention, control, and reporting of mdros and healthcare-associated infections (hais) benefit from effective data dissemination plans and harmonized surveillance systems. hais compromise patient safety because they are preventable and serve as a reservoir for mdros, which are associated with excess length of hospital stay, increased antimicrobial drug exposure, and excess costs [1] [2] [3] [4] [5] . in the asia-pacific region, the risks of hais have been estimated to be 2-20 times higher than in developed countries, with up to 25% of hospitalized patients reported to have acquired infections [4] . although infection prevention and control is well recognized in the asia-pacific region, there are inconsistencies in the quality of evidence and dedicated resources to enhance current infection prevention practices, surveillance, and patient safety [4] [5] [6] . existing evidence gaps include education, organizational and cultural barriers, physician and nursing champions, administrative support, infrastructure, fiscal resources, and key leadership that endorse implementation of infection prevention and control as a core component of patient safety programs across the asia-pacific region [6, 7] . this issue of clinical infectious diseases focuses on 3 key themes of infection prevention and control in healthcare settings across the asia-pacific regions: (1) epidemiology and evidence to support prevention and control interventions, (2) enhancements to infection prevention and control in healthcare settings, and (3) practices associated with the containment of emerging infectious diseases and outbreaks. the epidemiological data and evidence to support prevention and control interventions include 2 national survey studies for best practices and 4 epidemiology studies. a survey from thailand and japan relative to the united states compared evidence-based practices for prevention of hais and identified a modifiable gap associated with quality improvement for hospitals in these asian-pacific countries. a second national survey focused on policy, process, and outcomes associated with methicillin-resistant staphylococcus aureus (mrsa) and mdr acinetobacter baumannii interventions in thailand. higher compliance (>75%) with bundled approaches was associated with reduction in mrsa, where containment of mdr a. baumannii necessitated bundle compliance along with additional infection control interventions. four other publications describe the complex clinical factors, molecular epidemiology, and transmission dynamic associated with prevalent and emerging mdros in singapore and south korea (eg, mrsa, community-associated mrsa, and carbapenem-resistant enterobacteriaceae). together, these studies provide information on the epidemiology of infection prevention and control and the transmission dynamic of mdros in asia-pacific settings. the second theme in this issue on infection prevention and control in the asia-pacific region is the innovative use of molecular diagnostics, electronically linked administrative data, and information technology to enhance infection prevention and control. findings from 4 meta-analyses or network meta-analyses demonstrate the reduction in central catheter-associated bloodstream infection with the use of minocycline-rifampin-impregnated central venous catheters; the effects of antibiotic stewardship on outcomes including antibiotic consumptions, mortality rates, and the reduction in mdros; infection control-specific interventions associated with control of specific mdr gram-negative pathogens in intensive care units; and the potential role of the enteric microbiome in postoperative complications. together, these studies also provide strong methodological approaches for the use of network meta-analyses, which permitted assessment of these exposure-response relationships inclusive of potential confounding factors. in addition, a report from singapore describes the use of an electronic medical recordlinked administrative database that included language selectively of specific terms, using text parsing of chest radiographic reports to identify evidence of pneumonia. use of this electronic medical record-linked data source in a case validation of hospitalized adults with community-acquired pneumonia provides an enhanced method for conducting real-world evidence studies that can include enhanced case detection, diagnostic evaluations, and pathogen-specific treatment patterns in future studies. finally, the real-life feasibility of using the xpert mtb/ rif assay (cepheid) in clinical practice in a tuberculosis-prevalent country was reported from thailand. results suggest the potential role of this molecular diagnostic method in the containment of mdr tuberculosis in the asia-pacific region. the third theme is practices associated with the containment of emerging infectious diseases and outbreaks in asia-pacific countries. two publications describe successful containment for emerging infectious diseases, inclusive of lessons learned from the 15-year hong kong infectious diseases preparedness experience and the experience of zero secondary chain transmission of middle east respiratory syndrome coronavirus in thailand. together, these 2 studies emphasize the key message that control of emerging infectious diseases is feasible and sustainable in these geographic regions. finally, 2 interesting outbreaks are reported in this issue. the first is a large food-borne outbreak of streptococcus agalactiae infection in singapore. findings imply a potential role of regional collaboration to control s. agalactiae, because molecular epidemiological data were linked to the zoonotic spread from farmed freshwater fish. these fish share phylogenomic analysis of st238, similar to human and fish isolates from thailand and hong kong. a second report confirms a pseudo-outbreak of bacillus spp. bacteremia linked to contaminated hospital linen, with emphasis on the benefit of good hygienic practice in central supply and sterilization services. these collective efforts complement existing evidence for the advancement of infection prevention science in the asia-pacific region. healthcare in the asia-pacific region has had notable milestones in infection and prevention control. the publications in this issue support future work focused on continued generation of evidence and dissemination of data, ongoing microbial surveillance, and implementation, if not adoption, of effective prevention strategies to contain the spread of mdros and key emerging infectious diseases in the region. we thank the infectious diseases association of thailand for its partial sponsorship of this issue and share our hope that the collective experience from the study contributors reporting work in this issue will serve as a 2017 platform for improvement in the science and practice of infection prevention in the asia-pacific region and in other resource-limited settings. notes supplement sponsorship. this article appears as part of the supplement "infection prevention in asia pacific, " sponsored by the infectious diseases association of thailand (idat) with additional author sponsorship. potential conflicts of interest. all authors: no potential conflicts. estimating the proportion of healthcare-associated infections that are reasonably preventable and the related mortality and costs apsic guide for prevention of central line associated bloodstream infections (clabsi) apsic guidelines for environmental cleaning and decontamination the burden of healthcare-associated infections in southeast asia: a systematic literature review and meta-analysis european and asian guidelines on management and prevention of catheter-associated urinary tract infections feasibility and efficacy of infection-control interventions to reduce the number of nosocomial infections and drug-resistant microorganisms in developing countries: what else do we need? socioeconomic impact on device-associated infections in limited-resource neonatal intensive care units: findings of the inicc all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-272956-0yumc7em authors: gnavi, roberto; demaria, moreno; picariello, roberta; dalmasso, marco; ricceri, fulvio; costa, giuseppe title: therapy with agents acting on the renin-angiotensin system and risk of severe acute respiratory syndrome coronavirus 2 infection date: 2020-05-22 journal: clin infect dis doi: 10.1093/cid/ciaa634 sha: doc_id: 272956 cord_uid: 0yumc7em exposure to agents acting on the renin-angiotensin system was not associated with a risk increase of covid-19 infection in 2 italian matched case-control studies, 1 nested in hypertensive patients and the other in patients with cardiovascular diseases or diabetes. angiotensin-converting enzyme 2 (ace2) is a functional receptor for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that facilitates the entry of the virus into the cells [1] . as agents inhibiting the renin-angiotensin-aldosterone system (raas) could increase the levels of ace2, recent studies have raised concern over the association between these agents and both sars-cov-2 infection and illness severity [2] [3] [4] [5] [6] . consequently, patients treated with ace inhibitors (aceis) or angiotensin ii receptor blockers (arbs), in particular those with diabetes or cardiovascular disease, should be considered at higher risk of developing severe coronavirus disease 2019 (covid-19) infection (cvi), and of experiencing unfavorable outcomes [2, 3] . therefore, it has been suggested that these patients should be carefully monitored or even have their therapy substituted with calcium channel blockers [3, 7] . however, the hypothesis of a link between agents inhibiting the raas and cvi is still under debate [7, 8] , and whether patients should switch to other antihypertensive therapies is controversial. research to clarify the role of raas blockade therapies and cvi is highly needed [7, 9] . in the first half of february 2020, the first cases of covid-19 infection were recorded in italy [10] , that, at present, is one of the leading countries in the world for both the number of diagnosed cases and for case fatality. diabetes, hypertension, and cardiovascular diseases, conditions that are frequently treated with aceis or arbs, are among the most prevalent comorbidities in these patients [11, 12] . as, to the best of our knowledge, a relationship between acei or arb treatments and increased risk of cvi has never been demonstrated [8] , the aim of the present study was to determine whether an association exists between therapies based on agents acting on the raas and cvi in 2 populations at greater risk of being diagnosed with sars-cov-2 infection: hypertensive patients and patients who were affected by a cardio-cerebrovascular disease. we conducted 2 population-based case-control studies nested in 2 cohorts built using administrative data from the piedmont region (4 400 000 inhabitants in northwest italy, with a high rate of cvi). the population is covered by an automated system of databases, which record, among others, all drugs dispensed from all regional pharmacies, all hospital discharges, and a regional register of persons with diabetes. from the beginning of the cvi epidemics a surveillance system was implemented to collect all cases identified by reverse-transcription polymerase chain reaction testing for sars-cov-2. these archives can be linked together by a unique anonymous identifier that is encrypted to protect the patient's privacy. from the regional surveillance system, we obtained a random sample of 1000 confirmed cases of cvi occurring in the first month of the epidemic from 22 february 2020 (beginning of the epidemic) to 23 march 2020, who were linked to the 2 populations, to include only cases within cdd or hy. from the same population sources, we randomly selected 5 controls for each case, matched for year of birth and sex. cvi cases were ineligible for resampling as controls. exposure to aceis or arbs the regional drug database was used to identify cases and controls who had been prescribed aceis or arbs at any time from 1 june to 31 october 2019 (last available month), considering atc codes c09a or c09b for aceis, and c09c or c09d for arbs. the risks of cvi associated with drug dispensation of aceis or arbs, considered both separately and together (to take into account possible switch from one medication to the other), were estimated by fitting conditional logistic regression models, expressed as odds ratios (ors) and corresponding 95% confidence intervals (cis). to take into account the dose of medication prescribed (ie, to explore for a dose-response relationship), the number of boxes prescribed was grouped into 4 classes (0, 1-6, 7-12, and > 12). all calculations were made using sas version 9.4 software (sas institute, cary, north carolina). out of 804 909 hy and 337 059 cdd subjects, we identified 316 and 171 cases, respectively, of cvi, who were matched with 1580 and 855 controls. in the hy population, 58.5% were male and the mean age was 71.4 years, whereas the mean age of the cdd population was 74.5 years and 78.4% were male. among the hy population, 68.0% of cases and 73.0% of controls had at least 1 prescription of agents acting on the raas, while among the cdd population, 54.4% of cases and 55.6% of controls received at least 1 prescription. in both populations, there were no differences between cases and controls by size of the municipality of residence (supplementary table) . in neither of the 2 populations was the prescription associated with the risk of sars-cov-2 infection. in the hy population, ors for aceis, arbs, and the combination of the 2 were, respectively, 0.89 (95% ci, .70-1.15), 0.90 (95% ci, .70-1.17), and 0.78 (95% ci, .60-1.02). in the cdd population, ors for aceis, arbs, and the combination of the 2 were, respectively, 0.92 (95% ci, .64-1.32), 1.03 (95% ci, .70-1.50), and 0.95 (95% ci, .68-1.34). there was no association with the level of exposition, thus excluding a dose-response relationship ( table 1) . as a sensitivity analysis, we considered that some controls could be unknown cases of cvi and thus a differential misclassification could have occurred. we repeated our analysis for the hy population exposed to aceis or arbs (or, 0.78) considering both 10% and 20% misclassification of controls. ors showed a slight change only, to 0.81 and 0.89, respectively. the question whether therapy with agents acting on the raas increases the susceptibility to cvi has raised concern among practitioners and citizens alike [2, 7] , with conflicting opinions [8] but no sound evidence [9] . the key message of our analysis is that no association, regardless of causality, could be determined. our study has several strengths. we used a nested casecontrol design enrolling both cases and controls within 2 unselected populations of subjects at increased risk of developing cvi. the consistency of ors in these 2 independent populations can be viewed as a confirmation of our results. the study was conducted using data retrieved from the regional surveillance system of confirmed cvi cases. all agents acting on the raas were included, and, as they are dispensed and reimbursed only by prescription, we are confident to have included all dispensations. moreover, the potential confounding due to differences in access to diagnosis is probably low, given no differences in the size of municipality. our study also has potential limitations. given the high prevalence of undiagnosed or unknown cases of cvi in the general population, differential misclassification of controls is likely to have occurred. however, even assuming 20% of misclassification, the ors changed slightly, confirming the absence of association. second, the use of a database of dispensed drugs rather than usage data might have overestimated the use aceis and arbs; in addition, not considering the last 4 months closer to the onset of the disease could have slightly affected the prevalence of drug users; however, it is unlikely that these would have affected cases and controls differently. finally, given the small sample size, an association between agents acting on the raas and cvi cannot be ruled out. despite the above limitations, at the time of this writing, our study is the first contribution to explore the question whether agents acting on raas increase the risk of being infected by sars-cov-2, and the first available evidence of safety of this class of drugs. we are aware that further epidemiological research is desirable, ideally considering larger cohort studies to confirm our first findings. however, as studies gathering a large population of confirmed cases require time and may be demanding, smaller but more timely studies can contribute to give answers to urgent public health questions. furthermore, as our study was limited to explore whether raas therapy increases the risk of sars-cov-2 infection, the question whether raas therapy worsens the outcomes of cvi patients remains to be clarified. in conclusion, on the basis of our study, as also mentioned by the major cardiology societies [13, 14] , there is no reason to modify current antihypertensive therapy. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. .893 abbreviations: acei, angiotensin-converting enzyme inhibitor; arb, angiotensin ii receptor blocker; cdd, circulatory disease/diabetes; ci, confidence interval; or, odds ratio. sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor covid-19 and the cardiovascular system are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. what is the evidence? renin-angiotensin-aldosterone system inhibitors in patients with covid-19 rapid response: re: preventing a covid-19 pandemic: ace inhibitors as a potential risk factor for fatal covid-19 at present there is no evidence to abandon reninangiotensin system blockers is there an association between covid-19 mortality and the renin-angiotensin-system-a call for epidemiologic investigation case-fatality rate and characteristics of patients dying in relation to covid-19 in italy prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers hfsa/acc/aha statement addresses concerns acknowledgments. the authors thank the consorzio per il sistema informativo del piemonte for its essential contribution in prompt data gathering and data management, and dr chiara pasqualini of the regional unit for infective diseases, azienda sanitaria locale alessandria, for her valuable contribution in collecting and accessing data of the regional surveillance system.potential conflicts of interest. key: cord-265242-y8t37p0b authors: cui, wei; fan, ying; wu, wei; zhang, feng; wang, jun-ying; ni, an-ping title: expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome date: 2003-09-15 journal: clin infect dis doi: 10.1086/378587 sha: doc_id: 265242 cord_uid: y8t37p0b in a cohort of 38 patients with severe acute respiratory syndrome (sars), we observed leukopenia in 47% of patients, lymphopenia in 84%, and t lymphopenia in 95%. cd4(+) t lymphocyte levels were reduced in 100% of patients, cd8(+) t lymphocyte levels were reduced in 87%, b lymphocyte levels were reduced in 76%, and natural killer cell levels were reduced in 55%. our data suggested that these patients' immune systems were impaired during the course of sars. the absolute counts of lymphocyte subsets demonstrated a clinical significance for patients with sars. since november 2002, an outbreak of severe acute respiratory syndrome (sars) has been taking place throughout the world [1, 2] . the world health organization announced that a novel coronavirus is the cause of sars [3] [4] [5] . in the people's republic of china, beijing is the worst-affected city. as of 6 june 2003, there were 2521 cases of sars and 191 deaths reported in this city [6] . unfortunately, because sars is a new type of highly contagious disease in humans, there are no accurate and adequately field-tested laboratory diagnostic methods available yet. the diagnosis of sars remains based on clinical and epidemiological findings [7] [8] . because the immune response is likely to be a useful indicator in the development of effective methods of diagnosis and treatment of sars, we analyzed the expression of lymphocytes and lymphocyte subsets by flow cytometry in patients with sars. patients and methods. we enrolled a total of 38 patients with sars (20 male and 18 female; age range, 15-80 years) whose illnesses fit the authoritative definition of sars [9, 10] . all these patients were initially admitted to peking union medical college hospital (beijing) and had not received any ribavirin and corticosteroid therapy. in addition to the 38 patients with sars, we had previously tested 200 healthy blood donors to establish interlaboratory reference ranges for various parameters. we used these reference values as data for healthy control subjects in this study. after informed consent was obtained, 2-ml samples of edta anti-coagulated peripheral blood were collected from patients with sars before they received initial treatment. all the samples were prepared and tested within 6 h after being obtained. first, a complete blood count and a differential count were performed with an automated hematology analyzer (advia 120; bayer). second, 5 aliquots of 100 ml from each blood sample were placed in 5-ml polypropylene tubes. these blood specimens were then incubated in the dark for 20 min at room temperature with 20 ml of each of the following reagents: fluorescin isothiocyanate (fitc)-cd4/phycoerythrin (pe)-cd8/ peridinin chlorophyll protein (percp)-cd3 three-color antibody, fitccd3/pecd16+56 two-color antibody, pe-cd19 antibody, fitc-igg1/pe-igg1/percp-cd3 isotype control, fitc-igg1/pe-igg1 isotype control and pe-igg1 isotype control (all from becton dickinson). to remove contaminating erythrocytes and to fix the cells, 2 ml of lysing solution (becton dickinson) was individually added to 5 tubes. after 8 min, the samples were centrifuged (5 min at 300 g) and resuspended in 2 ml of pbs. after the second wash, samples were resuspended in 500 ml of pbs with 0.5% bovine serum albumin. the cell preparations were stored at 4њc in the dark until measurement, which was done within 4 h. every sample was measured using the epics elite esp flow cytometer (beckman coulter). before measurement, the optical path was adjusted by testing with the optics calibrator flow check (beckman coulter). the result of one-half of the value of the coefficient of variation should be !2%. we then tested cyto-trol (beckman coulter immunotech), as a control, to confirm that the results were within the target range. data acquisition and analysis were performed with the elite workstation. a count cycle contained 10,000 cells. by means of proper gating and compensation, we could distinguish lymphocytes from other clusters in a forward-versus side-scatter dot plot and then, by use of fluorescence signals, analyze the expression of cd4 + t cells (cd3 + cd4 + cd8 ϫ ), cd8 + t cells ). means ‫ע‬ sd the differences in the mean values between patients with sars and healthy control subjects were estimated with student's t test, with a limit of significance of 0.05. a p value of !.05 was considered statistically significant. results. the initial blood counts showed leukopenia (a total leukocyte count of ! cells/l) in 18 (47%) of 38 9 4 ϫ 10 patients with sars. the difference in this value between patients with sars and healthy control subjects was statistically significant ( ). leukocytosis was not observed. lympho-p ! .001 penia (an absolute lymphocyte count of ! cells/l) 9 1.00 ϫ 10 was noted in most of patients with sars. of the 38 patients, 32 patients (84%) showed a decreased lymphocyte count and 31 (82%) had a decreased lymphocyte percentage (a lymphocyte percentage of !20.6%). the mean lymphocyte count of patients with sars was significantly lower than that of healthy control subjects ( ; table 1) . the results of lymphocyte immunophenotyping demonstrated that the absolute counts of all lymphocyte subsets declined in patients with sars (table 2) . a sharply decreased t cell count was observed in most patients: 36 (95%) of the 38 patients had t cell absolute counts lower than the mean count for the healthy control subjects. decreased b cell counts were observed in 29 patients (76%). a moderate reduction in the nk cell count was noted in 21 patients (55%). among the t cell subsets, cd4 + t cell counts were found to have decreased in all patients; the lowest value was cells/l. a decrease 9 0.04 ϫ 10 in the cd8 + t cell count was observed in 33 patients (87%); the lowest value was only cells/l. there were sig-9 0.07 ϫ 10 nificant differences between patients with sars and healthy control subjects for these indices ( ). p ! .01 as for the percentages of lymphocyte subsets, the comparative percentage of t cells decreased in 22 patients (58%). in contrast, the b cell and nk cell percentages were normal or elevated in 37 patients (97.4%) and in 36 patients (94.7%), respectively. only 1 patient (2.6%) was noted to have a relative decrease in the b cell count, and 2 patients (5.3%) were noted to have a decreased nk cell count. with respect to the subsets of t cells, the percentage of cd4 + t cells relatively decreased in 31 patients (82%). the percentage of cd8 + t cells was also reduced in 13 patients (34%). seventeen patients (44%) had a decreased ratio of cd4 + t cells to cd8 + t cells. compared with healthy individuals, the t cell and b cell percentages were significant different ( ). the data are summarized in p ! .001 table 3. discussion. levels of lymphocytes and lymphocyte subsets are of great importance to keep the immune system functional. usually viral infection, immunodeficiency diseases, and other infectious diseases lead to abnormal changes in the levels of lymphocyte subsets [11] [12] [13] . at present, sars is spreading over the world as a new clinical entity. although a novel coronavirus has been identified as the cause of sars, we know little, as yet, about the mechanisms by which sars impacts the human immune system. in our study, we found that lymphopenia-in particular, t lymphopenia-was common among patients with sars, which suggests that the patient's immune system is impaired during the course of early infection with the sars virus. in addition to reductions in t lymphocyte level, reductions of b lymphocyte and nk cell levels are observed in patients with sars. because of the depletion of lymphocytes, leukopenia or a low-normal leukocyte count is noted in some patients. all these findings are quite different from those associated with pneumonia caused by common respiratory viruses, which usually is associated with a normal or elevated lymphocyte count [14] . our results with respect to the levels of t cell subsets support the hypothesis that, in patients with sars, the percentage of cd4 + t cells is much lower than that of cd8 + t cells. this implies that cd4 + t cells are more severely damaged by the sars virus than are cd8 + t cells. however, 87% of the patients with sars also show decreases in of the cd8 + t cell level, in contrast to the findings for patients with hiv infection. in general, the adaptive immune response to viral infections occurs by means of the cytotoxic t lymphocyte (ctl) response. ctls are generated in response to an invading pathogen, and they specifically recognize and kill virus-infected cells and/or release inhibitory antiviral soluble factors [12, 13] . therefore, cd8 + t cell counts should be sharply increased in patients with sars. the mechanism of reduction of cd8 + t cell counts in patients with sars needs further investigation. the human immune system shows a different response to the sars coronavirus than it does to hiv and other viruses. our study shows that the absolute counts of cd4 + t cells and cd8 + t cells have clinical significance in patients with sars and that surveillance of lymphocytes and lymphocyte subsets is helpful in the diagnosis and treatment of sars. study of lymphocytes and lymphocyte subsets is important in order to reveal the mechanism of sars infection. further study of lymphocyte levels in health care workers who were exposed to sars is ongoing in our laboratory. global update on sars cases faster…but fast enough? responding to the epidemic of severe acute respiratory syndrome coronavirus never before see in humans is the cause of sars a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome epidemic curves-severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong preliminary clinical description of severe acute respiratory syndrome severe acute respiratory syndrome (sars) updated interim case definition decreased ccr5 expression on cd4 + t cells of siv-infected sooty mangabeys acute primary infection with cytomegalovirus (cmv) in kidney transplant recipients results in the appearance of a phenotypically aberrant cd8 + t cell population epstein-barr virus specific cd8 + t cells that re-express cd45ra are apoptosis-resistant memory cells that retain replicative potential respiratory syncytial virus and other respiratory viruses we deeply thank mr. wenxin ma for his critical review of the manuscript. key: cord-252005-3ld5e7f5 authors: lewis, nathaniel m; chu, victoria t; ye, dongni; conners, erin e; gharpure, radhika; laws, rebecca l; reses, hannah e; freeman, brandi d; fajans, mark; rabold, elizabeth m; dawson, patrick; buono, sean; yin, sherry; owusu, daniel; wadhwa, ashutosh; pomeroy, mary; yousaf, anna; pevzner, eric; njuguna, henry; battey, katherine a; tran, cuc h; fields, victoria l; salvatore, phillip; o'hegarty, michelle; vuong, jeni; chancey, rebecca; gregory, christopher; banks, michelle; rispens, jared r; dietrich, elizabeth; marcenac, perrine; matanock, almea m; duca, lindsey; binder, allison; fox, garrett; lester, sandra; mills, lisa; gerber, susan i; watson, john; schumacher, amy; pawloski, lucia; thornburg, natalie j; hall, aron j; kiphibane, tair; willardson, sarah; christensen, kim; page, lindsey; bhattacharyya, sanjib; dasu, trivikram; christiansen, ann; pray, ian w; westergaard, ryan p; dunn, angela c; tate, jacqueline e; nabity, scott a; kirking, hannah l title: household transmission of sars-cov-2 in the united states date: 2020-08-16 journal: clin infect dis doi: 10.1093/cid/ciaa1166 sha: doc_id: 252005 cord_uid: 3ld5e7f5 background: although many viral respiratory illnesses are transmitted within households, the evidence base for sars-cov-2 is nascent. we sought to characterize sars-cov-2 transmission within us households and estimate the household secondary infection rate (sir) to inform strategies to reduce transmission. methods: we recruited laboratory-confirmed covid-19 patients and their household contacts in utah and wisconsin during march 22–april 25, 2020. we interviewed patients and all household contacts to obtain demographics and medical histories. at the initial household visit, 14 days later, and when a household contact became newly symptomatic, we collected respiratory swabs from patients and household contacts for testing by sars-cov-2 rrt-pcr and sera for sars-cov-2 antibodies testing by enzyme-linked immunosorbent assay (elisa). we estimated sir and odds ratios (or) to assess risk factors for secondary infection, defined by a positive rrt-pcr or elisa test. results: thirty-two (55%) of 58 households had evidence of secondary infection among household contacts. the sir was 29% (n = 55/188; 95% confidence interval [ci]: 23–36%) overall, 42% among children (<18 years) of the covid-19 patient and 33% among spouses/partners. household contacts to covid-19 patients with immunocompromised conditions had increased odds of infection (or: 15.9, 95% ci: 2.4–106.9). household contacts who themselves had diabetes mellitus had increased odds of infection (or: 7.1, 95% ci: 1.2–42.5). conclusions: we found substantial evidence of secondary infections among household contacts. people with covid-19, particularly those with immunocompromising conditions or those with household contacts with diabetes, should take care to promptly self-isolate to prevent household transmission. the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) outbreak that began in china in december 2019 has become a global pandemic. as of july 3, 2020, the united states has reported the greatest number of cases and deaths worldwide [1] . transmission has been reported in many settings, including health care facilities and family and community gatherings [2] [3] [4] . in china, household transmission and contact tracing investigations found that a range of 3% to 32% of household contacts acquired infection [5] [6] [7] [8] [9] [10] . however, published data on systematic household transmission investigations in us households are scarce. the centers for disease control and prevention collaborated (cdc) with state and local health departments in the milwaukee, wisconsin, and salt lake city, utah, metropolitan areas to identify persons with laboratory-confirmed sars-cov-2 infection captured by public health surveillance during march 22-april 25, 2020. in wisconsin, sars-cov-2 outpatient testing was limited to persons meeting cdc criteria for influenza testing [11] and excluded asymptomatic or mildly symptomatic a c c e p t e d m a n u s c r i p t 4 persons who were not health care workers [12] . outpatient testing in utah required clinical features (fever, cough, or shortness of breath) and an epidemiologic risk factor [13] . the investigation team defined persons identified by local health departments as "index patients." households were selected by convenience sampling and considered eligible if the index patient was not hospitalized at the time, lived with ≥1 additional person, and tested positive for sars-cov-2 rrt-pcr from a nasopharyngeal (np) swab collected ≤10 days prior to enrollment. all persons in the household were asked to participate; we excluded households where >1 person declined. prior to the first household visit, questionnaires capturing demographic characteristics, medical histories, and recent symptoms were administered to index patients and all household contacts by phone. a household-level questionnaire captured physical characteristics of the residence (e.g., square footage, number of bedrooms and bathrooms). for each household, the investigation team conducted an initial in-person visit (on day 0) and a visit 14 days later (day 14). the investigation period was defined as 14 days, the maximum duration of the sars-cov-2 incubation period [14, 15] . on day 0 and day 14, we collected an np swab, self-collected anterior nasal swab, and blood sample for all persons living in the household, including the index patient. each person completed a daily symptom diary during days 0-14; a newly symptomatic person prompted an interim visit during which repeat swabs were obtained for all household contacts. during day 0 and day 14 visits, we interviewed households on precautionary practices to reduce transmission. additional information regarding household selection and investigation protocols are in the supplemental appendix. swabs were tested by the milwaukee health department laboratory (mhdl) or the utah public health laboratory (uphl) using the cdc 2019 novel coronavirus real time rt-pcr assay [16] . blood samples were processed by mhdl or uphl; sera were subsequently shipped to cdc and tested using a cdc-developed sars-cov-2 enzyme-linked immunosorbent assay (elisa) [17] . for this analysis, we excluded households for which the primary patient could not be determined. we summarized household environment characteristics, as well as demographic characteristics and medical histories of primary patients and household contacts. we estimated the sir as the proportion of participating household contacts with secondary infection and estimated the serial interval as the number of days from symptom onset of the primary patient to symptom onset of the associated household contact with secondary infection. statistical tests of trends were calculated using the cochran-armitage trend test. two-sided p-values and 95% confidence intervals (ci) for binomial proportions were calculated with chi-square tests and wilson score intervals, respectively. a c c e p t e d m a n u s c r i p t 6 characteristics of household environments, primary patients, and household contacts were assessed as potential risk factors. we estimated unadjusted odds ratios (or) and 95% ci for potential risk factors of secondary infection among household contacts using a generalized estimation equation (gee) approach [19] . to account for within-household correlation, we specified an exchangeable correlation structure by households. to address potential misclassification of secondary infections among household contacts, we excluded contacts with evidence of sars-cov-2 infections by serology only and repeated sir calculations and risk factor analysis. we estimated risk ratios and its 95% ci for household environment characteristics as potential risk factors for whether any secondary transmission occurred in the household. we described precautionary practices reported by households on day 0 and day 14. data collected from questionnaires and symptom diaries were entered into a redcap electronic database hosted at cdc [20, 21] . analyses were conducted in sas enterprise guide, version 7.1 (sas institute, cary nc). this protocol was reviewed by cdc human subjects research officials and the activity was deemed non-research as part of the covid-19 public health response. we enrolled sixty-two households (utah, n = 36; wisconsin, n = 26), and excluded four households for which we could not identify the primary patient. among the remaining 58 households (utah, n = 34; wisconsin, n = 24), 58 primary patients and 188/197 (95%) household contacts were included in the analysis; 9 household contacts declined participation after their respective households were table 2 ). the median house size was 2,200 square feet (range: 600-8,000). demographic and clinical characteristics of primary patients and household contacts are shown in table 1 . the median age was 40 years (range: 16-90) for primary patients and 22 years (range: <1-76) for household contacts. among 58 primary patients, 21 (36%) had at least one underlying medical condition; the most common symptoms were respiratory (n = 56, 97%), followed by neurologic (n = 51, 88%), constitutional (n = 50, 86%), and gastrointestinal (n = 41, 71%) (supplementary table 3 ). one primary patient was asymptomatic and tested based on a known nonhousehold exposure. median intervals to the day 0 household visit were 11 days (interquartile range table 4 ). by the initial household visit, 43/52 (83%) had acquired secondary infection; 9 (17%) household contacts acquired secondary infection during the 14-day investigation period. eight (15%) of 52 household contacts with secondary infection had detectable sars-cov-2 antibodies but no positive rrt-pcr test during the investigation. among these 8 household contacts, 3 (38%) seroconverted between days 0 and 14, 4 (50%) were seropositive on day 0, and 1 (12.5%) did not have serology testing on day 0 but was rrt-pcr-negative and seropositive on day 14; all 8 were symptomatic with symptom onset occurring ≥2 days after symptom onset of the corresponding primary patient. table 6 ). repeat risk factor analyses excluding household contacts who were seropositive but rrt-pcr-negative produced similar results, with the exception that household contacts of primary patients with constitutional symptoms were found to be more likely to have a secondary infection table 8 ). among the 58 households, 55 (95%) reported precautionary practices at any point following symptom onset of the first laboratory-confirmed covid-19 patient in the household; 51 (88%) households reported ill persons sleeping in a separate bedroom and 33 (57%) reported use of cloth face covers or masks by ill persons (figure 4 ). household-level precautionary practices assessed were not associated with preventing household transmission (supplementary table 8 ). these findings from suggest that us household settings may lead to substantial sars-cov-2 transmission. by the day 0 household visit, 83% of the secondary infections among household contacts had already occurred, highlighting the importance of timely case identification and isolation. transmission occurred in 55% of households and the sir was 29% among household contacts. we demonstrate potentially increased infection risk among children and spouses of primary patients living in the same household, household contacts of primary patients with an immunocompromising condition, household contacts of male primary patients, and household contacts with diabetes mellitus. previously reported household sirs from china were 3-32% [5] [6] [7] [8] [9] [10] , and a recent point-prevalence study of household transmission in new york, united states estimated a 38% sir [22] . one study from china estimated a 28% sir for spouses of primary patients and 4% for household contacts aged a c c e p t e d m a n u s c r i p t 10 <18 years by sars-cov-2 rrt-pcr testing [6] . we observed similar sirs of 33% among spouses of primary patients and 23% among household contacts aged <18 years after limiting our results to detection by rrt-pcr alone. our sirs may vary from china and new york based on differences in sociocultural context and an investigation protocol (i.e., entire household testing,14-day follow-up, serologic testing) that likely captured infections missed in point-prevalence surveys, routine contact tracing, or rrt-pcr testing alone. people with diabetes mellitus within our investigation had increased odds of acquiring sars-cov-2 infection, although collinearity between diabetes mellitus and obesity could have confounded the association. among covid-19 patients, comorbidities such as diabetes, cardiovascular, cerebrovascular, and oncologic diseases have been identified as risk factors for severe disease and increased mortality [14, [23] [24] [25] [26] [27] . our findings illustrate that increased risk posed by diabetes for covid-19 could include susceptibility to sars-cov-2 infection as well as increased morbidity and mortality, particularly among people with poorly controlled diabetes [28, 29] . people with diabetes are at increased risk for infection generally [30, 31] investigation. reasons for this is unclear, although it may reflect behavioral differences between male primary patients and female primary patients or increased viral shedding as males have a higher likelihood of developing severe symptoms [7, 24] . most households demonstrated willingness to adopt precautionary practices, with most reporting ill persons sleeping in a separate bedroom and over half reporting use of cloth face covers or masks by ill persons. although we did not find transmission differences between households where ill persons isolated and those where they did not, we were unable to capture the extent, timing, and consistency of such precautionary practices. a household transmission study from china, however, demonstrated that mask use and self-isolation decreased risk of secondary sars-cov-2 infection in households [8] . further investigations are needed to identify measures that may be acceptable to and successful for us households. these results must be considered with respect to several limitations. first, we assumed that household transmission was responsible for infections among household contacts. the household sir could therefore be an overestimation, although concurrent stay-at-home orders should have limited community exposures. second, misclassification of primary patients would affect risk factor analysis. third, households were from convenience samples from 2 states, and not representative of all us households. future studies should assess, for example, households of primary patients with severe illness and transmission dynamics in apartment buildings. fourth, transmission had already occurred in some households by enrollment due to delays in testing and reporting at the time of the investigation. timelier enrollment would help differentiate between transmission generations and refine our risk factor analysis among household contacts. fifth, we may have missed infections as repeat respiratory swabs were not obtained within a 24-hour period to confirm or exclude sarsa c c e p t e d m a n u s c r i p t 12 period. sixth, timing and consistency of precautionary practices were not obtained, and we were unable to evaluate their efficacy. finally, our approach to assessing household-level risk factors for secondary transmission did not account for individual-level characteristics. our findings should thus be considered hypothesis-generating and suitable for evaluation in future analytic studies. households are likely major settings of sars-cov-2 transmission of in the united states. transmission dynamics are not uniform across or within households and some people, including spouses and children of primary patients, people living with immunocompromised primary patients, and people with diabetes, may be at higher risk of secondary infection. given public health guidance to isolate at home when sick or to quarantine at home when exposed, effective strategies to reduce a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t covid-19 response team utah department of health salt lake county health department city of milwaukee health department covid-19): cases in the first known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention household transmission of sars-cov-2 the characteristics of household transmission of covid-19 epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study household transmission of sars-cov-2 report of the who-china joint mission on coronavirus disease 2019 (covid-19): world health organization household secondary attack rate of covid-19 and associated determinants in guangzhou, china: a retrospective cohort study. the lancet infectious diseases guide for considering influenza testing when influenza viruses are circulating in the community urgent update -prioritization of covid-19 testing for hospitalized patients covid-19 testing criteria process and overview clinical characteristics of coronavirus disease 2019 in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia centers for disease control and prevention. cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel. available at covid-19): people who are immunocompromised. available at: validation of a sars-cov-2 spike protein elisa for use in contact investigations and sero-surveillance models for longitudinal data: a generalized estimating equation approach research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners characteristics and clinical outcomes of adult patients hospitalized with covid-19 -georgia hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan cerebrovascular disease is associated with an increased disease severity in patients with coronavirus disease 2019 (covid-19): a pooled analysis of published literature cancer patients in sars-cov-2 infection: a nationwide analysis in china association of blood glucose control and outcomes in patients with covid-19 and pre-existing type 2 diabetes newly diagnosed diabetes is associated with a higher risk of mortality than known diabetes in hospitalized patients with covid-19 risk of infection in type 1 and type 2 diabetes compared with the general population: a matched cohort study diabetes and the occurrence of infection in primary care: a matched cohort study risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia risk factors for mers-cov seropositivity among animal market and slaughterhouse workers plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars. diabetic medicine : a journal of the clinical features and short-term outcomes of 144 patients with sars in the greater toronto area a c c e p t e d m a n u s c r i p t 14 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 16 a c c e p t e d m a n u s c r i p t key: cord-287119-eo0evoog authors: kendall, emily a title: when infections don’t reflect infectiousness: interpreting contact investigation data with care date: 2020-08-08 journal: clin infect dis doi: 10.1093/cid/ciaa1144 sha: doc_id: 287119 cord_uid: eo0evoog nan a c c e p t e d m a n u s c r i p t contact tracing, besides being a useful public health tool for both finding superspreaders [1] and treating the exposed [2] , is often central to learning about the dynamics of disease transmission [3, 4] . in their article in this issue of cid, martinez and colleagues made use of contact investigation data to evaluate the association between tb and hiv from the perspective of tb transmission. by systematically reviewing studies of the contacts of index cases with tb, martinez and colleagues determined that the contacts of hiv-positive tb patients were approximately 33% less likely to be infected with m. tuberculosis than the contacts of hiv-negative tb patients. similar uses of contact tracing to estimate infectiousness have recently been discussed widely in the context of covid-19. in south korea, data from the country's thorough covid-19 contact tracing program [5] was used to analyze the relationship between the age of an index case and the prevalence of sars-cov-2 infection among his or her contacts. this study, which described a relatively high prevalence of covid-19 among the contacts of older child and adolescent index cases, has been widely interpreted as evidence that older children with covid-19 may be as infectious as adults. care is required, however, when using contact investigation data to evaluate infectiousness. although the prevalence of infection or disease among the contacts of an index case may reflect the infectiousness of that case, it is far from a direct measure of infectiousness. interpreting it as such invokes at least three assumptions about transmission: that transmission occurs over the same time period for all index cases, that the index case was always the first person infected within a contact pair, and that infections identified through contact investigation share a direct transmission link. in reality, each of these three assumptions may be violated in important ways. first, transmission depends on both the degree and the duration of infectiousness. contact investigation data can compare the cumulative amount of transmission that has occurred from two index cases, but it should not be interpreted as measuring their relative infectiousness on any given day unless their duration of infectiousness has been equivalent. as asymptomatic carriers illustrate [6, 7] , less infectious hosts can ultimately spread more disease than those with a higher pathogen burden, if their milder disease allows the natural duration of their infectiousness to be prolonged. similarly, characteristics that cause delays in diagnosis (and therefore in isolation or treatment) may increase cumulative transmission without affecting disease duration also increase transmission. for tb, the more rapid progression of hiv-associated cases to clinically diagnosable disease or death causes hiv-negative tb to be overrepresented among prevalent cases [8] . therefore, when martinez and colleagues present evidence that hiv-positive tb patients generate fewer secondary cases, the explanation for this finding might be that they are less infectious at any given moment, or it might be that their shorter duration of tb disease provides less opportunity to spread. a similar but opposite effect might be observed in contact-based estimates of the infectiousness of drug-resistant tb: because the detection and appropriate treatment of drug-resistant tb is often delayed, contactbased study designs may overestimate the relative infectiousness of drug-resistant cases (and thus under-estimated fitness costs associated with drug resistance). a second caveat to estimating infectiousness from contact data is that the first person diagnosed may not have been the first infected. "index case" describes the sequence of detection, and not necessarily that of transmission. sequences of detection and transmission may correspond poorly for diseases with variable incubation periods, low case detection rates, or high degrees of m a n u s c r i p t asymptomatic or presymptomatic transmission. in the case of tb, although hiv promotes rapid disease progression, many hiv-uninfected people experience months or years of protracted tb with minimal symptoms [9] . in some hiv-negative individuals, tb may even resolve without ever being diagnosed or treated [10] . thus, an hiv-negative person who spreads tb to their hiv-positive contact may persist with active tb until a contact investigation of the hiv-positive index case is conducted, or they may spontaneously improve prior to contact investigation (such that they appear to be a latently-infected contact). in studies such as those reviewed by martinez and colleagues, misclassifying the direction of transmission would tend to weaken any true association between hiv status and infectiousness. covid-19 contact investigation data may be prone to similar effects. in south korea's recent contact study, the authors noted they could not determine direction of transmission, and the data do not fully support the widespread interpretation of the proportion of contacts infected as a measure of index case infectiousness. in the rare instances that an older child or adolescent was the index case (2% of all clusters), they had a small number of total contacts, so although the proportion of contacts infected was relatively high, the absolute number of infected contacts was low (less than 1, on average). these young people had to be infected with covid by someone, even if that source had gone undiagnosed. such data would be consistent with an alternative scenario in which most or even all transmission originated from adults. the child and adolescent index cases would represent the occasions in which the adult source had asymptomatic disease but the child or adolescent whom they infected developed covid symptoms -leading the secondarily-infected child to become the cluster's index case. a third limitation of contact investigation data is that contact investigations cannot evaluate all possible exposures. an index case and his or her contact may both have been infected by an external source (either shared or distinct) -and the risk of exposure to external cases in the community may depend on other characteristics of the index case. for tb, martinez and colleagues previously estimated that >80% of transmission occurs outside of households [11] . moreover, data on drug resistance concordance suggest that even when two active tb cases develop within a household in rapid succession, their infections are unrelated up to 20% of the time [12] . thus, the prevalence of infection among an index case's contacts reflects, in part, the past and present risk of tb exposure within those contacts' broader networks. determinants of tb exposure include spatial and socioeconomic factors that are shared at the household level. where those household-level risk factors for tb exposure are also associated with hiv (such as vulnerable economic status or living in a high tb-and hiv-burden neighborhood), they may confound the relationship between index case hiv status and household prevalence of tb infection. in summary, the prevalence of infection among contacts is an imperfect measure of index case infectiousness. independent of index case infectiousness, the prevalence of infection in contacts may be increased by index case characteristics that extend the duration of disease over which transmission can occur, that allow secondary cases to be diagnosed sooner than the sources of their infections, or that increase the household-level risk of exposure to cases in the broader community. the magnitude of these effects on estimates of the infectiousness of hiv-associated tb is uncertain, but the biological plausibility of risk factors that martinez and colleagues identified for infectiousness within hiv populations (namely, high sputum bacillary burden and less advanced hiv disease) suggest that the bias may be small. more importantly, regardless of mechanism, the measured m a n u s c r i p t burden of infection and disease in contacts of hiv-positive tb index cases has clear policy implications: contacts of tb patients are at high risk for tb, and contacts of hiv-positive tb patients have nearly as high a risk of tb (and a much higher risk of hiv) than other tb contacts. whether or not their high risk is a direct result of index case infectiousness, they are an important target population for interventions to diagnose and prevent disease, and contact investigation is a useful tool for delivering that care. contact tracing strategies in heterogeneous populations contact investigation for tuberculosis: a systematic review and meta-analysis transmission of nipah virus -14 years of investigations in bangladesh household transmission of vibrio cholerae in bangladesh early release -contact tracing during coronavirus disease outbreak mr n the milker, and dr koch's concept of the healthy carrier contribution of asymptomatic plasmodium infections to the transmission of malaria in kayin state the prevalence of hiv among adults with pulmonary tb at a population level in zambia tuberculosis transmission during the subclinical period: could unrelated cough play a part? natural history of tuberculosis: duration and fatality of untreated pulmonary tuberculosis in hiv negative patients: a systematic review transmission of mycobacterium tuberculosis in households and the community: a systematic review and meta-analysis concordance of drug resistance profiles between persons with drug-resistant tuberculosis and their household contacts: a systematic review and meta-analysis this work was supported by the national institutes of health [k08ai127908 to e.a.k.]. dr. kendall had no conflicts to declare. a c c e p t e d m a n u s c r i p t key: cord-007064-nepgttxf authors: chemaly, roy f; dadwal, sanjeet s; bergeron, anne; ljungman, per; kim, yae-jean; cheng, guang-shing; pipavath, sudhakar n; limaye, ajit p; blanchard, elodie; winston, drew j; stiff, patrick j; zuckerman, tsila; lachance, silvy; rahav, galia; small, catherine b; mullane, kathleen m; patron, roberto l; lee, dong-gun; hirsch, hans h; waghmare, alpana; mckevitt, matt; jordan, robert; guo, ying; german, polina; porter, danielle p; gossage, david l; watkins, timothy r; marty, francisco m; chien, jason w; boeckh, michael title: a phase ii, randomized, double-blind, placebo-controlled trial of presatovir for the treatment of respiratory syncytial virus upper respiratory tract infection in hematopoietic-cell transplant recipients date: 2019-12-03 journal: clin infect dis doi: 10.1093/cid/ciz1166 sha: doc_id: 7064 cord_uid: nepgttxf background: hematopoietic-cell transplant (hct) recipients are at risk for severe respiratory syncytial virus (rsv) infection. we evaluated the rsv fusion inhibitor presatovir in a randomized, double-blind, phase ii trial in hct recipients with rsv upper respiratory tract infections. methods: patients were stratified by lymphopenia (<200/µl) and ribavirin use; were randomized, stratified by lymphopenia (<200/μl) and ribavirin use, to receive oral presatovir at 200 mg or a placebo on days 1, 5, 9, 13, and 17, and were followed through day 28. the coprimary efficacy endpoints were the time-weighted average change in the nasal rsv viral load between days 1 and 9 and the proportion of patients developing lower respiratory tract complications (lrtcs) through day 28. results: from 23 january 2015 to 16 june 2017, 189 patients were randomly assigned to treatment (96 to presatovir and 93 to the placebo). presatovir treatment, compared with the placebo treatment, did not significantly affect (prespecified α = 0.01) a time-weighted average decline in the rsv viral load from day 1 to 9 (treatment difference, −0.33 log(10) copies/ml; 95% confidence interval [ci] −.64 to −.02 log(10) copies/ml; p = .040) or the progression to lrtc (11.2% vs 19.5%, respectively; odds ratio, 0.50; 95% ci, .22–1.18; p = .11). in a post hoc analysis among patients with lymphopenia, presatovir decreased lrtc development by day 28 (2/15 [13.3%] vs 9/14 [64.3%], respectively; p = .008), compared with the placebo. adverse events were similar for patients receiving presatovir and the placebo. conclusions: presatovir had a favorable safety profile in adult hct recipients with rsv but did not achieve the coprimary endpoints. exploratory analyses suggest an antiviral effect among patients with lymphopenia. clinical trials registration: nct02254408; eudra-ct#2014-002474-36. adult recipients of autologous or allogeneic hematopoietic-cell transplants (hct) are at high risk for respiratory syncytial virus (rsv) infection and associated morbidity and mortality. up to 17% of hct recipients may develop an rsv infection [1] [2] [3] [4] [5] [6] [7] , of whom 17% to 84% progress from an upper respiratory tract infection (urti) to a lower respiratory tract infection (lrti) [2, 3, 5, [7] [8] [9] [10] [11] [12] [13] [14] . progression to a lrti often requires hospitalization, during which oxygen supplementation and intensive care may be required; rsv lrtis are associated with increased mortality rates, ranging from 6% to 35% [2, 4, 8, 9, [15] [16] [17] [18] [19] [20] . survivors of a respiratory viral infection after hct may have a long-term airflow decline [15, 21] . currently, there are no effective vaccines or approved antiviral agents for rsv infection in hct recipients. aerosolized ribavirin (virazole) is approved for the treatment of rsv infections in young children but is not used in general pediatric practice because of efficacy and tolerability concerns and the complexity of the required specialized aerosol delivery system [22] [23] [24] . a randomized, placebo-controlled trial of aerosolized ribavirin attempted in hct recipients recruited only 14 subjects in 5 years due to slow accrual [25] . epidemiologic studies and a single-center, retrospective analysis suggest that ribavirin-based therapy has some efficacy for preventing rsv-associated morbidity or mortality in high-risk hct recipients [6, 14, 18] . however, these results are from uncontrolled, retrospective studies and the ribavirin benefit remains unconfirmed. thus, there remains a significant, unmet medical need for safe, convenient, and effective treatments for rsv infection. presatovir (formerly gs-5806) is an oral rsv fusion inhibitor with potent and selective anti-rsv activity in vitro and a terminal half-life of ~34 hours [26] . when tested in a human challenge study of healthy volunteers, presatovir reduced the rsv viral load and the severity of clinical disease [26] . in the current study, we evaluated presatovir's safety, tolerability, and efficacy among hct recipients with rsv urtis. this phase ii, randomized, double-blind, placebo-controlled, 2-group, parallel study recruited allogeneic or autologous hct recipients with positive local rsv test results who were 18 to 75 years of age from 43 centers in 9 countries (supplementary appendix). patients with new or worsening respiratory symptoms for ≤7 days, who had been diagnosed with an rsv infection of the upper respiratory tract for ≤6 days, and who were without new abnormalities on a chest x-ray obtained <48 hours from the start of study treatment were eligible to participate. patients with a specified, documented respiratory virus coinfection within 7 days from the start of study treatment or with another significant respiratory or systemic infection were excluded. the full eligibility criteria are provided in the supplementary methods. this study followed international conference on harmonization requirements and the principles of the declaration of helsinki and was approved by local ethics committees. written informed consent was obtained from patients or their legally responsible representatives. the protocol amendments and data monitoring committee activities are described in the supplementary methods. the trial was registered at clinicaltrials.gov (nct02254408) and eudract (2014-002474-36) before enrollment began. patients were randomly assigned (1:1) to receive presatovir or a placebo, were stratified centrally by lymphopenia (lymphocyte count <200 cells/mm 3 within 6 days of screening), and were prescribed the use of ribavirin by any route of administration at randomization. the study treatment assignments were provided by an interactive web response system (bracket global, wayne, pa, usa). patients, all study staff, and the study sponsor were blinded to study treatment. allocation was concealed by the use of presatovir and placebo tablets that were identical in appearance. the patients received presatovir at 200 mg (4 × 50 mg tablets) or a placebo orally or by a nasogastric tube during study visits on days 1, 5, 9, 13 , and 17 (±24 hours), and were followed through study day 28. based on human pharmacokinetic and pharmacodynamic studies [26] , this regimen was predicted to provide plasma concentrations >4-fold over requirements to inhibit the replication of >95% of tested rsv isolates. patients with detectable rsv by reverse transcription quantitative polymerase chain reaction (rt-qpcr) on day 22 could participate in an optional extended, weekly follow-up through day 56. a detailed schedule of the study assessments and procedures is provided in supplementary table 1 . plasma pharmacokinetic methods are described in the supplementary methods. for virology assessments, bilateral intranasal swabs were obtained using mid-turbinate, adult, flocked swabs (copan diagnostics, murrieta, ca) at each study visit. samples were analyzed using rt-qpcr to measure the rsv viral load; rsv f gene sequencing, to detect the development of resistance; and a multiplex assay to identify respiratory viral coinfections. all nasal samples were analyzed at central laboratories; further methodological details are provided in the supplementary methods. chest x-rays or computed tomography scans were performed per standard care in patients with suspected lower respiratory tract complications (lrtc). imaging studies and results of local microbiology tests were collected for review by the endpoint adjudication committee. clinical safety assessments included vital signs, body weight, and oxygen saturation by pulse oximetry; laboratory safety assessments included complete blood cell counts and liver enzyme measurements. cardiac safety was assessed via electrocardiograms and troponin testing (per us food and drug administration cardiac monitoring requirements) on days 1, 17, and 28. additional safety assessments included the evaluation of adverse events (aes) and the documentation of concomitant medications. the coprimary endpoints were the time-weighted, average change in the nasal rsv viral load, measured by rt-qpcr (log 10 copies/ml) between day 1 and day 9, and the proportion of patients who developed lrtcs-defined as a primary rsv lrti, a secondary bacterial lrti, a lower respiratory tract infection due to unusual pathogens, or an lrtc of unknown etiology-from day 1 through day 28. the development of an lrtc was determined by an independent, blinded endpoint adjudication committee (details are in the supplementary methods). the secondary efficacy endpoint was the proportion of patients who died or developed respiratory failure requiring invasive mechanical ventilation from day 1 to day 28. safety was assessed from aes, vital signs, electrocardiograms, and clinical laboratory test results. assuming a time-weighted average change in the rsv viral load from day 1 to day 9 of −1 log 10 copies/ml with a standard deviation (sd) of 2 log 10 and an lrtc event rate of 30% in patients receiving the placebo, 100 patients per treatment group were planned to provide >80% power to detect a ≥1-log 10 decrease in the first coprimary endpoint with a 2-sided α of 0.01 and >90% power to detect a ≥20% reduction in the second coprimary endpoint with a 2-sided α of 0.04 in patients receiving presatovir, relative to the placebo. the efficacy population included patients who received ≥1 dose of presatovir with a quantifiable nasal rsv viral load on day 1. the coprimary and secondary endpoints were analyzed in the efficacy population and in prespecified subgroups defined by the randomization stratification factors (lymphopenia and ribavirin use on day 1), and were also analyzed post hoc in subgroups defined by the duration of rsv symptoms, hospitalization status, time after hct, and graft-vs-host disease (gvhd) status on day 1. the safety population included patients who received ≥1 dose of presatovir. the first coprimary analysis was performed by a parametric analysis of covariance with the baseline viral load and randomization stratification factors as covariates. the second coprimary analysis and secondary efficacy analysis were performed using 2-sided cochran-mantel-haenszel tests stratified by lymphopenia (<200 cells/mm 3 ) and the intent to use ribavirin at baseline. if the number of events was small, the fisher exact method was applied. a fallback approach was employed to control the type i error rate at 0.05 across the coprimary and secondary endpoints (details are in the supplementary methods). subgroup analyses were performed using the corresponding analysis of covariance model for the first coprimary endpoint and the fisher exact test, with a 95% confidence interval (ci) based on the clopper-pearson method, for the second coprimary and secondary endpoints. from 23 january 2015 to 16 june 2017, 213 patients were screened for eligibility; 24 patients were excluded, the majority (n = 14) of whom did not have a documented rsv infection of the upper respiratory tract. a total of 189 patients were randomly assigned to a study treatment (96 to presatovir and 93 to the placebo), and 185 received ≥1 dose of a study drug (95 received presatovir and 90 received the placebo; figure 1 ). the sponsor halted the study on 20 september 2017, before achieving the planned 200-subject enrollment, because an unplanned interim analysis before a database lock by an unblinded team indicated that results were unlikely to differ if enrollment was extended through another rsv season. important protocol deviations are described in the supplementary results and supplementary table 2 . overall, 168 (90.8%) patients (88 assigned to presatovir and 80 to the placebo) completed treatment with a study drug through day 17 ( figure 1 ). patient demographic and baseline clinical characteristics were generally well balanced between treatment groups, except for hospitalization of a larger number of patients receiving presatovir, compared with the placebo, at the beginning of study treatment (43.2% vs 26.7%, respectively; table 1 ). the majority of treated patients (146/185, 78.9%) underwent allogeneic hct, and 69/185 (37.3%) had gvhd at baseline. lymphopenia was noted in 29 (15.7%) patients, and 44 (23.8%) patients were treated with aerosolized or oral ribavirin at baseline (table 1) . efficacy figure 2a -b shows the absolute rsv viral load and change from baseline at each study visit. despite adequate plasma concentrations (supplementary results; supplementary table 3) , presatovir did not significantly (prespecified α = 0.01) reduce the time-weighted average change in the rsv viral load from day 1 to day 9, compared with the placebo (mean, −1.26 [sd, 0.964] log 10 copies/ml vs −0.91 [sd, 1.145] log 10 copies/ml, respectively; treatment difference, −0.33 log 10 copies/ml; 95% ci, −.64 to −.02 log 10 copies/ml; p = .040). the development of lrtcs through day 28 is shown in figure 3 . compared with the placebo, presatovir did not significantly reduce the proportion of patients in the efficacy population who developed an lrtc from day 1 through day 28 ( ). there were 2 events in each treatment arm that were attributed to a primary rsv lrti, and 1 event in the presatovir arm was adjudicated as a secondary bacterial infection. sensitivity analyses are reported in the supplementary results. death or respiratory failure requiring mechanical ventilation through day 28 occurred in 5/89 (5.6%) patients receiving presatovir and 5/87 (5.7%) patients receiving the placebo (p = .98; figure 4 ). in prespecified subgroup analyses, presatovir numerically decreased the proportion of patients who developed an lrtc from day 1 through day 28, relative to the placebo, among patients with baseline lymphopenia ( table 2; supplementary tables 4 and 5 ). the proportions of patients receiving presatovir who developed lrtc were similar among patients without baseline lymphopenia and in patients without ribavirin use at baseline, as compared to those receiving the placebo (supplementary tables 4 and 5 ). overall, ribavirin use was higher among patients who developed an lrtc (37.0%) versus those who did not (23.5%). patients hospitalized at baseline had a numerically higher rate of lrtcs, relative to those who for this value, n = 10 for presatovir, n = 11 for placebo, and n = 21 total. c for this value, n = 88 for placebo and n = 183 total. , and the hospitalization status was imbalanced between the presatovir and placebo arms at baseline. the effects of presatovir versus placebo treatment on the time-weighted average change in the viral load from day 1 to day 9 and the occurrence of death or respiratory failure requiring mechanical ventilation through day 28 were similar between patients hospitalized and not hospitalized on day 1 (supplementary table 6 ). however, treatment with presatovir, relative to the placebo, was associated with a 28% lower lrtc event rate among patients hospitalized on day 1 ( table 2; supplementary table 6 ). in other post hoc analyses, the proportion of patients who developed lrtcs was numerically lower following presatovir treatment, as compared to placebo treatment, among patients with shorter than median symptom durations (≤4 days) and ≤365 days since hct (table 2; supplementary tables 6-9) . a post hoc multivariate cox proportional hazard model for the time to an lrtc through day 28 in patients receiving presatovir, adjusted for lymphopenia and ribavirin use on day 1, enrollment site, and hospitalization status on day 1, yielded an adjusted hazard ratio of 0.44 (95% ci, .19-.99; p = .091), as compared to those receiving the placebo. optional extended rsv monitoring and serologic responses are presented in the supplementary results. patients with treatment-emergent substitutions in rsv f that were associated with presatovir resistance had a numerically smaller change in the time-weighted average rsv load, but not worse clinical outcomes, relative to those with wild-type f sequences; such substitutions occurred at a significantly higher frequency in patients with, versus without, lymphopenia (supplementary results; supplementary tables 10-11). overall, aes were reported in 76 (80%) of the patients receiving presatovir and 78 (86.7%) of the patients receiving the placebo, while 18 (18.9%) of the patients receiving presatovir and 23 (25.6%) of the patients receiving the placebo had serious adverse events (saes). the most common aes were diarrhea (15.8%), nausea (13.7%), and pyrexia (12.6%) in the patients receiving presatovir; and diarrhea (15.6%), vomiting (13.3%), and nausea (11.1%) in the patients receiving the placebo (table 3 ). most grade 3 or 4 aes and saes occurred less frequently in patients receiving presatovir, except for pyrexia as an sae in 4 (4.2%) patients and gvhd in the gastrointestinal tract as an sae, grade 3 pyrexia, and grade 4 pneumonia in 2 (2.1%) patients each (supplementary tables 12-13 ). there were no imbalances in new electrocardiogram findings or troponin abnormalities between the 2 groups. overall, 6 patients died during the study; 2 (2.1%) were treated with presatovir and 4 (4.4%) were treated with the placebo. there were 2 patients receiving presatovir who died from gastrointestinal hemorrhage and pneumonia (1 each), and 4 patients receiving the placebo died from an lrti, pneumonia, recurrent acute myeloid leukemia, and an intracranial hemorrhage (1 each). this is the largest randomized, double blind, placebo-controlled clinical trial to date for the treatment of allogeneic and autologous hct recipients with rsv urtis. presatovir treatment did not meet the coprimary endpoints of a greater time-weighted average change in the rsv viral load from day 1 to 9 and the reduced development of lrtcs through day 28, but was well tolerated, with a comparable safety profile relative to the placebo. in a post hoc analysis of patients with lymphopenia, the proportion who developed an lrtc through day 28 was 51% lower following treatment with presatovir, as compared to the placebo; other post hoc analyses also indicated trends toward a treatment effect on lrtcs. the results suggest lessons for the design of future clinical trials of drugs for rsv or other respiratory viruses in transplant recipients or other immunocompromised patients. among healthy adults with established experimental rsv infections, presatovir treatment, as compared to the placebo treatment, significantly reduced the rsv load and clinical severity [26] . the current study did not reproduce these findings, most likely because the challenge study participants received presatovir at or before symptom onset, whereas the current study patients were treated after a median of 4 days of symptoms. an exploratory analysis revealed trends toward reduced lrtc rates following presatovir treatment, versus placebo treatment, of patients with median or shorter symptom durations (table 2) . future studies of anti-rsv drugs, particularly fusion inhibitors, should explore whether earlier therapy improves treatment outcomes. some transplant centers treat rsv infections in immunocompromised patients with oral or aerosolized ribavirin, despite lacking randomized clinical trial evidence [1] . ribavirin use in rsv-infected hct recipients, especially those with urtis, has been associated with more favorable outcomes in retrospective studies [6, 8, 27 ]. in the current study, placebo-treated patients who received ribavirin had a higher lrtc progression rate, compared with those who did not (26% vs 18%, respectively), and all patients who developed an lrtc used ribavirin more frequently (37.0%), relative to those without progression (23.5%). as this was not a randomized, controlled study of ribavirin treatment, these observations require confirmation. the observed rate of lrtcs was lower than the expected 30% used for the sample size calculation, and the day 28 mortality was very low (~3%) relative to previous retrospective studies [2, 7, 10] , possibly due to the recruitment of less severely ill patients who would not typically undergo rsv testing. lymphopenia is a well-described risk factor for lrtcs in rsv-infected hct recipients [9, 12, 14, 28] , as observed in the current study (64% in placebo-treated patients with lymphopenia vs 11% in those without). treatment with presatovir reduced the development of lrtcs in patients with lymphopenia-a surrogate marker of impaired t-cell or humoral immunity-possibly because robust immune responses masked any treatment effect by improving outcomes regardless of the treatment. furthermore, lymphopenia could influence respiratory immunopathology, providing better evidence of presatovir's antiviral efficacy. perhaps the most important question is whether an all-cause lrtc rate is a clinically relevant endpoint and, if so, whether the observed trends are clinically meaningful. respiratory failure and mortality are more clinically significant, but their rates in this study suggest that the sample size required would be prohibitive, especially for hct recipients. the current study endpoint of lrtcs included multiple etiologies, because rsv urtis may predispose patients to secondary infections-for example, by disrupting mucociliary function [29, 30] -so treatment could prevent a secondary lrti, as well as a primary lrti. furthermore, any lrtc is a clinically significant event that may prolong hospitalization, necessitate intensive clinical care (including empiric antimicrobial treatment), and potentially result in death. only a minority of lrtcs in this study were adjudicated as primary rsv lrtis-likely due to other etiologies, as well as a lack of lower respiratory tract samples for the confirmation of rsv-underscoring the potential importance of nonviral pulmonary events in hct recipients with rsv infections. determining the cause of each lrtc event in a clinical trial, while ideal, requires invasive procedures (eg, bronchoscopy or lung biopsy) that could pose significant patient risks and are not globally mandated by the current clinical standard of care. thus, radiographic confirmation, corroborated by clinical data with central, blinded adjudication, as used here, may be the best approach to classify lrtis. whether the near-50% relative reduction in lrtc events is clinically meaningful, despite lacking statistical significance, is left to interpretation. the consistent trends toward a treatment effect in exploratory analyses need confirmation in future studies. in summary, this study provided important lessons for the design of future clinical trials of drugs for rsv and other respiratory virus infections in hct recipients. although the coprimary endpoints were not achieved, presatovir treatment was associated with trends toward an antiviral effect and clinical benefit. similar future trials should judiciously select suitable at-risk patients (ie, patients with lymphopenia, neutropenia, the median duration of respiratory symptoms on day 1 in the efficacy population was 4 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in stem cell transplant patients: the european experience ciliary dyskinesia is an early feature of respiratory syncytial virus infection respiratory syncytial virus and human bronchial epithelium financial support. this work was supported by gilead sciences inc. potential conflicts of interest. r. f. c. received research grants paid to his institution and personal fees from gilead sciences inc., during the conduct of the study; research grants paid to his institution from ablynx, aicuris, ansun, chimerix, merck, oxford immunotec, novartis, pulmotec, shire, and xenex; and consultant fees/honoraria from ablynx, achaogen, adma biologics, ansun, astellas, chimerix, clinigen, janssen, oxford immunotec, merck/merck sharp & dohme (msd), pulmotect, shionogi, shire, and xenex, outside the current study. s. s. d. received grants paid to his institution from gilead sciences inc., during the conduct of the study; grants to his institution and personal fees for consulting, advisory board, and speaker bureau participation from merck; personal fees for advisory board participation from clinigen and janssen; and grants paid to his institution from ablynx, aicuris, ansun, glaxosmithkline (gsk), oxford immunotec, and shire, outside the current study. a. b. received funding to her institution from gilead sciences inc., during the conduct of the study; research grants from sos oxygène and pfizer; and consultant/ speaker fees from gilead sciences inc., merck, therakos, shire, zambon, ablynx, and pfizer, outside the current study. p. l. received research grants or fees/honoraria paid to his institution from merck, gilead sciences inc., astellas, shire, and oxford immunotec; and consultant fees/honoraria from ablynx nv and aicuris, outside the current study. y.-j. k. received grants from gilead sciences inc., during the conduct of the study. g.-s. c. was a member of the endpoint adjudication committee (eac) for this trial and received payment from gilead sciences inc. for this work. s. n. p. was a member of the eac for this trial and received salary support paid to his institution by gilead sciences inc. for this work. a. p. l. was a member of the eac for this trial and received payment from gilead sciences inc. for this work; served on the data safety monitoring board for another trial sponsored by gilead sciences inc.; received grants and consulting fees as a site investigator from merck, site investigator payments from astellas and oxford immunotec, and consulting fees from helocyte and virbio, outside the current study. e. b. received speaker fees from gilead sciences inc., pfizer, novartis, roche, and boehringer ingelheim, outside the current study. d. j. w. received research grants from gilead sciences inc., during the conduct of the study; research grants from merck, oxford immunotec, shire, and chimerix; and consultant fees from merck, outside the current study. p. j. s. received grants from gilead sciences inc., during the conduct of the study. g. r. received grants and personal fees from msd and pfizer and personal fees from astellas, outside the current study. c. b. s. received grants paid to her institution from gilead sciences inc., during the conduct of the study, and from gsk, viiv, merck, shire, schering, abbott, chimerix, and ablynx, outside the current study. k. m. m. received grants as a clinical trial investigator from gilead sciences inc., during the conduct of the study; clinical research grants from ansun, astellas, merck, rebiotix, scynexis, and shire; and consultant fees/honoraria from chimerix, gsk, merck, and scynexis, outside the current study. r. l. p. received research grants paid directly to his institution by gilead sciences inc., during the conduct of the study. d.-g. l. received research grants and consultant fees or speaker honoraria from pfizer, msd, and astellas, outside the current study. a. w. received clinical trial support from gilead sciences inc., during the conduct of the study. m. m., y. g., p. g., d. p. p., and t. r. w. are employees of gilead sciences inc., and may hold stock. r. j. is a former employee of and holds stock in gilead sciences inc. d. l. g. is an employee of sanofi genzyme and a former employee of gilead sciences inc., and may hold stock in both companies. f. m. m. received research grants paid to his institution and consulting fees for clinical trial design from gilead sciences inc., during the conduct of the study; research support paid to his institution and consulting fees for clinical trial design from gsk and chimerix; research grant support paid to his institution from ansun; and personal fees from visterra and roche molecular diagnostics, outside the current study. j. w. c. is an employee and stockholder of janssen biopharma and a former employee and current stockholder of gilead sciences inc. m. b. received clinical trial support and consulting fees from gilead sciences inc., during the conduct of the study; grants and personal fees from ablynx, ansun, chimerix inc., gsk, merck, shire, and vir bio; and personal fees from bavarian nordic, humabs inc., janssen, modema therapeutics, pulmocide, and pulmotect, outside the current study. all other authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-291726-8670s4st authors: che, xiao-yan; di, biao; zhao, guo-ping; wang, ya-di; qiu, li-wen; hao, wei; wang, ming; qin, peng-zhe; liu, yu-fei; chan, kwok-hong; cheng, vincent c. c.; yuen, kwok-yung title: a patient with asymptomatic severe acute respiratory syndrome (sars) and antigenemia from the 2003–2004 community outbreak of sars in guangzhou, china date: 2006-07-01 journal: clin infect dis doi: 10.1086/504943 sha: doc_id: 291726 cord_uid: 8670s4st an asymptomatic case of severe acute respiratory syndrome (sars) occurred early in 2004, during a community outbreak of sars in guangzhou, china. this was the first time that a case of asymptomatic sars was noted in an individual with antigenemia and seroconversion. the asymptomatic case patient and the second index case patient with sars in the 2003–2004 outbreak both worked in the same restaurant, where they served palm civets, which were found to carry sars-associated coronaviruses. epidemiological information and laboratory findings suggested that the findings for the patient with asymptomatic infection, together with the findings from previously reported serological analyses of handlers of wild animals and the 4 index case patients from the 2004 community outbreak, reflected a likely intermediate phase of animal-to-human transmission of infection, rather than a case of human-to-human transmission. this intermediate phase may be a critical stage for virus evolution and disease prevention. suggested that sars-cov was reintroduced to humans from animals [2, 3] . although it was reported that none of these patients were involved in the secondary spread of symptomatic sars-cov infection [1] , we describe a patient with asymptomatic sars-cov infection who was identified from 115 contacts of the index case patients in this community outbreak. this asymptomatic case of sars-cov infection was confirmed by a sensitive method of viral antigen detection and by serological tests. the asymptomatic case also highlights some issues associated with disease prevention. the present study was performed retrospectively. seventeen serum specimens were collected from 4 index case patients who exhibited recurrence of sars with laboratory-confirmed sars-cov infection in guangzhou city, china, from 22 december 2003 through 30 january 2004. an additional 118 serum specimens were collected from 115 contacts of the 4 index case patients with sars. to identify the nucleocapsid (n) protein of sars-cov in serum specimens, 2 antigen-capture assays were performed: an n antigen-capture elisa and an n antigen-capture chemiluminescent immunoassay (cia). the development of these 2 assays was based on the detection of 3 monoclonal antibodies directed against the n protein of sars-cov, as described elsewhere [4] [5] [6] . serum igg and igm antibodies to sars-cov were detected using commercially available indirect elisa kits (bgi-gbi; biotech) according to the manufacturer's instructions [6] . the sensitivity and specificity of elisa for the detection of igg antibodies to sars-cov were 99%-100% and 89%-97.2%, respectively, and the sensitivity and specificity of elisa for the detection of igm antibodies to sars-cov were 89.8% and 97.6%, respectively [6] [7] [8] . an indirect immunofluorescent assay (ifa) for the detection of igg antibodies to sars-cov was performed using a commercially available ifa kit (euroimmun), according to the manufacturer's instructions [9] . this ifa has a specificity of 100% (with detection of igg antibodies to sars-cov in 200 healthy blood donors) and a sensitivity of 97%-100% (with detection of igg antibodies to sars-cov in 150 serum specimens obtained, at least 10 days after the onset of symptoms, from patients with sars). in-house ifas for the detection of igg specific to human coronaviruses 229e and oc43 were performed as described by us elsewhere [10] . a microneutralization assay was performed according to procedures described elsewhere, with modifications [1] . in brief, a 96-well microtiter plate that contained confluent fetal rhesus kidney-4 cells in 100 ml of maintenance medium was prepared. two-fold dilutions of patient serum samples (50 ml), which started at 1:10 and increased to 1:320, were premixed with 50 ml tcid 50 of sars-cov (the hku-39849 isolate) and were incubated at 37њc for 1.5 h. then, 100 ml of the virus-serum mixture was inoculated in duplicate wells with fetal rhesus kidney-4 cells and was further incubated at 37њc. a cytopathic effect was observed at 72 and 96 h. the neutralization titer was determined to be the reciprocal of the highest serum dilution that produced 50% cytopathic effect on cells. a neutralizing antibody titer of у10 was considered to be positive for sars-cov. all serum specimens were tested for the n protein of sars-cov by use of both n antigen-capture elisa and n antigencapture cia. the findings of these 2 assays had been validated previously with the use of serum specimens obtained from patients with serologically confirmed sars, and the sensitivity and specificity of the n antigen-capture elisa were documented [4] [5] [6] . although the characteristic of the n antigencapture cia was shown to be equivalent to that of the n antigen-capture elisa (data not shown), the n antigen-capture cia seemed to be more sensitive than n antigen-capture elisa in the present study. both n antigen-capture assays detected n protein in serially obtained serum specimens that were collected from 3 patients 6-9 days after the onset of symptoms; cia was also able to detect n protein in serum samples collected from the second index case patient on the ninth and 10th days after the onset of symptoms, although these samples had negative results according to the n antigen-capture elisa (table 1) . this result was not surprising, because enzyme-amplified chemiluminescent chemical analysis is generally more sensitive than conventional elisa [11] . for index case patient 4, the first serum sample was obtained on day 17 after the onset of symptoms, and n protein was not detectable in serially obtained serum specimens during this patient's illness. of the 118 serum specimens obtained from the 115 contacts of the index case patients, 1 specimen, which was collected on 6 january 2004, was from a female contact of the second index case patient, who experienced onset of symptoms on 26 december 2003. this female contact was found to be positive for the n protein by means of n antigen-capture cia, and the presence of sars-cov infection was further confirmed by serological tests (table 2) . ifa detected a 16-fold increase in the sars-cov antibody titer in paired serum samples collected over a 1-week period. igm and igg antibodies to sars-cov were also detected by elisa. antibodies to sars-cov were also detected in the convalescent-phase serum sample obtained from this female contact case patient 12 weeks later, and the results of the sars-cov microneutralization assay were also found to be positive, with a titer of 20 determined for the convalescent-phase serum sample obtained from this female contact case patient. the serological results were verified by the center for disease control and prevention of guangzhou, which is one of the sars reference laboratories in china. to exclude the possibility of a false-positive result, serum specimens from the contact case patient were tested for antibodies against the human coronaviruses 229e and oc43, by use of ifa. paired serum samples showed no increase in the titer of antibodies to either 229e or oc43. these tests were also performed for serially obtained serum specimens from the 4 index case patients (table 1) . although the paired serum samples obtained from the first and second index case patients did show an increase in the titers of antibodies to 229e and oc43, no neutralizing antibodies against 229e and oc43 were detected in these index case patients during a previous study [1] . although none of the 4 index case patients showed evidence of secondary spread of the infection [1] , the direct detection of sars-cov n protein by the highly sensitive cia a serum samples in a serial 2-fold dilution (from 10-fold to 5120-fold). b the highest ratio of acute-phase to convalescent-phase absorbance values at the same serum dilution tested. illness, her case is the first confirmed and documented asymptomatic case of sars in this outbreak. this asymptomatic case of infection occurred in a 31-year-old woman who was working as a service chief in the local restaurant tdlr [2] , where the second index case patient worked as a waitress [2, 3] . although the asymptomatic case patient had only indirect contact with the second index case patient, she had a history of clear and close animal (i.e., palm civet) contact. she often showed the wild animals to customers who were ordering exotic food. subsequently, sars-cov n protein was detected, by use of n antigen-capture elisa, in 5 of a total of 6 palm civet serum samples obtained during the same period from the restaurant tdlr; results revealed a high level of n protein, with a mean od 450 value of 1.556, compared with the cutoff value of 0.21. viral rna was also detected in all throat and rectal swab specimens obtained from 6 palm civets, by use of rt-pcr for the detection of polyprotein and n genes of sars-cov, and 3 full genome sequences and 2 complete s gene sequences of sars-cov were identified in the specimens obtained from 5 of 6 palm civets [3] . when this information is considered together with the viral genomic information from the palm civets that was collected from the same restaurant during the same period [2] [3] , the clear conclusion is that the palm civets used for exotic food in the restaurant must have been heavily contaminated with sars-cov. with the limited samples available, the kinematics of the antigenemia and antibody responses of the second index case patient were compared with those of the asymptomatic case patient. the asymptomatic case patient had antigenemia (i.e., detection of n antigen in serum; antigenemia was detected in the asymptomatic case patient on 6 january 2004) disappear at almost the same time as it disappeared in the second index case patient (who had detectable antigenemia up to 5 january 2004). meanwhile, the second index case patient had seroconversion occur on 3 january 2004, whereas the asymptomatic case patient had a positive antibody result from 6 january 2004; however, because serum samples were unavailable before this date, the exact date of seroconversion cannot be determined. nevertheless, because the mean incubation period for sars is 6 days [12] , it seems that the asymptomatic case patient was unlikely to have acquired infection directly from the second e4 • cid 2006:43 (1 july) • che et al. index case patient. combining the epidemiological information regarding the contact history with the molecular diagnostic profiling of either the animals (the palm civets) or the human (the second index case patient), we infer that this asymptomatic case of sars was more likely to have resulted from an animalto-human infection (likely due to sars-cov carried by the palm civets from the restaurant) than from the second index case patient. sars is a zoonotic disease. sars-cov evolved consistently and rapidly within its animal and human hosts, while both the infectivity of the virus and the severity of the disease varied, along with the variation/adaptation of the virus to its hosts [2, 13] . it is particularly significant that even in the "early phase" of the 2002-2003 sars epidemic [13] , human-to-human transmission of infection was observed, and the symptoms were so severe that the disease was eventually named after its primary symptomatic characteristics. however, in the 2003-2004 community outbreak of sars, none of the 4 index case patients with confirmed sars had severe illness, and they all seemed to have acquired infection with sars-cov directly from animals. although sars-cov-specific antibodies previously had been detected at a relatively high rate among the population handling wild animals or had been observed in health care workers [14] [15] [16] , it was impossible to correlate serological results with the corresponding onset of clinical symptoms in these retrospective screenings. therefore, the asymptomatic case patient described in the present article is the first patient with asymptomatic sars-cov infection with detectable antigenemia and seroconversion caused by animal-to-human infection. this finding, together with the previously reported findings of serological analysis of the handlers of wild animals and the 4 index case patients from the community outbreak of sars in 2004, reveals that there likely is an intermediate epidemiological phase, which might be critical for genetic adaptation of the virus to its new hosts before critical mutation enables it to eventually cause severe symptomatic sars, as was seen in early 2003. although it is usually difficult to observe the virus in the intermediate phase, sars-cov is one of the rare viruses for which both genotypes [2, 13] and the antigen/antibody corresponding to the intermediate phase were detected. this finding may also be significant for the prevention of sars epidemics. although the most contagious human epidemic strain of sars-cov from the middle and late phases of the 2002-2003 pandemic has not been seen in nature since june 2003 [13] , the risk of asymptomatic or mildly symptomatic infection with sars-cov should not be ignored. to date, molecular epidemiological data have indicated that, although it might take decades for sars-cov to evolve into a contagious strain for human infection, it would not be too difficult for sars-cov to reach the stage in which it might cause infection either without symptoms or with mild symptoms in humans. this process could be accelerated if appropriate susceptible animal hosts, such as palm civets, were largely present together with the human population. on the other hand, if the highrisk population with infection can be properly monitored at the asymptomatic stage and the disease can be promptly controlled at the mildly symptomatic stage (both objectives are technically feasible now), future threats of a reemerging sars epidemic could be much lower than once was surmised. laboratory diagnosis of four recent, sporadic cases of community-acquired sars cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human sars-cov infection in a restaurant from palm civet nucleocapsid protein as early diagnostic marker for sars sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome detection of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in serum: comparison with results of other viral markers evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus anti-sars-cov immunoglobulin g in healthcare workers novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 comparison of chemiluminescent assays and colorimetric elisas for quantification of murine il-12, human il-4 and murine il-4: chemiluminescent substrates provide markedly enhanced sensitivity the severe acute respiratory syndrome the chinese sars molecular epidemiology consortium. molecular evolution of the sars-coronavirus during the course of the sars epidemic in china isolation and characterization of viruses related to the sars coronavirus from animals in southern china asymptomatic sars coronavirus infection among healthcare workers mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of health-care workers in a teaching hospital in singapore potential conflicts of interest. all authors: no conflicts. key: cord-299274-gnbp7so5 authors: bodkin, claire; mokashi, vaibhav; beal, kerry; wiwcharuk, jill; lennox, robin; guenter, dale; smieja, marek; o’shea, timothy title: pandemic planning in homeless shelters: a pilot study of a covid-19 testing and support program to mitigate the risk of covid-19 outbreaks in congregate settings date: 2020-06-08 journal: clin infect dis doi: 10.1093/cid/ciaa743 sha: doc_id: 299274 cord_uid: gnbp7so5 we tested 104 residents and 141 staff for covid-19 who failed daily symptom screening in homeless shelters in hamilton, canada. we detected one resident (1%), seven staff (5%) and one case of secondary spread. shelter restructuring to allow physical distancing, testing and isolation can decrease outbreaks in shelters. a c c e p t e d m a n u s c r i p t sars-cov-2, the novel coronavirus responsible for causing covid-19, has particularly affected those in congregate settings such as nursing homes, prisons and homeless shelters (1, 2) . in canada, there have been several outbreaks documented in homeless shelters (3, 4) . preventing and minimizing outbreaks in shelter settings using limited resources protects residents and staff within shelters as well as those they may interact with in the broader community. furthermore, the higher prevalence of comorbidities amongst shelter residents places them at higher risk of severe covid-19 disease (5) . preventing transmission in this population may have a greater impact on reducing hospital admissions and burden on critical care resources. the objective of this report is to describe our experience with shelter facility restructuring, daily symptom screening and rapid testing to mitigate the risk of covid-19 in the homeless shelter setting in hamilton, ontario, canada. the homeless shelter system in the city of hamilton is operated as a partnership between the municipality and social service agencies. healthcare within the shelters is provided by the hamilton shelter health network, a group of physicians (family medicine, internal medicine and psychiatry), nurse practitioners, nurses and midwives who are funded through an alternate funding plan by the provincial government of ontario. a c c e p t e d m a n u s c r i p t at the start of the pandemic, there were approximately 341 shelter beds across eight shelters in hamilton. collaboration between the local public health unit, municipal government, shelter operators and the shelter health network began in march of 2020. the collaboration allowed an increase from 341 to 395 shelter beds spread across the preexisting shelters, three additional hotel sites and one additional temporary men's shelter. this enabled increased physical spacing between shelter beds and lower density within each shelter. furthermore, a temporary isolation center (from a repurposed recreation center) was created for covid-19 positive homeless individuals. through a partnership with the hamilton regional laboratory medicine program (hrlmp) we were able to access covid-19 testing with rapid turnaround time. this allowed us to use existing spaces within shelters for short-term isolation while awaiting covid-19 testing results and to immediately arrange two-week isolation for those who tested positive. between march 17 and april 30, 2020, covid-19 testing was performed on all shelter residents and staff who failed daily screening for potential covid-19 related symptoms as well as staff and residents identified as close contacts of positive cases. symptom screening was conducted according to direction given by the local public health unit, and was updated as new information regarding potential symptoms of covid-19 were uncovered (e.g. residents awaiting results were isolated within their shelter in single room areas. residents who tested positive for covid-19 and did not require hospitalization were transferred to our prepared isolation center for further monitoring. transportation was organized by the city of hamilton and involved the use of a dedicated transportation vehicle with enhanced infection control measures including a barrier between patient and driver and surgical mask, face shield, gloves and gown for the driver. isolation continued for a total duration of fourteen days, with the ultimate decision to end isolation made in conjunction with our local public health unit. nurses performing tests and shelter staff caring for isolated residents wore a disposable gown, surgical mask, face shield and disposable gloves. staff who tested positive were excluded from work and provided with information related to self-isolation and symptom monitoring. all positive results were reported to the local public health unit for appropriate case management and contact tracing. for context, the provincial government declared a state of emergency on march 17, limiting public gatherings to 50 people or less, and subsequently closed non-essential businesses on march 23. gatherings were further limited to five people on march 28. no specific limitations were imposed upon shelter residents during the study period although movement between shelters was discouraged. universal masking within common areas in the shelter system was initiated on april 18. a c c e p t e d m a n u s c r i p t between march 19 and april 30, a total of 245 nps were obtained from 141 staff and 104 residents (figures 1a and 1b) . of the 88 total tests (59 residents and 29 staff) completed on the multiplex pcr platform prior to april 17, 12 (13.6%) were positive for a viral pathogen. ten of 59 residents (16.9%) were diagnosed with rhinovirus/enterovirus infection and two of 29 staff (6.8%) were diagnosed with covid-19 infection. a total of 157 (44 residents and 113 staff) tests performed after april 17 were tested exclusively for covid-19; of these 1 resident (2.3%) and 5 staff (4.4%) tested positive. overall during our study period, 1 of 104 residents (1.0%) and 7 of 141 staff (5.0%) were diagnosed with covid-19 infection, and a total of 17 tests (6.9%) identified a viral pathogen. all positive cases were detected only through the daily symptomatic screening protocol or contact tracing. follow up contact tracing and testing revealed no secondary spread linked to the one positive shelter resident. of the seven staff diagnosed with covid-19, four were part of a cluster that was cohabiting at the same location distant from the shelter and were presumed to be community acquired. the remaining three staff worked at two separate shelter sites. during the time period of our study, one additional shelter resident was diagnosed with covid-19 after presentation to a local emergency room (outside of our surveillance protocol). for comparison, during the study time period, the city of hamilton reported 422 patients with covid-19 and a positivity rate of approximately 5-7%. approximately 10% of the cases m a n u s c r i p t there are several limitations which should be taken into account in interpreting our data. our testing program provided evaluation of those staff and residents who were identified as symptomatic through active screening within the shelters. we are aware of instances where shelter residents and staff have presented to other settings where testing has been performed, and is not captured in our data. secondly the test characteristics of a nps can be influenced by testing technique, and as such the sensitivity of our test in the real world setting of a mobile testing unit has not been clearly established. however, the lack of large scale outbreaks in area shelters suggests that we have not had a large number of false negative tests thus far. efforts to mitigate the risk of outbreaks of covid-19 in high risk congregate settings such as long term care facilities, homeless shelters, and prisons are essential in moving towards the broader goal of managing covid-19 risk in the general community. our group has recently initiated a cluster randomized control trial to examine the effectiveness and acceptability of various surveillance testing strategies of asymptomatic individuals within our shelter system in an ongoing effort to enhance our ability to rapidly detect and isolate covid-19 infected individuals. however, such efforts should be conceptualized as secondary prevention. there is also a pressing need to consider primary prevention -that is, upstream actions to end homelessness. our study demonstrates that accessible shelter housing that allows for rapid testing, isolation and physical distance is imperative to outbreak prevention in the shelter setting. the strategy presented here should be considered as part of the covid-19 pandemic response alongside other homelessness prevention and reduction interventions. m a n u s c r i p t epidemiology of covid-19 in a long-term care facility in king county, washington outbreak among three affiliated homeless service sites -king county, washington, 2020. mmwr morb mortal w rep testing of asymptomatic homeless shelter clients underway after four cases confirmed in calgary. calgary herald covid-19 outbreak spikes among homeless in toronto with 135 cases morbidity and mortality in homeless individuals, prisoners, sex workers, and individuals with substance use disorders in high-income countries: a systematic review and meta-analysis. the lancet we thank the hamilton regional laborary medicine program (hrlmp), hamilton public health, city of hamilton and staff working within the hamilton shelter health network.none of the authors has any potential conflicts of interest to disclose. m a n u s c r i p t were in congregate settings (mainly long term care homes). in the province of ontario, there were 15973 reported covid-19 infections and 1080 deaths during our study period.test turnaround time throughout our study period averaged 14 hours from time of specimen arrival in the lab to time of reported results and 89 per cent of results were reported within 24 hours. we have thus far been successful in preventing large outbreaks in the shelter setting despite identifying positive cases in both staff and residents. our results emphasize the importance of taking a proactive, aggressive approach to outbreak mitigation in high risk settings. while there has certainly been some random chance, we postulate that four factors have been particularly important in increasing our chances of success: a c c e p t e d m a n u s c r i p t key: cord-294718-n3gx862b authors: tam, patrick c k; ly, kathleen m; kernich, max l; spurrier, nicola; lawrence, diana; gordon, david l; tucker, emily c title: detectable severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in human breast milk of a mildly symptomatic patient with coronavirus disease 2019 (covid-19) date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa673 sha: doc_id: 294718 cord_uid: n3gx862b sars-cov-2 is a novel coronavirus and causative pathogen to the pandemic illness covid-19. although rna has been detected in various clinical samples, no reports to date have documented sars-cov-2 in human milk. this case report describes an actively breastfeeding patient with covid-19 infection with detectable viral rna in human milk. m a n u s c r i p t severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has emerged as the virus causing coronavirus disease 2019 (covid-19) following an outbreak of the novel pathogen in a seafood market in wuhan, china, in late 2019 [1] . with over four million confirmed cases worldwide to date, the world health organisation (who) has declared covid-19 a global pandemic [2] . the precise transmission dynamics of sars-cov-2 remain uncertain but human-to-human transmission through respiratory droplets is the presumed leading mechanism [3] . there are other proposed routes of transmission, given detection of viral shedding in serum and faecal specimens although no confirmed cases of transmission through these routes have been established [4] . to date, sars-cov-2 has not been detected in human milk. we report a case of detectable sars-cov-2 rna in a woman with mild symptoms of covid-19. a forty-year old female with no significant medical problems returned to australia with her eight-month old son in early march 2020 from a covid-19 endemic region after a two-month stay. three days following her return, she developed a sore throat, myalgias, a minimally productive cough and fevers up to 38.9 degrees celsius. a combined oro/nasopharyngeal swab was performed, exhibiting detectable rna for sars-cov-2 on real time polymerase chain reaction (rt-pcr) testing. a c c e p t e d m a n u s c r i p t 3 the patient was recalled to hospital for further management along with her son who was still breastfed until the day of maternal symptom onset. on presentation to hospital, she had a normal respiratory examination and did not require supplemental oxygen. initial investigations demonstrated a white cell count of 5 x 10 9 /l with reactive lymphocytes and a c-reactive protein of 0.6mg/l (normal limits: 0-8 mg/l). routine biochemistry including liver function tests and lactate dehydrogenase were within normal limits. radiological imaging was not performed due to the patient's clinical stability. on admission to hospital, the infant, with no previously known medical problems underwent nasopharyngeal testing given mild coryzal symptoms and a non-productive cough beginning one day following his mother's symptom onset. his nasopharyngeal swab was also positive for sars-cov-2 on rt-pcr testing. both patients had daily nasopharyngeal samples collected to monitor viral shedding on rt-pcr. samples of the mother's human milk and stool samples from the infant were also collected and analyzed throughout their hospital stay. the results of these samples on rt-pcr testing for sars-cov-2 rna targeting the envelope protein gene (e-gene) are depicted in figure 1 . the mother's serum, urine and saliva were also tested for sars-cov-2. none of these samples had detectable rna. prior to hospitalisation and onset of maternal symptoms, the infant was breastfed up to three times per day in addition to small meals in-between. breastfeeding was discontinued by the mother once she developed symptoms due to concerns of covid-19 transmission to the infant. the first sampling of human milk occurred five days following maternal symptom onset with no episodes of breastfeeding in those five days prior to collection of the sample. during collection of the first human milk sample, the patient was not using a facemask given resolution of her respiratory symptoms. self-expression of human milk occurred after breast decontamination with soap and water as well as appropriate hand hygiene. human milk was expressed directly into a sterile specimen container. upon confirmed covid-a c c e p t e d m a n u s c r i p t 4 19 infection in the infant, breastfeeding was resumed with nil adverse effects. subsequent samples of self-expressed human milk collected for analysis were obtained prior to breastfeeding in an attempt to reduce contamination from the infant's oropharynx. specific recommendations on cleaning of the breast were not imposed during collection of subsequent specimens. the mother remained diligent with hand hygiene prior to human milk expression. an additional six samples of human milk were collected with one further sample demonstrating detectable sars-cov-2 rna (figure 1 ). faecal samples from the infant were also collected from the third day of admission. these samples continued to have detectable rna sixty-six days following infant symptom onset (figure 1 ). after resolution of respiratory symptoms and documented viral clearance from the nasopharynx of both patients, they were discharged from hospital after an uneventful fifteen-day stay. to our knowledge, this is the first case of detectable sars-cov-2 rna from human milk in a patient with covid-19. despite mild clinical symptoms, our patient had detectable virus in human milk in two separate samples taken ten days apart but interspersed with a number of negative results. there have been limited studies examining sars-cov-2 presence in human milk, with no peer-reviewed reports to date demonstrating detectable rna in human milk [5] . faeces up to twenty-nine days following symptom onset [6] . the detection of viral rna in faeces sixty-six days after initial symptom onset found in our report appears to exceed the longest duration found in the literature to date [7] . the lack of detectable virus in the mother's urine and serum is similar to reports in the literature that viral detection in urine is rare and viremia similarly uncommon [4] . although the maternal oropharyngeal samples had undetectable rna by day seven of infant symptom onset, the human milk demonstrated detectable rna at day fifteen. this discordance is also similar to other studies showing a c c e p t e d m a n u s c r i p t 6 continued rna shedding in other bodily fluids such as serum or faeces despite a negative respiratory sample [8] . the mechanism by which sars-cov-2 viral shedding occurs in breast tissue leading to detectable rna is however uncertain. the implications of recoverable rna in human milk remains unclear. although sars-cov-2 rna was identified in our samples, whether this translates to viable virus or degraded residual nucleic acid could not be ascertained. whilst contamination of the patient's human milk was considered unlikely, if the detectable rna in the human milk samples was a result of contamination remains an important finding as it poses a potential and unanticipated source of exposure for an uninfected infant. this emphasizes the importance of appropriate breast hygiene prior to feeding and cleaning of breast pumps if used as per recommendations by the american academy of pediatrics [5] . the infant in this report however was already infected at presentation with acquisition likely through overseas travel as well as close contact with the mother through respiratory transmission. transmission of virus via breastfeeding is uncertain but presumed unlikely. given this uncertainty and detectable viral rna in human milk, the benefits of human milk likely greatly outweigh risks associated with maternal covid-19 infection, due to conferring protection to other respiratory illnesses [9] . this is in line with current recommendations for breastfed infants to continue human milk consumption during maternal covid-19 infection [5] , [10] . our patient despite having positive rna in human milk continued to feed her infant with no adverse outcomes when breastfeeding was resumed. although we did not test for the presence of sars-cov-2 antibodies in the human milk, there have been previous reports of detectable sars-cov-1 antibody in human milk of a patient with severe acute respiratory syndrome (sars) collected 131 days after onset of illness [11] . breastfeeding a c c e p t e d m a n u s c r i p t cycle thresholds (ct) values for the envelope protein gene (e-gene) target for collected specimens. a single flock swab was used in the mother to collect combined oral and nasopharyngeal samples. a ct value ≥40 is interpreted as undetectable. a c c e p t e d m a n u s c r i p t 12 figure 1 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding covid-19 map -johns hopkins coronavirus resource center the sars-cov-2 outbreak: what we know detection of sars-cov-2 in different types of clinical specimens initial guidance: management of infants born to mothers with covid-19 characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding prolonged presence of sars-cov-2 viral rna in faecal samples molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes breast feeding and respiratory morbidity in infancy: a birth 10 cohort study q&a on covid-19, pregnancy, childbirth and breastfeeding a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-277410-lt19mijb authors: salvatore, phillip p; dawson, patrick; wadhwa, ashutosh; rabold, elizabeth m; buono, sean; dietrich, elizabeth a; reses, hannah e; vuong, jeni; pawloski, lucia; dasu, trivikram; bhattacharyya, sanjib; pevzner, eric; hall, aron j; tate, jacqueline e; kirking, hannah l title: epidemiological correlates of pcr cycle threshold values in the detection of sars-cov-2 date: 2020-09-28 journal: clin infect dis doi: 10.1093/cid/ciaa1469 sha: doc_id: 277410 cord_uid: lt19mijb background: detection of sars-cov-2 infection has principally been performed through the use of real-time reverse-transcription pcr (rrt-pcr) testing. results of such tests can be reported as cycle threshold (ct) values, which may provide semi-quantitative or indirect measurements of viral load. previous reports have examined temporal trends in ct values over the course of a sars-cov-2 infection. methods: using testing data collected during a prospective household transmission investigation of outpatient and mild covid-19 cases, we examined the relationship between ct values of the viral rna n1 target and demographic, clinical, and epidemiological characteristics collected through participant interviews and daily symptom diaries. results: we found ct values are lowest (corresponding to higher viral rna concentration) soon after symptom onset and are significantly correlated with time elapsed since onset (p<0.001); within 7 days after symptom onset, the median ct value was 26.5 compared with a median ct value of 35.0 occurring 21 days after onset. ct values were significantly lower among participants under 18 years of age (p=0.01) and those reporting upper respiratory symptoms at the time of sample collection (p=0.001) and were higher among participants reporting no symptoms (p=0.05). conclusions: these results emphasize the importance of early testing for sars-cov-2 among individuals with symptoms of respiratory illness and allows cases to be identified and isolated when their viral shedding may be highest. since its emergence in 2019, the sars-cov-2 virus has caused over 19 million cases of covid-19 and over 700,000 deaths globally [1] . diagnosis of covid-19 relies primarily on the use of real-time reversetranscription pcr (rrt-pcr) tests, including the cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel [2] . such rrt-pcr tests amplify and detect target viral genetic sequences and generate cycle threshold (ct) values [3] . qualitative rrt-pcr tests do not measure the viral load within a clinical specimen, but ct values offer a semi-quantitative assessment of viral rna concentration: lower ct values correspond to higher viral rna concentrations. while quantitative interpretation of ct values is dependent on multiple factors including reaction conditions and amplification efficiency, a common theoretical value can provide a useful benchmark for interpreting ct values: an increase of 3.3 units in ct value corresponds to 10-fold less target rna under optimum conditions [3] . as a result, ct values can serve as an indirect indicator of relative viral load in diagnostic samples of persons tested for sars-cov-2. however, relatively few studies have found significant associations between epidemiological factors and sars-cov-2 ct values [4] [5] [6] [7] [8] [9] . given the paucity of data examining associations of these factors with ct value at the time of diagnosis, we sought to identify relationships between ct values and time since onset, demographic factors, and symptoms among laboratory-confirmed covid-19 cases identified in a multistate investigation of sars-cov-2 household transmission. and wisconsin between march 23 and may 13, 2020 who tested positive for sars-cov-2 on an nasopharyngeal (np) swab at enrollment or during follow-up, including index cases and household members [10] [11] [12] . briefly, households identified by local health authorities as having a member with laboratory-confirmed covid-19 were approached for participation. np specimens were collected from household members on the day of enrollment and repeatedly during follow-up. all participants completed questionnaires that included questions on demographics, medical history, timelines for exposure, and symptom assessment. symptom assessments were conducted by cdc investigators at the time of specimen collection and by participants daily using symptom a c c e p t e d m a n u s c r i p t 4 diaries. household visits were made to collect np specimens from all participating household members on the day of enrollment, 14 days later, and on any day in between when a household member reported the onset of a new symptom. all participants were outpatients at the time of data and specimen collection. np specimens were collected by cdc clinicians using flexible minitip flocked swabs (becton, dickinson, and company, franklin-lakes, new jersey, usa). specimens were transported in three milliliters of viral transport media. for participants who tested positive more than once during the investigation period, ct values from only the first positive test were included. all specimens were tested at the utah public health laboratory or the milwaukee public health laboratory using the cdc 2019-ncov real-time rt-pcr diagnostic panel [2] . briefly, this assay amplifies and detects two targets (n1 and n2) of the viral nucleocapsid gene with a limit of detection in the range of 1000-3,162 viral rna copies per ml. the human housekeeping gene target rnase p (rp) was also measured in each sample for use in normalization. viral rna extraction procedures varied by instrument (using either the qiagen ez1 advanced xl or the biomérieux emag) and followed manufacturer's instructions; rt-pcr was performed using the abi 7500. results were considered positive if signal was detected (ct<40) for rp, n1, and n2 genes. results were classified as "not detected" if rp was detected but no signal was observed (ct≥40) from either n1 or n2. results were classified as inconclusive if rp was detected (ct<40) and either n1 or n2 was detected (but not both). results were classified as invalid if no rp was detected in the sample. any specimens for which results were inconclusive were re-tested; specimens which produced inconclusive results after re-testing were excluded from the analysis (n=17 specimens, 6 participants). ct values for amplification of both viral targets (n1 and n2 probes) and the human housekeeping gene rnase p (for specimen quality assessment) were analyzed. symptoms at the time of np swab collection were classified into categories. respiratory symptoms included runny nose, nasal congestion, or sore throat (upper respiratory); cough, discomfort while breathing, shortness of breath, wheezing, or chest pain (lower respiratory). covid-19-like symptoms were defined as fever, cough, or shortness of breath [13] . the council for state and territorial epidemiologists case definition for covid-19 (adopted by several public health organizations) included any of cough, shortness of breath, or a c c e p t e d m a n u s c r i p t 5 discomfort while breathing, or, alternatively, two or more of the following: fever, myalgia, headache, chills, sore throat, loss of taste, and loss of smell [14] . the criteria used by cdc for surveillance and monitoring of seasonal influenza-like illness activity included fever and either cough or sore throat [15] . the world health organization (who) surveillance definition of acute respiratory infection included shortness of breath, cough, sore throat, nasal congestion, or runny nose [16] . further details on symptom categorization can be found in supplemental table s1 . categories of examined comorbid conditions were diabetes, any immunosuppressive condition, and any other reported comorbidity. because ct values are semi-quantitative, differences in ct values between groups were assessed nonparametrically using the mann-whitney u test. correlations between ct value and time since symptom onset were assessed nonparametrically using tie-corrected spearman rank correlation coefficients. statistical tests for which p<0.05 were reported as statistically significant. cdc collaborated with state and local health departments in the public health response to covid-19 cases. this activity involved identification, control, or prevention of disease in response to an immediate public health threat; it was determined not to be public health research. therefore, this activity did not require irb review. characteristics of the population evaluated in that investigation have been described previously [10] [11] [12] . a total of 93 household members (including index cases) who tested positive for sars-cov-2 by np swab were included in this analysis. a majority of those with a positive np swab were female (53%), white (78%), and non-hispanic (87%). the median age of participants was 37 years (interquartile range, iqr: 21-53 years) and 13 participants were under 18 years of age. all participants reported symptoms before, after, and/or on the day the day their positive np swab was collected. among participants reporting symptoms on or before the day their positive np swab was collected (n=90), a median of 8 days (iqr: 6-11 days) elapsed between the day of the participant's symptom onset and the day of collection. ct values ranged from 14.4 to 38.4 (median, 29.7) for the n1 probe, were similar to those obtained for the n2 probe and did not vary by rnase p value (supplemental figure s1 ). for this analysis, we focused on a c c e p t e d m a n u s c r i p t 6 values of the n1 probe. among participants who developed symptoms on or before their positive np was collected (n=90), ct values were significantly and positively correlated with time elapsed since earliest symptom onset (p<0.001; figure 1a , 1c). among individuals who tested positive within 7 days of symptom onset, the median ct value was 26.5 (iqr: 20.8-29.9). by comparison, among individuals who tested positive 21 days or more after symptom onset, the median ct value was 35.0 (iqr: 33.5-35.7). ct values were not significantly correlated with age as a continuous variable due to variability in ct values among adults ( figure 1b) . however, when dichotomized, ct values were significantly lower among participants under 18 years of age (mann-whitney p=0.01, figure 1c ). age was significantly correlated with time from onset to collection (spearman p=0.002). ct values were not significantly associated with sex, race, or ethnicity ( figure 1c figure 2b ). similarly, ct values were significantly lower among those reporting one or more respiratory symptoms compared to those with no respiratory symptoms (median ct values of 29.3 and 33.4, respectively; p=0.012; figure 2b ). the significant difference noted among those reporting any respiratory symptoms was likely due to significantly lower ct values among those reporting upper respiratory symptoms (p<0.001). additionally, median ct values were lower among participants falling into several symptom categories and syndromes compared to participants who did not fall into these categories, but these were not statistically significant differences ( figure 2b ). ct values were higher among participants reporting no symptoms at the time of collection than among those reporting any symptoms (median ct values: 33. 3 a c c e p t e d m a n u s c r i p t 7 p=0.0496); the p-value for this relationship approaches, but falls under, the a priori alpha threshold of 0.05, driven by wide confidence intervals among participants reporting any symptoms (95% ci: 16.4-37.5). among participants reporting no symptoms at the time of collection (n=13), all reported symptoms either prior to collection (n=6), after collection (n=7), or both (n=4). although these patients did not report symptoms at the time of np collection, they represent a mixed group with some being post-symptomatic and some being presymptomatic. among these participants, ct values were lower among pre-symptomatic participants (median: 23.1) than among participants who were post-symptomatic (median: 35.9; p=0.036, figure 3 ). of these 13, three were children under 18 years of age; one was pre-symptomatic and two reported symptoms both before and after symptom collection. for symptoms and symptom combinations significantly associated with ct value, medians, 95% confidence intervals, and associated p-values are presented in table 1 . in this analysis, we examined associations between sars-cov-2 ct values and epidemiological characteristics of participants with confirmed covid-19. among participants who were symptomatic on or before the day of their positive np, we found that ct values were significantly correlated with time since symptom onset. while ct values are not direct quantitative measures of viral load, these results suggest that viral rna levels in the nasopharynx (and, by extension, possible infectiousness) are highest soon after symptom onset. additionally, we identified several clinical presentations significantly associated with lower ct values, including upper respiratory symptoms and the who definition of acute respiratory infection [16] . of note, we found that participants without symptoms at the time of collection exhibited higher ct values than those with symptoms. however, this relationship was influenced by high ct values among post-symptomatic participants. these observations illustrate the importance of rapid quarantine and testing of individuals with high-risk exposures (such as close contacts of known cases) who may be infectious while pre-symptomatic or asymptomatic. our results also support recent findings that children are likely to contribute to transmission of sars-cov-2 [17, 18] . simultaneously, the household-based nature of this investigation captured a population of outpatient and mild covid-19 cases and therefore was not powered to detect correlations between ct values and more severe covid-19 symptoms or presentations (which occur more rarely in outpatient/mild cases). as a result, this household transmission investigation may have captured some individuals who would not have sought care otherwise (e.g., those with mild symptoms) but may still contribute to household transmission [12] . we excluded six participants for whom only inconclusive results were obtained. finally, though some subgroups exhibited significantly higher ct values, all participants had positive np swabs, and associations between high ct values and symptoms or syndromes should be interpreted cautiously. despite these limitations, this approach provides unique insight using prospective specimen collection among household members and the testing of individuals who were not seeking care. this has the potential to provide a more complete description of infection beyond healthcare settings. some report no correlation or association between viral load (or, separately, ct value) and time since onset of symptoms [4, 5] , while others observed signals of greatest viral rna amounts closest to the onset of symptoms [6] [7] [8] 19] or in the first week following symptom onset [20] [21] [22] . there is some evidence that the timing of peak viral load may be dependent on the anatomical source of the specimen tested (e.g., peaking earlier in throat swabs than in sputum specimens [23] ) or extent of disease (e.g., peaking later in patients showing signs of lung infection [23] or with more severe disease [24] ). fewer studies have examined possible relationships between clinical presentation and ct value. while one analysis found higher loads in patients with severe disease [24] , multiple studies in which these associations were examined found no significant relationship between viral load a c c e p t e d m a n u s c r i p t 9 (or, separately, ct value) and patient demographics [4, 8] , comorbidities [4] , symptom status [5, 7, 9] , disease severity [8] , or medical intervention [6] . our findings -that ct values are significantly lower among individuals with respiratory symptoms -represent a novel addition and enhanced granularity to this growing body of evidence. our results bolster the importance of early access to sars-cov-2 diagnostic testing for individuals who develop symptoms of respiratory illness or who have high-risk exposures (even while pre-symptomatic) and reinforce existing guidance for testing [25] . by quickly identifying such individuals early (when ct values are lowest and infectiousness may be high) and implementing isolation protocols, public health practitioners might more effectively interrupt transmission of sars-cov-2. a c c e p t e d m a n u s c r i p t 10 m a n u s c r i p t 13 figure legends cycle threshold (ct) values for sars-cov-2 rrt-pcr target probes n1 and n2 are plotted against the time elapsed between symptom onset and np specimen collection in panel 1a. ct values for the n1 probe are plotted against participant age in panel 1b. distributions of n1 probe ct values among participants categorized by time between symptom onset and np collection, age, sex, race, and ethnicity are displayed in panel 1c. age was significantly correlated with time between symptom onset and np collection. "other" race includes american indian/alaska native, native hawaiian/other pacific islander, and multiracial, which were grouped due to low sample sizes. boxes illustrate interquartile ranges, whiskers illustrated 95% distributions, and medians are highlighted in white. distributions of n1 probe ct values among participants exhibiting specific symptoms or combinations of symptoms are displayed in panels 2a-2b. boxes illustrate interquartile ranges, whiskers illustrated 95% distributions, and medians are highlighted in white. each column of paired data (colored and grey) depicts the set of participants that reported the symptom(s) labeled on the x-axis (colored distribution) compared with the set of participants that did not report the same symptom(s) (grey distribution). mann-whitney p-values for each comparison are displayed beneath data pairs. panel points illustrate individual cycle threshold (ct) values among participants reporting no symptoms on the day of positive np specimen collection. "no symptoms at collection" refers to all such participants. "pre-symptomatic" refers to participants who reported no symptoms at or before the day of collection but reported symptoms later in the follow-up period. (participants whose positive np was collected on the final day of follow-up were excluded from this group.) "post-symptomatic" refers to participants who reported symptoms prior to collection but none on or after the day of collection. "mid-symptomatic" refers to participants who reported symptoms prior to and following the day of collection but not on the day of collection a c c e p t e d m a n u s c r i p t 14 table 1 supplementary table s1 . ‡ for the category of "no symptoms at collection", participants meeting the symptom/syndrome criteria includes participants who did not report symptoms at the time of collection; participants not meeting symptom/syndrome criteria includes participants who reported any symptoms at the time of collection. world health organization. coronavirus disease (covid-19) situation report -204 2019-ncov) real-time rt-pcr diagnostic panel instructions for use to interpret the sars-cov-2 test, consider the cycle threshold value saliva is a reliable tool to detect sars-cov-2 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore sars-cov-2 viral load in upper respiratory specimens of infected patients temporal dynamics in viral shedding and transmissibility of covid-19 asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility -king county loss of taste and smell as distinguishing symptoms of covid-19 a prospective cohort study in non-hospitalized household contacts with sars-cov-2 infection: symptom profiles and symptom change over time household transmission of sars-cov-2 in the united states centers for disease control and prevention. covidview: purpose and methods standardized surveillance case definition and national notification for 2019 novel coronavirus disease (covid-19) centers for disease control and prevention. u.s. influenza surveillance system: purpose and methods world health organization. rsv surveillance case definitions age-related differences in nasopharyngeal severe acute respiratory syndrome coronavirus 2 (sars-cov-2) levels in patients with mild to moderate coronavirus disease 2019 (covid-19) contact tracing during coronavirus disease outbreak, south korea quantitative detection and viral load analysis of sars-cov-2 in infected patients clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (covid-19) in the united states viral load kinetics of sars-cov-2 infection in first two patients in korea viral load of sars-cov-2 in clinical samples virological assessment of hospitalized patients with covid-2019 viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china all authors have completed icmje conflict of interest disclosure statements have no competing interests to declare. key: cord-007047-7ty9mxa9 authors: reller, l. barth; weinstein, melvin p.; baron, ellen jo title: implications of new technology for infectious diseases practice date: 2006-11-15 journal: clin infect dis doi: 10.1086/508536 sha: doc_id: 7047 cord_uid: 7ty9mxa9 new assays for the diagnosis of infectious diseases—particularly those that use molecular technologies—will revolutionize infectious diseases practices, but the fulfillment of the promise is several years away. problems with currently available molecular assays include a lack of knowledge about the extent of microbial nucleic acid in “normal” hosts, concentration of agent material in small volume samples, lack of microbiologist expertise, lack of adequate reimbursement, and difficulty with validation based on conventional methods. clinicians must appreciate the shortcomings of new technology to use it effectively and appropriately. however, we are realizing tangible progress in our ability to detect new etiological agents; the availability of rapid, accurate diagnostic tests for previously difficult infections; and advances into new, human response—based paradigms for diagnostic testing. the promise of a star trek-inspired hand-held infectious diseases diagnostic device that scans a patient's body and immediately provides the information needed for diagnosis and treatment is probably years away. until that time, when clinicians and microbiologists become unemployed, we can use the amazing technologies that are already available. this overview will discuss a selection of technologies and provide an opinion about their utility, their pitfalls (summarized in table 1) , and their potential. although the day when all diagnostic microbiology laboratories will routinely use molecular tools has not yet come [1, 2] , many clinicians believe that a plethora of such tests is readily available. laboratorians are regularly asked to perform a pcr test to detect an agent that only a few published research studies have reported, with those studies usually describing only preliminary results. physicians often seem surprised that the laboratory cannot provide this service. a survey by the american society for microbiology published in 2003 provides a snapshot of the current environment in the united states. of the 612 laboratories (representing community, academic, and commercial laboratories) that responded to the survey, 95% performed bacteriology tests, but only 17% performed any molecular tests for infectious diseases [3] . the bulk of the latter laboratories offered only molecular testing for chlamydia/neisseria gonorrhoeae (as determined on the basis of college of american pathologists surveys participant summaries). for the foreseeable future, because of staffing and financial issues, many nonconsolidated or nonacademic hospital laboratories will not perform new, high-technology, expensive, and skill-demanding assays. there is a national shortage of clinical laboratory scientists. the number of training programs has decreased over the past decade, and qualified students are now choosing careers in medicine or computer science. nurses are paid dramatically more than clinical laboratory scientists-a further disincentive for students to consider a laboratory career-although the same level of professionalism and dedication to patient care is required. the overall vacancy rate among microbiology technologists is 57%, and almost one-half of all vacancies require at least 3 months to fill [3] . the staffing shortage in the pipeline in the united states today becomes more worrisome when combined with the aging of the current experienced staff. thirty-four percent of microbiologists working today are 150 years old [3] ; most of these will retire in the next 10 years. their competence in molecular methods is miniscule. however, these senior microbiologists can interpret a gram stain and deliver an educated guess as to the identification of a pathogen at an early stage. infectious diseases clinicians have relied on these expert workers, and reliable molecular diagnostic tests are not readily available for many infectious agents commercial tests should only be used for validated specimen types transportation problems, low concentrations of infectious agent, primer binding site genetic changes, final assay volume, inhibition, contamination, nonspecific amplification, and operator error lead to false-negative and false-positive amplification results genomic bacterial sequencing is subject to error because of sequence homology among different bacteria, database problems, and mutations unknown extent of microbial dna in "normal" host tissues lack of necessary resources (human and economic) reimbursement often not sufficient to cover costs today's infectious diseases trainees are still being trained in laboratories utilizing such resources. the new, younger laboratory workforce is not highly skilled in either the conventional or the high-tech environment, and microbiologists in general are in short supply. we are facing a knowledge and practice gap for the next 10-15 years, during which time molecular tools will gradually fulfill their promise. even in those laboratories that are staffed with skilled microbiologists during the daytime, many critical test results are needed on the evening and night shifts, when the laboratories are staffed by nonspecialized laboratorians. as a result, physicians will have less trust in test results and will need to make a greater number of empirical decisions and use a greater number of broad-spectrum antimicrobial agents. another aspect of the dearth of experienced technologists is that testing has become more instrument based than discipline based. this is apparent in the migration of hepatitis and hiv serological testing from microbiology or virology departments to the immunochemistry laboratory, where generalists or chemistry technologists perform these tests along with tests for other serum factors, such as toxins and proteins. the testers are, in general, not knowledgeable about the agents for which they are testing, which could lead to interpretation errors or failure to recognize inappropriate results. it is difficult, at best, to validate test results (assuring the clinical utility, reliability, and reproducibility of results) using new technologies. traditional culture-or antigen-based comparator methods often yield results that fail to correlate with the newer, more sensitive tests. finding clinical criteria or alternate molecular targets to verify which result is correct can be tedious, expensive, and occasionally impossible. obtaining adequate standards or positive patient samples to validate test results and to train performing technologists is also challenging. one example is pcr of csf specimens for herpes simplex virus meningoencephalitis; this is considered to be the best diagnostic assay available, because cultures are insensitive, and brain biopsy is too invasive [4] . the difficulties in finding enough samples and in creating seeded samples with known viral loads for validation studies have prevented or delayed development of this and other tests in many laboratories [5, 6] . in fact, identical problems have impeded submission and us food and drug administration (fda) approval of commercial assays for herpes simplex virus. some analyte-specific reagent test kits are available for both herpes simplex virus and enteroviruses, but they still require extensive in-house validation studies. the threat of an avian influenza pandemic may help spur the development of multiplex molecular assays for respiratory viruses [7] . the use of laboratory tests for diagnosis of enteroviral cns disease was shown to be cost effective long ago [8] , but the availability of rapid molecular tests for this purpose has been long coming. in many circumstances, tests that infectious diseases physicians have heard about (e.g., at national meetings, such as that of the infectious diseases society of america) will be available only as send-out tests to referral laboratories, accompanied by the attendant transportation problems and possible specimen degradation, delayed results, and costs. if an available molecular test is not performed in the local laboratory, one of the most important benefits-rapid results-is not realized, because the sample must be sent to a distant reference laboratory. even after the newest generation of real-time molecular test systems, such as the genexpert test for group b streptococci (cepheid), slated for fda clearance as moderately complex, becomes available [9] , their initial cost may delay widespread implementation. there are, of course, some widely performed molecular methods, such as hiv load [10] and nucleic acid amplification (naa) assays for chlamydia trachomatis and n. gonorrhoeae that use genital samples or urine specimens [11, 12] . however, many physicians assume that one can just as easily test an eye swab as a cervical swab, without appreciating that the laboratory must perform an extensive validation of a sample not included in the original fda clearance of the device if a report is to be issued (and the test is to be paid for) [13, 14] . many molecular tests are limited to reference laboratories or for research investigations only. of course, not all new technology is nucleic acid based, and i will also discuss other methods. a number of nucleic acid hybridization and amplification methods are now in use, including direct probe hybridization (advandx fish for staphylococcus aureus [advandx] and genprobe for group a streptococci [genprobe]), hybrid capture (digene for human papillomavirus; digene), pcr, branched-chain dna (bdna; bayer diagnostics), and transcription-mediated amplification (probe-tec for chlamydia and n. gonorrhoeae; becton dickinson). sample volume (which encompasses both original sample volume and number of nucleic acid sequences present in the final amplified sample portion), transport conditions, dilution factors, inhibitory factors, genome changes, instrument problems, and operator error all contribute to false-positive and false-negative test results [6, [15] [16] [17] [18] . even closed systems at the detection end do not prevent cross-contamination from positive sample processing at the front end. for example, current fda restrictions require that papanicolaou smears be first performed when a single suspension of cervical cells is received in preservative, with requests for both cytology and for detection of human papillomavirus, chlamydia species, and n. gonorrhoeae; this contributes to the common finding that insufficient sample size remains for the infectious diseases tests after the papanicolaou sample has been removed [19] . unfortunately, unlike direct visual examinations in which sample quality can be assessed, a negative result of such an assay includes no comment on the quality of the specimen. although molecular tests that use whole blood samples for detection of the agents of sepsis are not yet available, several such assays are in development. volume will be a factor in the sensitivity of these tests. the amount of bacteria in blood obtained from a patient with bacteremia is often !1 cfu/ml [20] , yet the volume used in most naa reaction vials is !10 ml. even if the sample is concentrated in some way, the chance that microbial genetic material will reach the reaction vial could be very small, resulting in false-negative results. in samples containing human neutrophils or erythrocytes-the very samples most likely to harbor the infectious agent-it is challenging to concentrate microbial dna without also adding overwhelming amounts of human somatic dna, and the available frontend methods (extraction and filtration columns, magnetic particle capture, and silica-based systems) still lack efficiency [21, 22] . manufacturers are spending most of their energy on backend technology (detection and reporting). front-end processing is urgently needed, ideally resulting in the development of a creative new method for concentrating cell-rich specimens (such as purulent fluid, blood, and sputum) from humans to allow for sensitive detection of microbial nucleic acids. diagnosis of tuberculous meningitis is another example of the problems inherent in amplification technology using pcr [23] . patients may have confounding signs and symptoms, the number of organisms in the csf sample may be very low, and conventional tests are insensitive and/or slow. direct acid-fast staining of csf specimens has a sensitivity of ∼52% [24] . the volume of csf needed for adequate culture is at least 5 ml (preferably 10 ml), because mycobacterium tuberculosis is extremely buoyant in csf as a result of its waxy cell wall; thus, concentration by centrifugation is inefficient [25] . smears for acid-fast bacilli made from the sediment must be layered and examined exhaustively-sometimes for 30 min-in the hope of detecting the rare m. tuberculosis rod [23] . naa would seem to be the answer. sadly, naa tests display an overall sensitivity of 71% (range, 25%-100%) and a specificity of 95% (range, 92%-97%), as determined by a meta-analysis of 49 published studies, 35 of which used home-brewed (individual laboratorydeveloped and -validated) methods [26] . when results from the 14 laboratories that used commercially available assays were evaluated separately, the overall sensitivity decreased to 56% (range, 46%-66%). these are the assays that are likely to be available to most clinicians. the naa results were equal to those for the acid-fast bacilli smear. clinicians must realize that unwanted reliance on new technology may lead to diagnostic errors. another highly favored technology for laboratory diagnosis of infectious diseases, whether testing tissue directly or testing isolated colonies, is identification by nucleic acid sequencing. there are also pitfalls in this approach. for example, many organisms, although they may be antigenically or biochemically distinct, are virtually genetically identical. members of the bacteroides fragilis group, for example, are highly related and cannot be separated easily by standard sequencing methods [27] . most clinicians realize that escherichia coli and shigella species are highly related [28] , but they have not considered the difficulty that this relatedness would pose if we relied on sequencing to identify these genera directly in patient samples. the extent of shared genes and taxonomically (versus phenotypically) shared traits among most microbes is unknown. another issue that vexes clinicians is the taxonomic confusion caused by genetic investigations. rhinosporidium seeberi, for example, is an agent of mass lesions in the sinus and mucous membranes. this organism has often been observed in histopathologic sections, but it has never been grown in culture. it resembles the spherule of coccidioides immitis, however, so it has long been considered an "unculturable" fungus. recently, 18s rrna sequencing placed the organism into a group of protozoan fish parasites [29] . we are constantly faced with new names for familiar organisms-and even with new organisms that may or may not be important in familiar syndromes. before more-extensive testing was performed, the newly discovered mycoplasma fermentans (originally called mycoplasma incognitus) had been implicated in both gulf war syndrome and hiv infection [30, 31] . it was later found to be quite common in humans [32] . after effective therapy, results of pcr of genital samples obtained from patients with c. trachomatis remain positive for 12 weeks [33] . nucleic acid is detectable but probably not viable. we do not know the extent of carriage of unculturable microbial or "normal" microbial dna in other healthy hosts. the cost of equipment, expensive reagents, and additional skilled personnel needed is not easy to justify. for example, the fda has cleared a real-time pcr test for group b streptococcal vaginal/rectal colonization during labor [34] . widespread use of this test could negate the need to perform cultures for pregnant women during weeks 35-37 of gestation, and it would solve the problems associated with treating women who present in labor without screening test results [35] . currently, 25% of women receive prophylactic penicillin during labor [36] . this number would decrease if only women with pcr results positive for group b streptococci at labor were treated [36, 37] . the problem, however, is that current recommendations [38] have already reduced the incidence of early-onset neonatal group b streptococcal disease to less than the 2010 national health objective of 0.5 cases per 1000 live births, and additional improvement is not cost-effective [39] . lucile packard childrens hospital johnson center, a stanford university medical center affiliate on the same campus, delivers ∼1800 babies annually. to perform the pcr assay immediately after each sample is received (a necessity to assure that prophylaxis is administered for at least 4 h before delivery), we would have to staff 1 technologist at all times. test performance takes ∼15 min of hands-on time, and the test itself runs 45 min, so a technologist must perform continuous sample management. twenty-four/seven staffing for 365 days per year would require 3.9 clinical laboratory scientists, at a labor and overhead cost of $316,875. the reagents-not counting the cost of the instrument itself-cost $46,800 annually, for a total yearly cost of $363,675. stanford university medical center (stanford, ca) patients have a colonization rate of 17% among women in labor, resulting in 306 babies at risk per year. to prevent 3 potential group b streptococcal infections using this test, the cost per infection avoided would be $121,225. this is a hard sell to hospital administrators who are focused on the bottom line. potential laboratory staff labor savings with the use of random-access, moderately complex molecular systems, such as genexpert (mentioned above), may help to bring down costs. costs for new technologies are often added onto existing testing, so overall diagnostic costs increase. centers for medicare and medicaid services reimbursement is lagging dramatically behind the use of new tests. in the northern california reimbursement area, our laboratory receives only $98.08 for a pcr test for bordetella pertussis and bordetella parapertussis, although it costs the laboratory more than $150 to test each sample, because the tests are always performed one specimen at a time, which necessitates testing an additional 3 controls simultaneously. the future is not all bleak, of course. new agents of important diseases are being discovered with the help of molecular technologies. among those to have been elucidated recently are tropheryma whipplei, bartonella henselae, ehrlichia chaffeensis, and mycoplasma genitalium [40] . infectious disease-related challenges are ready for solutions with our new molecular tools. the etiology of 42% of cases of pharyngitis is unknown [41] , and the etiology of 20%-40% of cases of diarrhea is unknown [42] , as is the etiology of 40%-60% of cases of pneumonia [43] . we cannot cultivate ∼25% of the organisms in the subgingival crevices of patients with periodontal disease [40] . all of these mysteries regarding vexing syndromes will eventually yield to molecular technologies. proteomics (i.e., profiling the proteins generated by human cells in response to various stimuli, including components and products of infectious agents) is in its infancy, but exciting developments are enticing. the spots of dna or rna on "gene chips" can be designed to be homologous with nucleic acid sequences in the sample, representing nucleic acids found in both the normal situation and the activated state of transcription or representing various genetic sequences unique to a vast array of microbes for detection of infectious agents [44] . samples are treated to break down their dna or rna into small discrete sequences, for which an identical sequence exists on the chip. if the sample nucleic acid finds its match, it binds and emits an electronic signal [45] . such a human-response chip can be used to determine a patient's response to potentially toxic antimicrobial treatments in advance of delivery. if the patient lacks enzymes to break down toxins or cannot metabolize a drug effectively, alternative therapies can be used to avoid dangerous adverse effects [46] . in another prospective microarray-based test, human leukocyte response to various infectious agents may be used to pinpoint the etiology before the agent itself can be detected [47] . development of this sort of technology is on a fast track, to facilitate detection of bioterrorism agents before the symptomatic phase of disease renders widespread preventive measurements inadequate [48] . proteomics is being used to fine-tune the development of new anti-infectives as well [49] . one aspect of microarray technol-ogy yet to be standardized and codified is the monumental problem of data analysis [50] . an entire new discipline of bioinformatics has developed for this purpose. this challenge of interpreting the vast volume of data points generated by microarray studies is acquired along with the technological advances, and its resolution will require a collective and extensive effort. in summary, the future potential utility of these incredible technologies-and of numerous others not mentioned herewill revolutionize infectious diseases diagnostics. however, the shortcomings of these technologies must be recognized. present dangers include failure to support the need for traditionally trained microbiologists, allowance of expertise to be moved to sites distant from patient care activities, inappropriate trust of new technology, and underestimation of the value of clinical diagnosis based on the acumen of experienced laboratorians and infectious diseases practitioners. dna probes for infectious diseases molecular and cellular microbiology: new tools of the trade survey of clinical microbiology laboratory workloads, productivity rates, andstaffing vacancies herpes simplex encephalitis: children and adolescents cumitech 31-verification and validation of procedures in the clinical microbiology laboratory verification of infectious disease molecular assays: a self-study module (cd-rom or web-based) a multiplex rt-pcr for detection of type a influenza virus and differentiation of avian h5, h7, and h9 hemagglutinin subtypes the clinical relevance of 'csf viral culture': a two-year experience with aseptic meningitis in a rapid and highly accurate assay for the detection of enterovirus infections in cerebrospinal fluid samples using the genexpert dx system impact of human immunodeficiency virus type 1 (hiv-1) genetic diversity on performance of four commercial viral load assays: lcx hiv rna quantitative, am-plicor hiv-1 monitor v1.5, versant hiv-1 rna 3.0, and nuclisens hiv-1 qt comparing first-void urine specimens, self-collected vaginal swabs, and endocervical specimens to detect chlamydia trachomatis and neisseria gonorrhoeae by a nucleic acid amplification test comparison of methods for detection of chlamydia trachomatis and neisseria gonorrhoeae using commercially available nucleic acid amplification tests and a liquid pap smear medium validation of molecular-diagnostic techniques in the parasitological laboratory diagnostic pcr: validation and sample preparation are two sides of the same coin failure of commercial ligase chain reaction to detect mycobacterium tuberculosis dna in sputum samples from a patient with smear-positive pulmonary tuberculosis due to a deletion of the target region reproducibility of positive test results in the bdprobetec et system for detection of chlamydia trachomatis and neisseria gonorrhoeae false-positive gen-probe direct mycobacterium tuberculosis amplification test results for patients with pulmonary m. kansasii and m. avium infections the current status and potential role of laboratory testing to prevent transfusion-transmitted malaria characteristics of apparently false-negative digene hybrid capture 2 high-risk hpv dna testing occurrence and documentation of low-level bacteremia in a community hospital's patient population comparison of 9 different pcr primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 rna extraction methods comparison of six dna extraction methods for recovery of fungal dna as assessed by quantitative pcr rapid diagnosis of tuberculous meningitis: what is the optimal method? comparison of conventional bacteriology with nucleic acid amplification (amplified mycobacterium direct test) for diagnosis of tuberculous meningitis before and after inception of antituberculosis chemotherapy the bacteriological diagnosis of tuberculous meningitis diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis rapid identification of the species of the bacteroides fragilis group by multiplex pcr assays using group-and species-specific primers sequence analysis of four shigella boydii o-antigen loci: implication for escherichia coli and shigella relationships rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites lack of serological evidence for mycoplasma fermentans infection in army gulf war veterans: a large scale case-control study mycoplasma fermentans in individuals seropositive and seronegative for hiv-1 serological responses to mycoplasmas in hiv-infected and non-infected individuals monitoring of chlamydia trachomatis infections after antibiotic treatment using rna detection by nucleic acid sequence based amplification multicenter study of a rapid molecular-based assay for the diagnosis of group b streptococcus colonization in pregnant women comparison of rapid intrapartum screening methods for group b streptococcal vaginal colonization perinatal screening for group b streptococci: cost-benefit analysis of rapid polymerase chain reaction risk factors for early-onset group b streptococcal sepsis: estimation of odds ratios by critical literature review prevention of perinatal group b streptococcal disease: revised guidelines from cdc centers for disease control and prevention. diminishing racial disparities in early-onset neonatal group b streptococcal disease-united states discovering new pathogens: culture-resistant bacteria etiology of acute pharyngitis in children: is antibiotic therapy needed? the management of acute diarrhea in children: oral rehydration, maintenance, and nutritional therapy practice guidelines for the management of community-acquired pneumonia in adults. infectious diseases society of america dna microarray technology: devices, systems, and applications molecular signatures for diagnosis of infection: application of microarray technology use of dna microarrays to monitor host response to virus and virus-derived gene therapy vectors comparative dna microarray analysis of host cell transcriptional responses to infection by coxiella burnetii or chlamydia trachomatis applications and challenges of dna microarray technology in military medical research using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance iterative group analysis (iga): a simple tool to enhance sensitivity and facilitate interpretation of microarray experiments potential conflicts of interest. e.j.b. has been on the speakers' bureau for becton dickinson and cepheid and has received research support materials from cepheid, roche molecular diagnostics, and advandx. key: cord-310476-mhm7r3qc authors: yi, huiguang title: 2019 novel coronavirus is undergoing active recombination date: 2020-03-04 journal: clin infect dis doi: 10.1093/cid/ciaa219 sha: doc_id: 310476 cord_uid: mhm7r3qc nan (shown as a pie chart node in fig. 1 , see tab. s1 for the accessions), then the network is constructed using the median join [4] method in popart [5] . we found the 2019-ncov haplotype network has obvious characteristics of single origin from haplotype hap_011: first, the network is star-like, centralized on the haplotype hap_011; second, hap_011 has the largest sample size and majority of the samples are from hubei province-where outbreak originated (tab. s1); third, most of satellite haplotypes are also from hubei (fig. 1) ; fourth, the average collection dates of hap_011 (has 0 mutation relative to mn908947) is earlier than all other mutation groups (fig. s1 ). the single origin of 2019-ncov indicates a persistent animal to human transmission is unlikely, otherwise, multiple nodes with above characteristics should be observed. we found five haplotypes (hap_009, hap_017, hap_023, hap_048 and hap_050) forming loops ( fig. 1) , which typically indicate existing of genetic recombination. however, in rare cases, loops can also be formed by recurring sequencing error or parallel/back mutations. further examination of the differences pattern among the five haplotypes found three sets of recurrent differences: c29095t, g11083t and c8782t/t28144c (fig. 1) , most of which are nonsynonymous changes except c29095t (tab. s2). such highly frequent recurrent differences looks extremely unlikely raised by rare events like recurring sequencing error/mutations. to quantify the significance of recombination over recurring sequencing error/mutations, phi [6] and max chi-squared tests [7] in the software phipack [8] were performed. it showed p-values of 0.03 and 0.005 for phi and max chi-squared, respectively, indicating the presence of recombinants in 2019-ncov population. 3 since all the haplotypes originate from hap_011, the formation mechanisms of the five haplotypes could be inferred as follow: first, hap_048 and hap_050 mutated from the common ancestry hap_011 independently, hap_009 then mutated from hap_050; next, hap_048 donated the genome region harboring 11083t to hap_050 generated the recombinant hap_017; similarly, hap_048 or hap_017 donated the same region to hap_009 generated the recombinant hap_023 susceptibility directly from other strains [12, 13] ; the adaptability of 2019-ncov to human immune system might be significantly strengthened through genetic recombination; the accuracy of diagnosis based on serologic and molecular biology assays might be compromised [14] ; and the transmission tracking based on phylogenetics tree could be misleading since the topology of mutation route is a network rather than a tree. we think the communities of infectious disease physicians and disease control specialists should arouse particular concerns about this finding and its potential implications. and we expect this 4 findings may provide a valuable perspective for strategies design for secondary transmission prevention and drug treatments for 2019-ncov pneumonia. each haplotype node is represent by a pie chart, with size indicating the sample size (tab s1), and colors indicating collection regions. each hatch mark along the edge corresponds to a mutation between the two haplotypes linked. the mutation notation along a loop edge shows the position and nucleotide status change between the two haplotypes linked. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia. 2020. 2. confirmed 2019-ncov cases globally global map real-time, portable genome sequencing for ebola surveillance median-joining networks for inferring intraspecific phylogenies full-feature software for haplotype network construction a simple and robust statistical test for detecting the presence of recombination analyzing the mosaic structure of genes phipack: phi test and other tests of recombination ncov's relationship to bat coronaviruses & recombination signals (no snakes) -no evidence the 2019-ncov lineage is recombinant full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding possible involvement of cell fusion and viral recombination in generation of human immunodeficiency virus variants that display dual resistance to azt and 3tc recombination leads to the rapid emergence of hiv-1 dually resistant mutants under selective drug pressure human immunodeficiency virus superinfection and recombination: current state of knowledge and potential clinical consequences we acknowledge the authors and the originating and submitting laboratories of the nucleotide sequences from betacov2019-2020 of gisaid epiflutm database (9 feb 2020, 84 isolates). all authors: no reported conflicts. key: cord-258724-1qhen1bj authors: young, barnaby e; ong, sean w x; ng, lisa f p; anderson, danielle e; chia, wan ni; chia, po ying; ang, li wei; mak, tze-minn; kalimuddin, shirin; chai, louis yi ann; pada, surinder; tan, seow yen; sun, louisa; parthasarathy, purnima; fong, siew-wai; chan, yi-hao; tan, chee wah; lee, bernett; rötzschke, olaf; ding, ying; tambyah, paul; low, jenny g h; cui, lin; barkham, timothy; lin, raymond tzer pin; leo, yee-sin; renia, laurent; wang, lin-fa; lye, david chien title: viral dynamics and immune correlates of covid-19 disease severity date: 2020-08-28 journal: clin infect dis doi: 10.1093/cid/ciaa1280 sha: doc_id: 258724 cord_uid: 1qhen1bj background: key knowledge gaps remain in the understanding of viral dynamics and immune response of sars-cov-2 infection. methods: we evaluated these characteristics and established their association with clinical severity in a prospective observational cohort study of 100 patients with pcr-confirmed sars-cov-2 infection (mean age 46 years, 56% male, 38% with comorbidities). respiratory samples (n=74) were collected for viral culture, serum samples for measurement of igm/igg levels (n=30), and plasma samples for levels of inflammatory cytokines and chemokines (n=81). disease severity was correlated with results from viral culture, serologic testing, and immune markers. results: 57 (57%) patients developed viral pneumonia, of whom 20 (20%) required supplemental oxygen including 12 (12%) invasive mechanical ventilation. viral culture from respiratory samples was positive for 19 of 74 patients (26%). no virus was isolated when the pcr cycle threshold (ct) value was >30 or >14 days after symptom onset. seroconversion occurred at a median of 12.5 days (iqr 9-18) for igm and 15.0 days (iqr 12-20) for igg; 54/62 patients (87.1%) sampled at day 14 or later seroconverted. severe infections were associated with earlier seroconversion and higher peak igm and igg levels. levels of ip-10, hgf, il-6, mcp-1, mip-1α, il-12p70, il-18, vegf-a, pdgf-bb and il-1ra significantly correlated with disease severity. conclusion: we found virus viability was associated with lower pcr ct value in early illness. a stronger antibody response was associated with disease severity. the overactive proinflammatory immune signatures offers targets for host-directed immunotherapy which should be evaluated in randomised controlled trials. a c c e p t e d m a n u s c r i p t the coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused great harm to health and the global economy [1, 2] . understanding of sars-cov-2 pathogeneisis has advanced at an unprecedented speed, but key gaps remain and preliminary findings require validation. studies of covid-19 patients have described the inflammatory milieu in severe infections, with raised neutrophils, suppressed lymphocytes and elevated inflammatory mediators [3, 4] . however, most studies are limited to comparing severe against non-severe infections and lack serial data [5, 6] . a clearer definition of disease pathogenesis will support the development of risk stratification tools and therapeutics targeting critical pathways in the inflammatory cascade. early seroconversion has been reported with igg to the receptor binding domain (rbd) detected at day seven [7] [8] [9] [10] . however, evidence of correlation between antibody titres and disease severity is conflicting [8, [10] [11] [12] . there remains a need for more detailed assessment of antibody kinetics to help determine the impact of antibody-dependent enhancement in covid-19 pathogenesis, as well as guide convalescent plasma harvesting and use of serological assays for diagnosis [13] . sars-cov-2 can be detected from the nasopharynx for a median of 2-3 weeks following onset of symptoms [14] . several studies have reported that in immunocompetent individuals, virus is typically only cultured from respiratory samples during the first week of illness when viral loads are highest [8, 15, 16] . this suggests transmission risk declines in the second week. this finding requires confirmation in larger cohorts as it has important implications for infection control and isolation protocols [17] . a c c e p t e d m a n u s c r i p t in this multi-pronged study, we describe the serologic evolution, inflammatory response and pattern of viral shedding and viability in patients with virologically confirmed covid-19 in singapore, and analyse the contributions these make to severe infections. all individuals confirmed to have covid-19 by sars-cov-2 real-time reverse transcriptasepolymerase chain reaction (rt-pcr) and admitted to any of seven public hospitals in singapore were eligible for inclusion in this study. rt-pcr was performed on respiratory samples as previously described [18] (details in supplementary appendix). clinical information was extracted from the medical record using a standardized data collection form adapted from the international severe acute respiratory and emerging infection consortium (isaric) case record form [19] . serial blood and respiratory samples were collected during hospitalisation and follow-up post-discharge (days 1, 3, 7, 14, 21 and 28 after enrolment). a stool and urine sample was also collected for sars-cov-2 pcr and culture on day 1. at the time of the study, all patients with covid-19 in singapore were admitted to airborne infection isolation rooms regardless of disease severity. supportive therapy including supplemental oxygen and symptomatic treatment were administered as required. patients with moderate to severe hypoxia were transferred to icu for further management and invasive mechanical ventilation as required. a c c e p t e d m a n u s c r i p t patients were discharged from hospital only after resolution of symptoms and when two consecutive nasopharyngeal swabs >24 hours apart were negative for sars-cov-2 by rt-pcr. follow-up visits were arranged on day 28 of enrolment. material from nasopharyngeal swabs was collected in universal transport media and used to inoculate vero-e6 cells (atcc®crl-1586tm) for virus isolation in an animal biological safety level 3 laboratory. urine and stool samples were collected and transported fresh for virus culture. stools were filtered before inoculation. cells were cultured at 37 o c for seven days or less if cytopathic effect (cpe) was observed, and three blind passages were performed. cpe consisted of rounded cells and extensive cell death, usually by day four post-inoculation. positive isolation was confirmed by the observation of cpe and virus-specific pcr. total rna was extracted from all samples using e.z.n.a. total rna kit i (omega bio-tek inc.) according to the manufacturer's instructions and samples were analyzed by rt-pcr for the detection of sars-cov-2 as previously described [20] . serum collected during the acute and convalescent phases of infection were tested for sars-cov-2 receptor binding domain specific igm and igg using capture elisa (details in supplementary appendix). plasma samples were collected during acute and convalescent phases and treated with a solvent/detergent based on triton tm x-100 (1%) for virus inactivation [21] . immune mediator levels were measured using cytokine/chemokine/growth factor 45-plex human procartaplex tm panel 1 (thermofisher scientific) (details in supplementary appendix). cytokine levels were also measured in 23 healthy donor plasma as baseline controls. a c c e p t e d m a n u s c r i p t patients were categorised into three groups for comparison: no pneumonia on chest radiographs (cxr) throughout admission; pneumonia on cxr without hypoxia; pneumonia with hypoxia (desaturation to ≤94%) needing supplemental oxygen. day one was defined as the first day of symptom onset. fold change (fc) of antibody titres compared with negative controls were calculated. kruskal-wallis test followed by dunn's multiple comparison test was used to determine significance of antibody levels. unpaired t-test was applied to ascertain significant difference in the immune mediator levels between the covid-19 patients and healthy controls at different time points post illness onset. oneway anova analysis with post-hoc t-test corrected using the method of bonferroni was used to discern the differences in immune mediator levels between the various disease severity groups. one-way anova results were corrected for multiple testing using the method of benjamini and hochberg. a c c e p t e d m a n u s c r i p t written informed consent was obtained from all participants. the study protocol at all sites was approved by the national healthcare group domain specific review board, study reference 2012/00917; additional study protocol at singapore general hospital approved by the singhealth centralised institutional review board, study reference 2018/3045. 130 covid-19 patients were diagnosed in singapore from the first known case on 22 january 2020 to 6 march 2020 ( figure 1 ). among the 100 (77%) enrolled in this study, viral pneumonia was diagnosed by cxr in 57, of whom 20 required supplemental oxygen for hypoxia, and 12 required invasive mechanical ventilation (table 1) . following resolution of infection and viral shedding, 97 patients have been discharged and after 90 days of follow up, no re-infection or recrudescence has been detected. three patients have died. deaths occurred 27 days or more after hospital admission, and while all were assessed by the managing clinican as related to covid-19 they followed viral clearance and prolonged invasive mechanical ventilation. sars-cov-2 was detectable from nasopharyngeal swabs by pcr up to 48 days after symptom onset. further network analyses showed association between the aforementioned immune mediators and clinical parameters (pneumonia, hypoxia and icu admission). the intertwined relationship of the cytokines is shown in figure 5a . in addition, ingenuity pathway analysis (ipa) revealed canonical pathways associated with these immune mediators and severity, with the top ten canonical pathways involved in inflammatory diseases and cell signalling ( figure 5b ). the top canonical pathway highlights the common immune mediators between influenza and sars-cov-2 infection, including mcp-1, il-1ra, ip-10 and il-6 ( figure 5c ). in addition to ipa, string prediction of proteinprotein interactions identified il-6 as a direct interacting partner with other severity-associated immune mediators ( figure 5d ). longitudinal comparison of these immune mediators associated with severity in 12 icu patients was performed to explore their role as prognostic markers for severe covid-19 (supplementary figure 6 ). four of these twelve patients (ct009, ct032, ct037 and ct057) recovered and were discharged at the time of study. interestingly, hgf and vegf-a were distinctly separated into two levels, with the four discharged patients having lower hgf and vegf-a levels, and hgf approaching healthy baseline levels during convalescence, which further indicates that high levels of these cytokines were this study of 100 patients in the first few months of the covid-19 pandemic in singapore provides a detailed overview of clinical presentation, progress and outcomes. case detection and contact tracing in singapore is rigorous with the main gap possibly in missing asymptomatic cases [22] . from the cohort, 43% of patients never developed pneumonia, 37% developed pneumonia without hypoxia and 20% pneumonia with hypoxia. we found no relationship between illness severity and duration of viral shedding or pcr ct values. the central role of the immune response to sars-cov-2 in covid-19 was evident from the strong correlation between disease severity and levels of igg/igm and inflammatory immune mediators in our cohort. viral shedding of sars-cov-2 from the respiratory tract has been observed to persist for several weeks and potentially up to months [18, [23] [24] [25] . whether the virus remains infectious throughout this prolonged viral shedding is an important question to resolve. smaller studies have found that viable virus was readily isolated from immunocompetent individuals during the first week of illness, but were unable to successfully isolate viruses in culture from day 8 onwards despite detectable viral load by pcr [8, 25, 26] . however, a case report from taiwan showed it was possible to culture the virus up till day 18 [27] , while positive cultures up to day 20 has been reported from a study of patients with severe infection [15] . we found that successful virus culture was associated with pcr ct value ≤30. virus was isolated up to day 14 post-symptom onset, though the majority were cultured at day 10 or earlier. using pcr ct value to guide decision making on de-isolation may be an a c c e p t e d m a n u s c r i p t alternative to using day of illness and provide an additional level of reassurance. however, this requires further validation. we observed seroconversion of igm at a median of 12.5 days and igg at 15.0 days. similar to a hong kong study of 16 patients, there were no significant differences of igm and igg titres by age or comorbidities [10] . we found igm and igg titres correlated with disease severity, similarly found in two chinese studies of 173 [7] and 285 patients respectively [28] . different sample sizes and antibody assays may account for differing seroconversion rates, which merits further investigation in a large cohort over a longer period. serological testing is vital for determining the prevalence of infection in sero-surveys and as part of epidemiological investigations to understand transmission of clusters [29] . the timing of seroconversion may guide the timing of plasmapheresis for convalescent plasma [30, 31] . the role of antibodies in long-term immunity after infection needs further investigation. cytokine storm in a subset of patients with markedly increased levels of pro-inflammatory cytokines, chemokines and growth factors [32, 33] . notably, higher levels of il-6, mcp-1, ip-10, il-18, il-1ra, pdgf-bb, hgf, vegf-a, il-12p70 and mip-1 were associated with severe disease in our study. this robust induction of pro-inflammatory mediators indicates that innate immune cell responses and anti-viral t-cell responses are responsible for sars-cov-2 pathogenesis in covid-19 patients [34] . in addition, elevation of growth factors, including hgf [35] , plgf-1 [36] and lif [37] , illuminates the a c c e p t e d m a n u s c r i p t repair mechanisms following acute lung injury during sars-cov-2 infection. previous studies showed that icu patients had more significant cytokine activation compared with non-icu patients [1] . our longitudinal cytokine profiling in icu patients further evaluated prognostic values of specific cytokines. we observed better prognosis in critically ill patients with lower levels of acute lung injury associated growth factors, hgf and vegf-a at the time of admission to icu. our data suggest that the differences in degree of lung injury could reflect recovery rate of patients in icu. hgf and vegf-a may serve as early indicators of poor prognosis and may provide guidance to make pre-emptive clinical decisions in critically ill patients. a central role for il-6 in lung injury has been postulated with higher levels associated with mortality in two separate studies and severe immune-mediated injury in lung tissues of patients who died from covid-19 [38, 39] . similarly, the il-1 pathway has been highlighted to contribute towards sars-cov-2 pathogenesis [40] . importantly, our study highlighted the upregulation of il-1 pathway mediators il-18, mcp-1, and vegf-a in critically-ill patients, providing further evidence that dysregulation in the il-1 pathway could contribute to the hyperinflammatory state, especially in fatal cases. string analysis revealed potential protein-to-protein interactions in severe covid-19 infection, in which il-6 is the direct interacting partner of other cytokines associated with disease severity. thus, several approved il-6 receptor antagonists could be repurposed to treat severe sars-cov-2 infection. given the elevated level of vegf-a, jak inhibitors should also be investigated as a potential therapeutic option [41, 42] . however, the serial data presented here indicates that the therapeutic window for intervention is narrow. immune modulators will need to be active before the inflammatory cascade causing acute lung injury develops. our study has several limitations. active case finding through contact tracing is likely to have identified the majority of symptomatic infections in singapore over this time period. however, a c c e p t e d m a n u s c r i p t atypical or subclinical infections may have been missed. no infections were detected in children (<18 years) or residents of long term care facilities. individuals were enrolled as soon as possible following admission, but while biological samples were collected serially, they were not all acquired at the same timepoint after symptom onset. additionally, some laboratory data were incomplete, and clinical data such as date of symptom onset is subject to recall bias. we also did not investigate the effect of different sars-cov-2 lineages or mutations on study outcomes [43] . finally, samples were processed consistently but numerous factors determine the success of viral cultures, and correlation between culturability and infectiousness is unclear. in conclusion, we found virus viability was associated with lower pcr ct value in early illness. this dr young had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. the funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. by reports personal fees from roche and sanofi, outside the submitted work. all other no interest declared. m a n u s c r i p t m a n u s c r i p t 130 patients diagnosed in singapore as of 6 march 2020. *1 excluded as diagnosis was retrospective via serology rather than pcr. the 100 were enrolled from the following hospitals in singapore: national centre for infectious diseases (77); national university hospital (8); singapore general hospital (8); ng teng fong general hospital (3); changi general hospital (2); alexandra hospital (1); khoo teck puat hospital (1). fold of change (fc) is calculated by dividing optical density (od) reading of a test sample by the average od reading of negative controls. samples with fc > 3 are considered positive. as sampling time and numbers were not uniform for all patients, we plotted the highest igm/igg from a single serum sample for each patient when multiple samples were available. clinical features of patients infected with 2019 novel coronavirus in wuhan, china interventions to mitigate early spread of sars-cov-2 in singapore: a modelling study clinical and immunological features of severe and moderate coronavirus disease 2019 dysregulation of immune response in patients with coronavirus longitudinal analyses reveal immunological misfiring in severe covid-19 longitudinal covid-19 profiling associates il-1ra and il-10 with disease severity and rantes with 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an analysis of data of 150 patients from wuhan, china pathological findings of covid-19 associated with acute respiratory distress syndrome interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study the trinity of covid-19: immunity, inflammation and intervention covid-19: combining antiviral and anti-inflammatory treatments discovery and genomic characterization of a 382-nucleotide deletion in orf7b and orf8 during the early evolution of sars-cov-2 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t duration of symptoms before admission 6 (2 to 9) 5 (2 to 9) 6 (4 to 9) 0. a c c e p t e d m a n u s c r i p t key: cord-010570-ytv7dwr0 authors: casadevall, arturo; scharff, matthew d. title: return to the past: the case for antibody-based therapies in infectious diseases date: 1995-07-17 journal: clin infect dis doi: 10.1093/clinids/21.1.150 sha: doc_id: 10570 cord_uid: ytv7dwr0 in the preantibiotic era, passive antibody administration (serum therapy) was useful for the treatment of many infectious diseases. the introduction of antimicrobial chemotherapy in the 1940s led to the rapid abandonment of many forms of passive antibody therapy. chemotherapy was more effective and less toxic than antibody therapy. in this last decade of the 20th century the efficacy of antimicrobial chemotherapy is diminishing because of the rapidly escalating number of immunocompromised individuals, the emergence of new pathogens, the reemergence of old pathogens, and widespread development of resistance to antimicrobial drugs. this diminishment in the effectiveness of chemotherapy has been paralleled by advances in monoclonal antibody technology that have made feasible the generation of human antibodies. this combination of factors makes passive antibody therapy an option worthy of serious consideration. we propose that for every pathogen there exists an antibody that will modify the infection to the benefit of the host. such antibodies are potential antimicrobial agents. antibody-based therapies have significant advantages and disadvantages relative to standard chemotherapy. the reintroduction of antibody-based therapy would require major changes in the practices of infectious disease specialists. the antibiotic era is about 60 years old. as we approach the 21st century, the progress made in controlling infections is threatened by the growing numbers of immunosuppressed individuals; the emergence of new pathogens; the reemergence of old pathogens; and the widespread resistance of organisms to antimicrobial drugs. each development poses its own set of challenges; in combination, these developments are already undermining the efficacy of antimicrobial chemotherapy. there is a worldwide epidemic of immunosuppression due to malnutrition, aids, medical therapies for cancer and autoimmune diseases, and organ transplantation. immunosuppressed hosts provide ecological niches for low-virulence microorganisms [1] . in addition, antimicrobial drugs are less effective and sometimes are unable to eradicate infection in individuals with impaired immunity. classical pathogens such as mycobacterium tuberculosis and treponema pallidum have reemerged and are difficult to treat in immunosuppressed individuals [2, 3] . the increasing frequency of drug-resistant organisms is an equally alarming development, and resistance may be developing faster than new antibiotics can be introduced [4] . for example, in one new york city hospital, the percentage of vancomycinsusceptible enterococcus faecium decreased from 86% to 26% in 2 years [5] . the problem of resistance is not limited to bacteria; there are numerous reports of fluconazole-resistant candida albicans [6] . we briefly review the use of antibody-based therapy in the early 20th century and make the case for reintroducing passive antibody administration for the treatment ofinfectious diseases. advances in monoclonal antibody (mab) technology provide new opportunities for developing antibody-based therapies. in the case of pathogens for which antimicrobial chemotherapy already exists, antibody-based therapies could be used with existing chemotherapy to improve outcome and possibly reduce the development of resistance. in the case of pathogens for which no effective chemotherapy exists, antibody-based therapies could be used for primary therapy. given the diminishing efficacy of existing antimicrobials because of widespread resistance and the difficulties of treating infections in immunosuppressed individuals, the reintroduction of antibody-based therapies is an option that should be given serious consideration. this reintroduction would require major changes in the practice of infectious disease medicine. the administration of immune serum (usually animal) for the treatment of infections was called serum therapy and was introduced in the 1890s for the treatment of diphtheria. by the 1930s, serum therapy was widely used for the treatment of table 1 . infectious diseases that were treated with antibody-based therapies in the preantibiotic era. bacterial and viral infections (table 1) . for example, in the late 1930s, 86% of patients with type i pneumococcal pneumonia admitted to boston city hospital were treated with type-specific serum [36] . from 1927 to 1932 at bellevue hospital (new york), > 3,000 patients with erysipelas were treated with serum [37] (figure 1). the efficacy of serum therapy varied with the type or severity of infection. several large controlled studies revealed that type-specific serum reduced the mortality ofpneumococcal pneumonia [7] . serum therapy also appears to have significantly reduced mortality due to meningococcal meningitis in some epidemics [7, 38] . serum therapy reduced mortality due to haemophilus irif/uenzae meningitis, but the effect was small [8] [9] [10] . serum therapy for erysipelas reduced mortality in comparison to historical controls [37, 39] . serum therapy reduced mortality in diphtheria, and antibody therapy continues to be used today to treat this disease [40] . the efficacy of serum therapy for whooping cough, anthrax, dysentery (shigella dysenteriae), and gas gangrene was uncertain. human convalescent serum was effective for prophylaxis ofmeasles, which had a mortality rate of 6%-7% in some populations [29] [30] [31] . the effectiveness of serum in the pre-paralytic stage of poliomyelitis was uncertain [31, 32] . no consistently effective sera were developed against many pathogens, including staphylococcus [41] , mycobacterium, and salmonella species [42] . the historical use of serum therapy provides lessons for the effective use and development of antibody-based therapies. accurate microbiological diagnosis was essential for successful treatment, and serum therapy was most effective when used for prophylaxis or for therapy early in the course of infection. note. this is not a complete list. [7] [7] [8] [9] [10] nevertheless, it was possible to treat established bacterial infections such as meningococcal meningitis and pneumococcal pneumonia if antibody was administered shortly after symptoms began [7] . antibodies functioned as direct antibacterial agents (e.g., pneumococcal antisera) or antitoxins (e.g., diphtherial antisera). nonphysiological and nontherapeutic animal models of infection allowed investigators to successfully identify clinically useful antibody reagents [7] . a considerable amount of basic immunologic and microbiological research was required to develop each serum, and many fundamental discoveries were made in the search for better sera. (the contributions of pneumococcal research to basic science are discussed in [43] .) sulfonamides were introduced in 1935 and rapidly became the standard therapy for many infections [44] . because antimicrobial chemotherapy had significant advantages over serum therapy, the latter was largely abandoned. systemic administration of animal sera caused fevers, chills, and allergic reactions [23, 45] ; "serum sickness," a self-limited syndrome characterized by rash, proteinuria, and arthralgias, occurred in 10%-50% of patients because of immune complex disease. in addition, strain typing was necessary for choosing pneumococcal antisera; there was significant lot-to-lot variation in serum efficacy [46] ; and dosing was based on clinical experience. inadequate dosage, delays in treatment, errors in typing, mixed infections, and complicated infections (e.g., empyema) could result in failure of serum therapy [47] . serum was expensive because of the costs of animal husbandry, antibody purification, refrigeration, and reliance on mouse protection tests for standardization. chemotherapy was less toxic and more effective than serum therapy. treatment with type-specific serum reduced the mortality of pneumococcal pneumonia from 30%-40% to 10%-20% [7] , whereas the mortality rate among patients treated with sulfonamide was 7% [48] . serum therapy reduced the mortality rate of erysipelas from 10% to 7% [37] , whereas the mortality rate for patients treated with sulfonamides was 1%-2% [49] . for meningococcal meningitis, the efficacy of serum depended on the epidemic. in the mid-1930s, waghelstein [50] reported that the mortality rate for patients with meningococcal meningitis who were treated at sydenham hospital (baltimore, md) with sulfonamide was 15.3% and for those treated with serum it was 27%. in controlled trials, the superiority of chemotherapy over serum therapy was lessevident. for example, the mortality among patients with meningococcal meningitis who were treated early with adequate serum therapy was 16.7% vs. 11.6% for sulfanilamide therapy [50] . the mortality among patients with pneumococcal pneumonia treated with antimicrobial drugs or serum was 11% and 16.7%, respectively, but there was no difference in the mortality rates among younger .. literature sent on request. is used at bellevue hospital. it is supplied in packages of one therapeutic dose of approximately ten cubic centimeters. the data cited were published in [37] . patients, and younger patients who received serum recovered faster than those who received antimicrobials [51] . in the early days of the antibiotic era there was interest in using combination therapies with antibiotics and serum. sulfonamides and serum had a synergistic or additive effect against streptococcus pneumoniae [52] [53] [54] [55] , streptococci [56] , and meningococci [57] . the finding that sulfonamides made pneumococci more susceptible to antibody-mediated phagocytosis supported the idea of combination therapy [58] . it was similarly recognized that sulfonamides, unlike antibody, did not neutralize bacterial toxins or modify the course of disease caused by diphtheria or tetanus toxins [59] . combination therapy was recommended for scarlet fever [60] , pneumococcal pneumonia [44, 54, 61] , whooping cough [20] and meningitis due to pneumococcus [8, 62] , meningococcus [8, 50, 63, 64] , or h. influenzae [8] . however, the side effects of serum made the potential benefits of combination therapy marginal [50] . in summary, serum therapy was abandoned because of toxicity, difficulty in administration, narrow specificity, lot-to-lot variation, and expense. chemotherapy was less toxic and easier to use, lots were more consistent, and it was more effective in eradicating infection. in addition, there was no need for strain typing with chemotherapy. however, it is ironic that the broad antimicrobial activity of these drugs used for chemotherapy might have contributed to widespread resistance, and today susceptibility testing is essential to the selection of appropriate antimicrobial drugs. although antibodies are no longer used directly as antibacterial drugs, they continue to be used in infectious diseases (for recent reviews see [65] [66] [67] ). the majority of antibody preparations are now human. antibody administration reduces infection in individuals with immunodeficiencies [65] [66] [67] [68] and is effective for postexposure prophylaxis against measles, hepatitis a and b, rabies, and varicella viruses. specific antibody is used for the treatment of botulism [22] , diphtheria [40] , tetanus [22] , snake bites [69] , and spider stings [70] . passive antibody administration has been used for prophylaxis and/or therapy of several viral infections, including cytomegalovirus (cmv) [71, 72] , rotavirus [73] , lassa virus [74] , varicella [75] , enterovirus [76, 77] , and parvovirus b19 infections [78] . in recent years, there has been renewed interest in the use of antibody preparations to prevent infection in high-risk groups. intravenous administration of polyclonal antibody has been reported to reduce the number of infections in patients with aids [79] [80] [81] , patients in surgical intensive care units [82] , organ transplant recipients [83] , and neonates [84, 85] (for critical reviews and commentary on this practice see [86] [87] [88] [89] ). antibody therapy is used for a variety of illnesses that may or may not be infectious, including autoimmune thrombocytopenia, kawasaki disease, and autoimmune neuromuscular diseases [65, 66] . passive antibody administration is used for prevention of rh-hemolytic disease [90] . equine lymphocyte antiserum [91] and murine mabs to lymphocytes are used to prevent organ rejection [92] . digitalis-binding antibodies are useful for the treatment of digoxin toxicity [93] . thus, antibody therapy is still widely used in medicine, but its role in the treatment of infections is limited largely to viral and toxin neutralization and replacement therapy in patients with immunoglobulin deficiencies. in reconsidering the antibody option it is clear that heterologous antibody (i.e., serum therapy) is a poor choice because of toxicity. human immune sera have fewer side effects, but there are concerns about availability, potency, and consistency. a major disadvantage of all immune sera is that specific antibodies make up a small minority of the antibody present. there is significant variation in pathogen-specific opsonic activity of commercially available immunoglobulin preparations [94] . there is also concern about the potential of human sera to transmit infectious agents [95] . the discovery of hybridoma technology in 1975 [96] and recent advances in mab technology, including the generation of humanized antibodies [97] , make mab-based therapies a more attractive therapeutic option. in contrast to polyclonal sera, mabs are homogeneous and reproducible reagents that can be generated in large amounts. generation of mabs from mice and rats is still easier and more efficient than production of human mabs. however, several technologies are available for the humanization of murine mabs and generation of human mabs [97] . mouse-human chimeric antibodies can be constructed by linking the genes expressing the mouse variable region to human constant region genes [97] . the result is a molecule that is mostly human and has a longer half-life than the murine precursor [98] . as an alternative, the antigen binding regions of murine mabs can be grafted into human antibody frameworks by molecular techniques [97] , resulting in molecules that are almost completely human in origin. other strategies to generate human antibodies are the transformation of human b cells, the generation ofrecombinant antibody libraries from human b cells, and the use of transgenic mice that have the human immunoglobulin locus. recent setbacks for mab-based therapies: historical perspective clinical trials of antiendotoxin mabs for gram-negative sepsis have produced inconclusive results [99, 100] . when the difficulties encountered in developing antiendotoxin mab strategies are considered in the context of the development of serum therapy, they do not seem unusual or unexpected. for example, passive protection with pneumococcal antisera was demonstrated in 1891 by the klemperers [101] , but 30-40 years elapsed before serum therapy for pneumococcal pneumonia was widely accepted. for pneumococcal antiserum therapy to be consistently successful, several developments were necessary. these included the appreciation of antigenic variation in pneumococci [102] , the need for type-specific sera [103] , the development of rapid in vitro assays to establish antigenic type (i.e., capsular swelling and agglutination reactions) [104] , the development of methods to standardize potency (mouse protection test) [46] , and improved purification techniques that would reduce the toxicity of serum. for meningococcal antisera the situation was reversed; flexner's serum significantly reduced mortality in early epidemics of meningitis [38] but was subsequently less effective, possibly as a result of antigenic changes in neisseria meningitidis [7] . research into better meningococcal antisera was then hampered by the lack of useful animal models, loss of strain virulence in vitro, and poor understanding of the antigenic diversity of n meningitidis [105, 106] . a lesson from the preantibiotic era is that a considerable amount of basic research on the immunology and pathogenesis of each infection is usually necessary before antibody therapy can be developed that will be successful in clinical practice. spectrum ofactivity. antibodies can modify bacterial, fungal, parasitic, and viral infections and are a class of biological agents with broad antimicrobial activity against diverse pathogens. antibody-based therapies are usually pathogen-specific and have the theoretical advantage that they should not affect the normal flora of the patient or select for resistance in nontargeted microbes. nevertheless, narrow specificity is a disadvantage because mixed infections may not be treated by a single antibody preparation. pneumonia due to s. pneumoniae with more than one serotype was recognized as a reason for the failure of type-specific serum therapy [107] . one solution is to use cocktails of mabs active against common antigenic types. cocktails may be designed to include mabs to multiple serotypes and mabs with multiple isotypes to enhance antibody effector function. pathogen-specific drugs (such as mabs) are less attractive to the pharmaceutical industry because the narrow specificity reduces the potential market for the drug. however, the increasing incidence of resistant organisms resulting from the use of broad-spectrum antibiotic regimens could make pathogen-specific drugs attractive to drug companies. precedents for the development of pathogen-specific drugs already exist in the area of antiviral drug research. mechanisms ofaction. antibodies mediate protection by a variety of mechanisms. direct antibody mechanisms of action include inhibition of attachment, agglutination (and immobili-cm 1995;21 (july) zation), viral neutralization, toxin neutralization, antibodydirected cellular cytotoxicity, complement activation, and opsonization [108] . antibody therapy has the potential to enhance immune function in immunosuppressed hosts. however, since the mechanism of action of many antibodies involves promoting microbial clearance through nonspecific cellular immunity, antibody-based therapies may be less effective in individuals with defective macrophage, neutrophil, and natural killer cell function. in this regard, it is encouraging that antibodies are effective against pseudomonas aeruginosa in neutropenic mice [109] and that they can reduce the number of infections in pediatric patients with aids [79] . antibodies are usually considered to be "protective" effector molecules of the immune system. however, not all antibody responses to pathogens are protective and some may be deleterious to the host. for example, some viral-specific antibodies are capable of enhancing infection [110] . thus, antibody molecules being considered for clinical development will require extensive testing in vitro and in vivo. studies of the mechanisms of antibody action are important for understanding the mode of protection and for designing clinical trials. pharmacokinetics. the pharmacokinetics of human igg suggests several useful characteristics for its role as an antiinfective agent. these include a long half-life (~20 days [111]), good tissue penetration [112] , and, depending on the isotype, the ability to either cross the placenta to provide antibody protection to fetuses and newborns [113] or to be excluded from the placenta if fetal toxicity is a concern. heterologous mabs (i.e., murine mabs) have shorter half-lives in humans, but chimeric and humanized mabs have longer half-lives than their murine precursors. mabs with a longer half-life can, in principle, be engineered by altering the regions of the constant domain that regulate clearance. a disadvantage of antibody-based therapies is the need for systemic administration. oral administration is unlikely to be effective, with the possible exception of therapy for enteric pathogens [73, 114] . the blood-brain barrier is a potential obstacle for antibody therapy for infections of the brain. however, in infections of the brain in which there is inflammation, there is increased antibody penetration, and intravenous administration of antibodies appears to have been successfully used for therapy of meningococcal meningitis [115] . antibodybased therapies can also be administered directly into the subarachnoid space, as has been done for the treatment ofmeningococcal and h. influenzae meningitis [8, 38] . enhanced penetration of the brain can be achieved by modifying the charge of the molecule [116] or by linking it to carrier proteins that cross the blood-brain barrier [117] . antibody therapy for brain infections may be feasible if the blood-brain barrier is leaky, if antibody is administered directly into the subarachnoid space, or if antibodies with greater brain penetration are used. additional research into the pharmacokinetics of antibodies in infection is likely to be required for optimal use of antibody-based therapies. studies of antibody binding to tumors have revealed complex pharmacokinetics [112] . there may be problems with antibody penetration into abscesses as have been found with tumors. toxicity. immunoglobulins are generally safe drugs [67] . nevertheless, serious side effects have been reported with highdose (0.5-2 g/kg) antibody therapy, including rare cases of renal failure [118] , aseptic meningitis [119] , and thromboembolic events [120, 121] . high-dose immunoglobulin therapy is unlikely to be required in antiinfective mab therapy. in the past, serum therapy was effective against various pathogens despite the fact that immune sera contained only small amounts of specific antibody. mabs have significantly higher specific activity than polyclonal preparations. for example, 0.7 mg of two anti-tetanus toxin human mabs provides the same activity as 100-170 mg of immune globulin [122] . thus, the toxicities described for high-dose immunoglobulin therapy may not be relevant in antiinfective therapies with mabs. until fully human mabs are available, rodent, mousehuman chimeric, or humanized mabs are therapeutic options. for over a decade, murine mabs directed against t cells have been used to prevent organ graft rejection in humans [92] . a murine igm to endotoxin (e5) was tested in 247 patients with sepsis and appeared to be a safe treatment [123] . although administration of murine mabs is generally well tolerated in humans, allergic reactions occur in 1%-2% of patients [123] and most patients develop antibody responses to the murine mabs that may interfere with their therapeutic function [43, 124] . experience with human-mouse chimeric antibodies and humanized mabs is accumulating rapidly as clinical trials with several compounds progress. a chimeric anti-cd4 mab for the treatment of rheumatoid arthritis has been well tolerated [125] , but approximately half the patients had infusion-related side effects (headaches, nausea, fever, and chills), which were diminished by slowing the infusion rate [125] . mouse-human chimeric and humanized mabs are less immunogenic than their murine precursors [98, 126] , but antiidiotypic responses occur after repeated treatments [127] . overall, the experience with chimeric and humanized mabs suggests that they are relatively safe compounds [98, [125] [126] [127] [128] . antigenic variation and antibody resistance. the efficacy of antibody-based therapies may be diminished by antigenic changes in the pathogen. this could be minimized by antigenic surveillance systems, as is presently done for influenza virus. antibody-resistant mutants can be generated in the laboratory [129] , and it is likely that the same can occur in patients. mechanisms of antibody resistance include mutations that change the antigenic site and protease production. antibody use may select for antigenically distinct variants. evidence for the horizontal transfer of iga protease genes exists in neisseria gonorrhoeae and it is conceivable that widespread antibody use would result in selection of protease-producing strains for many pathogens [130] . thus, large-scale use of antibody-based therapies may result in rapid emergence of antibody resistance in a manner analogous to that of antibiotic resistance. however, one advantage of antibody-based therapies is the versatility of antibody compounds. for example, emergence of an antibodyresistant strain could be countered by a new antibody directed toward the mutated epitope or another antigenic target. emergence of protease-producing strains could be countered by designing antibodies without protease cleavage sites (although these may then be recognized as foreign and be immunogenic) or by addition of antiprotease mabs. selection of antibodyresistant organisms could be minimized by using cocktails of mabs directed at multiple antigenic targets or by using a combination of antibodies and antimicrobials. cost. cost effectiveness is a significant concern that is likely to be a major obstacle for the development of passive antibody therapies. antibody prophylaxis for cmv infections can cost $4,000 to $9,000 per patient [71] . the cost effectiveness of antibody therapy for prevention of infection in leukemic patients has been questioned [88, 131] . however, in selected populations such as premature infants, antibody therapy may be cost effective [85] . furthermore, the cost of a drug when first introduced may not reflect its long-term costs given the potential for improvements in technology and production. mabs are presently made in tissue culture, so the cost of production is high. mabs can be made in bacteria or yeast, and less costly means of production might be developed [97] . in the past, serum therapy was used despite its high cost because it was believed to be effective. new antibody-based therapies are likely to be used if they prove to be effective. for pathogens such as s. pneumoniae, n. meningitidis, and h infiuenzae, there is a large body of experimental and clinical evidence to support the development of passive antibody therapy. however, for many pathogens, antibody immunity has not been proven to be important. for example, cryptococcus neoformans is a fungus for which the importance of antibody immunity is uncertain. however, administration of preformed antibodies can modify the course of cryptococcal infection in various animal models [132] [133] [134] [135] [136] , and the combination of antibody and amphotericin b is a more effective treatment than the use of either agent alone [137] [138] [139] . studies with mabs have shown that there are protective, nonprotective, and deleterious antibodies to the c. neoformans capsule polysaccharide [136] . heavy chain isotype [136, 140] and epitope specificity [141] are important determinants of protective efficacy. the existence of protective and nonprotective antibodies against c. neoformans, a fungus for which antibody immunity mayor may not be important, provides a paradigm that may be applicable to other pathogens. we propose the hypothesis that for every pathogen there exists an antibody that will modify the course of infection to the benefit of the host; such antibodies are candidates for development as antimicrobial drugs. our proposal includes the use of antibodies for intracellular pathogens; for example, anti-bodies may exist that could prevent cell entry of intracellular bacteria and/or shift the intracellular location of such bacteria by promoting internalization through fe receptors. an mab that inhibits intracellular toxoplasma gondii infection has recently been described [142] . in addition, antibodies have been described that can neutralize viruses intracellularly [143] or penetrate the nuclear membrane [144] . our proposal is not limited to those pathogens for which antibody immunity has been demonstrated to be protective. conclusions on the importance of antibody immunity are usually based on observations made with polyclonal sera (i.e., passive protection or correlation of immunity with the presence of antibody). since polyclonal sera might contain protective antibodies, nonprotective antibodies, and disease-enhancing antibodies, the existence of protective antibodies cannot be ruled out on the basis of absence of protection in experiments involving passive transfer of polyclonal sera or immunizations. conversely, experiments that demonstrate that polyclonal sera can mediate protection indicate the existence of protective antibodies. for pathogens for which polyclonal antibody has shown no protection, the question of whether useful antibodies exist must be reevaluated with mabs since the presence of nonprotective or deleterious antibodies in polyclonal sera could create confounding variables. a corollary of our proposal is that antibody-based therapies may be developed against pathogens for which antibody immunity is not considered to be important. infections that are difficult to treat and that can be modified by antibody immunity provide a logical starting point for the development of antibody-based therapies. antibodies have historically been more effective in prophylaxis than in therapy. antibody-based therapies have traditionally been most effective in infections where viral and toxin neutralization modifies the course of the disease. however, most of the historical experience was gained with polyclonal preparations of uncertain composition and mayor may not be applicable to mab preparations with high activity. opportunistic infections. infections with low-virulence organisms in immunosuppressed individuals are often difficult to treat and are sometimes incurable. since the problem is deficient immunity, antibody therapy is an attractive option because antibodies can enhance immune function. table 2 lists examples of opportunistic pathogens for which antibody can modify the course of infection. antibiotic resistance. drug-resistant organisms are targets for development of antibody-based therapies. the spread of penicillin-resistant s.pneumoniae, methicillin-resistant staphylococcus aureus, and multiply-drug-resistant e. faecium has limited the useful antibiotic arsenal against these pathogens [4, 6, 153] . for pneumococcus, antibody therapy has been shown to be useful [7] . antibody may be useful against staphylococcal infection [154, 155] . for e.faecium, the role of antibody immu-ern 1995;21 (july) table 2 . opportunistic pathogens for which experimental data suggest a potential role for antibody-based therapies. nity is uncertain, but some patients make opsonic antibodies, which might be protective [156] . significant work has already been done in the development of pseudomonal antibodies for resistant gram-negative organisms (table 2). the addition of antibody-based therapies to chemotherapy may slow the development of resistance and improve outcome. toxin neutralization. the ability to neutralize toxins is a unique characteristic ofantibody-based therapies. antitoxin antibodies are useful for the treatment of diphtheria, tetanus, botulism, snake bites, and black widow bites (see above). recent attempts to develop antibody therapy for gram-negative sepsis have focused on mabs to neutralize endotoxin [123, 157, 158] and cytokines [159] . pseudomonas aeruginosa sepsis, combination therapy with mabs to endotoxin and tumor necrosis factor was superior to therapy with a single mab [160] . examples ofpotential targets for antibody-based therapy include the toxins and proteases associated with toxic shock syndromes [161, 162] . combination therapy with antibodies to exotoxins could improve outcome, but cocktails of mabs may be necessary because of toxin heterogeneity [162] . intravenous immunoglobulin administration has been associated with dramatic clinical improvement in a patient with streptococcus pyogenes toxic shock syndrome [163] . antivirals. virus neutralization is an established property of antibodies. in the preantibiotic era, serum was used for prophylaxis ofmeasles, chickenpox, mumps, and poliomyelitis. recently, the potential of antibody therapy against many viruses, including hepatitis b [164] , hiv [165] , respiratory syncytial virus [166] , cmv [150] , and parvovirus [167] , has received considerable attention. newly identified viral illnesses are targets for antibody drug research. combination ofantibody therapy and chemotherapy. antibody-based therapies are unlikely to be used alone unless they are the only available therapy or are being used for prophylaxis of infection. combinations of antibody therapy and chemotherapy offer theoretical advantages. antibodies promote microbial killing directly by a complement-mediated lytic process or indirectly by enhancing nonspecific immune mechanisms. table 3 lists examples of bacterial, fungal, and viral pathogens for which combination therapy has shown promise. combination therapy could reduce the amount of either agent required to table 3 . pathogens for which the combination of chemotherapy and antibody based therapy has shown some promise. pathogen chemotherapy antibody [57, 168] neisseria meningitidis sulfanilamide horse immune sera [55] streptococcus pneumoniae rabbit immune sera [8, 9] haemophilus infiuenzae sulfonamide rabbit, horse immune sera [169] staphylococcus aureus sparftoxacin human igg1 mab [172] p. aeruginosa human igm mab [173] p. aeruginosa murine e5 lps antibodies [170] p. aeruginosa human gamma globulin [172] escherichia coli murine e5 lps antibodies [174] shistosoma mansoni praziquantel rabbit immune sera [175] candida albicans amphotericin b human gamma globulin [137] cryptococcus neoformans amphotericin b rabbit immune sera [138, 139] c. neoformans murine igg1 mab [176] c. neoformans murine igg1 mab [177] lassa virus ribavirin monkey immune sera [72, 178] cytomegalovirus ganciclovir murine immune sera [179] herpes simplex virus adenine arabinoside rabbit and human sera [180] herpes simplex virus acycloguanosine human immune globulin note. this is not a complete list. achieve a therapeutic effect. for example, some antibiotics are quite toxic, and the ability to reduce doses could lessen side effects. in a similar vein, the addition of chemotherapy to antibody therapy could reduce the amount of antibody necessary to achieve a therapeutic effect, which would lessen the cost of therapy. the availability of broad-spectrum antibiotics has diminished the need for making exact microbiological diagnoses. for example, gram-negative sepsis or presumed fungal infections can be treated with empirical therapy without identifying the pathogen. this situation is in contrast to the practice of infectious diseases in the 1930s when identification of the pathogen (and its serotype) was necessary for choosing the correct antiserum. for antigenically diverse pathogens such as s. pneumoniae, rapid protocols were developed for typing strains. the present practice of infectious diseases is not unlike a gambling strategy where the probability of infection by a given microbe is matched to the likelihood of activity by the available antibiotics. this has resulted in great emphasis on broad-spectrum antibiotic "coverage" and less emphasis on making an exact microbiological diagnosis. although the relative merits of this practice are beyond the scope of this article, widespread antimicrobial resistance is decreasing the effectiveness of existing antibiotics, and increased caution is warranted when designing broad-spectrum combinations. a 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infection protection of mice against meningococcus infection by polyvalent antimeningococcic serum combined protective action of human gamma globulin and antibiotics when administered simultaneously in experimental staphylococcal infections synergism between human gamma globulin and chioroamphenicol in the treatment of experimental bacterial infections combined serum and sulphanilamide in the treatment of streptococcal infections in mice monoclonal antibodies for treatment of gram-negative infections efficacy of anti-endotoxin monoclonal antibody e5 alone or in combination with ciprofloxacin in neutropenic rats with pseudomonas sepsis praziquantelinduced exposure of schistosoma mansoni alkaline phosphatase: drugantibody synergy which acts preferentially against female worms. parasite immuno11994 effects of immunoglobulin g and low-dose amphotericin b on candida albicans infections in burned mice combination of 5-flucytosine and capsule-binding monoclonal antibody in therapy of cryptococcus neoformans infection enhanced treatment of lassa fever by immune plasma combined with ribavirin in cynomolgus monkeys -3-dihydroxy-2-propoxymethyl) guanine prevents death but not immunity in murine cytomegalovirus-infected normal and immunosuppressed balb/c mice combined effects of acycloguanosine and humoral antibodies in experimental encephalitis due to herpesvirus hominis synergistic antiviral effects of adenine arabinoside and humoral antibodies in experimental encephalitis due to herpesvirus hominis unequalled but unobtainable key: cord-278084-km04sz2s authors: martino, rodrigo; rámila, elena; rabella, núria; muñoz, josé manuel; peyret, mercé; portos, josé manuel; laborda, rosario; sierra, jorge title: respiratory virus infections in adults with hematologic malignancies: a prospective study date: 2003-01-01 journal: clin infect dis doi: 10.1086/344899 sha: doc_id: 278084 cord_uid: km04sz2s during a 2-year period, 157 consecutive episodes of respiratory virus infections that occurred in 130 patients with upper or lower respiratory tract infection were analyzed for respiratory viruses. a respiratory virus was identified in 75 episodes (48%), and several viruses were found in 13 episodes: there were a total of 56 influenza a virus infections, 14 respiratory syncytial virus infections, 8 adenovirus infections, 8 infections with parainfluenza virus types 1 or 3, and 7 enterovirus infections. on multivariate analysis, the only variable that predicted progression to pneumonia in patients with an upper respiratory tract infection was the presence of respiratory syncytial virus, whereas lymphocytopenia had a nonsignificant trend. also, among the 38 patients who had pneumonia at any time during the episode, both respiratory syncytial virus and lymphocytopenia were commonly found. for both epidemiological and therapeutic considerations, frequent screening for respiratory viruses should be incorporated into the routine diagnostic study of patients with hematologic malignancies. respiratory viruses have been recognized as potential causes of severe pneumonia in patients with hematologic malignancies [1] [2] [3] [4] [5] [6] [7] . in this patient population, these viruses cause upper respiratory tract infections (urtis), as in the general population; however, in patients with hematologic malignancies, these infections appear to have a higher tendency to progress to severe lower respiratory tract infections (lrtis). respiratory syncytial virus (rsv), influenza a and b viruses, and parainfluenza viruses have been well described as causes of severe respiratory morbidity and mortality, especially among recipients of hematopoietic stem cell transplants (hscts) [1] [2] [3] [4] [5] [6] [7] . adenoviruses cause both isolated res-piratory infections and disseminated visceral syndromes [8] . despite an increasing number of reports, there have been few prospective studies of respiratory virus infections in adults with hematologic malignancies. we performed a prospective 2-year study to assess the role of respiratory virus infections in the pathogenesis of urti and lrti and risk factors for severe lrti in an adult clinical hematology department. this prospective study was performed at the division of clinical hematology of the hospital de la santa creu i sant pau (barcelona, spain) from 1 october 1999 through 31 may 2001. all adult patients with a hematologic malignancy (including hsct recipients) who had signs and symptoms of a urti or lrti in the inpatient and outpatient settings underwent a detailed clinical evaluation, and samples from the upper and/or lower respiratory tracts were screened for the presence of respiratory viruses. patients with symptoms of urti underwent nasopharyngeal aspiration, whereas patients with lrti (i.e., pneumonia) underwent bronchoalveolar lavage (bal), when clinically possible. patients with pneumonia but no signs of urti did not undergo nasopharyngeal aspiration. patients (both outpatients and inpatients) whose nasopharyngeal aspirate (npas) specimens tested positive for respiratory viruses were asked to return to the clinic (or undergo an inpatient sampling) at least weekly for further clinical assessment and microbiological analysis until all symptoms resolved and the virus was not detected in a npa. microbiological methods. all samples were processed for respiratory viruses by antigen detection with use of direct immunofluorescence and culture. for direct immunofluorescence, the specimens were spotted onto glass slides and processed by use of standard techniques [9] . the presence of viral antigen in respiratory cells was indicated by the appearance of characteristic intracellular apple-green fluorescence in у1 cell. we used labeled antibodies to influenza a and b viruses; parainfluenza viruses 1, 2, and 3; rsv; and adenoviruses. if the samples did not contain respiratory epithelial cells, the finding was considered to be inconclusive. for viral culture, specimens were inoculated into each of 4 cell lines: human fibroblasts (mrc5), human epithelial cells (hep-2 and a-549), and madin-darby canine kidney cells. cultures were incubated for 2 weeks (4 weeks for detection of cytomegalovirus) on a roller drum at 35њc. viruses were identified on the basis of cytopathic effect in cell cultures and confirmed by staining with fluoresceinconjugated monoclonal antibodies [10] . bal samples were also processed for routine aerobic, anaerobic, mycobacterial, and fungal culture and for parasite examination. definitions. a first episode of infection was defined as the period during which the patient had symptoms of urti and/ or lrti, regardless of whether a respiratory virus was isolated. a further episode(s) of infection required the presence of a symptom-free period and negative results of testing of the npa for any respiratory viruses isolated during the previous episode. a polymicrobial infection was defined as the isolation of 11 respiratory virus during the same episode or another clinically relevant pathogen from a bal fluid specimen or a lung sample. a diagnosis of nosocomial respiratory virus infection was considered if a patient had been in the hospital for у3 days and if symptoms of the infection developed during hospitalization. a urti was defined by the new onset of nasal, pharyngeal, or laryngeal irritation, and an lrti (or pneumonia) was defined by the development of a new pulmonary infiltrate in patients with signs and symptoms of lrti (cough, rales, wheezing). death due to pneumonia was defined as death due to respiratory failure during the episode of respiratory virus infection. progression of a urti to an lrti was defined as onset of pneumonia in patients with a prior or concurrent urti, whereas patients who had pneumonia without a urti were considered to have an isolated lrti. the recommended therapy for lrti due to rsv was inhaled ribavirin (6 g per day for 5 days); for urti and lrti due to influenza a virus, the recommended therapy was orally administered amantadine (100 mg t.i.d. for 7 days). however, physicians were free to decide whether to treat patients who were ambulatory and had an isolated urti, according to their own clinical judgement. for inpatients, strict isolation was done in all cases to avoid spread of the virus. statistical analysis. univariate analyses of the risk factors for development of an lrti were done with use of the x 2 test or fisher's exact test, for discontinuous variables, and student's t test or the mann-whitney u test, for continuous variables. multivariate analysis of variables predictive of development of an lrti was done by logistic regression. age and neutrophil and lymphocyte counts were included as continuous variables and as binary variables (more than or less than a fixed value). the other factors that were analyzed were sex, underlying disease, disease status (early vs. advanced), type of therapy (receipt of an allogeneic hsct vs. other therapies), nosocomial infection (yes vs. no), use of corticosteroids (defined as у1 mg/kg of prednisone per day or equivalent for 11 week; yes vs. no), and virus isolated (influenza virus vs. other/none and rsv vs. other/none). tests of significance were 2-sided, and was p ! .05 considered to be statistically significant. during the study period, 157 episodes of infection were studied in 130 patients (17 patients had 11 independent episode). patient characteristics are shown in table 1. the study period was divided into 3 periods: first winter, summer, and second winter. as expected, most episodes were identified during winter months. ninety-seven patients (62%) had received autologous or allogeneic hscts, whereas most other patients were treated with intensive chemotherapy for leukemia, lymphoma, or myeloma. at the onset of infection, 23 patients (15%) had neutropenia, and 70 (45%) had lymphocyte counts of !10 9 lymphocytes/l. eighty-nine patients (57%) had fever at the onset of the infection. most patients (131 [83%]) had only symptoms of urti at inclusion in the study, 14 (9%) had both urti and lrti, and 12 (8%) had only pneumonia without clear symptoms of a urti. thus, 145 patients (92%) had symptoms of urti at inclusion in the study. initial findings of microbiological studies. the initial samples analyzed were npas in 146 cases, a bal sample in 9 cases, and both npa and bal fluid samples in 2 cases. table 2 details the results of the initial sample analysis. overall, a respiratory (15) 28 (18) hodgkin disease 5 (7) 1 (7) 7 (10) 13 (8) other 3 (4) 1 (7) 1 (1) 5 (3) previous treatment initial chemotherapy 21 (28) tract or a follow-up npa sample for several patients (table 3, figure 2 ). the same virus was isolated again from 16 (30%) of 54 for a median of 2 weeks. patients who were treated with amantadine for influenza a virus infection and who provided follow-up samples ( ) shed the virus longer (median, 3 n p 24 weeks; range, 1-9 weeks) than did those who were not treated and who had follow-up samples ( ; median, 1 week; n p 18 range, 1-4 weeks; ). however, it should be emphasized p p .03 that this was a prospective observational study and not a controlled treatment study, and, thus, it is probable that physicians were more prone to subjectively treat patients who were more ill or at higher risk. a new respiratory virus was isolated during the same clinical episode in 13 cases; these include 7 rsvs and 4 adenoviruses (table 3, figure 2) . thus, the total number of infections with the different respiratory viruses during the 157 episodes studied (including initial polymicrobial infections and later superinfections) was as follows: influenza a virus, 56 infections; rsv, 14 infections; adenoviruses, 8 infections; parainfluenza virus type 1, 4 infections; parainfluenza virus type 3, 4 infections; and enteroviruses, 7 infections. other pathogens. clinically relevant pathogens were isolated from bal samples obtained from 5 patients with lrti: cytomegalovirus isolates were recovered from 2 patients, and isolates of pseudomonas aeruginosa, pneumocystis carinii, and aspergillus fumigatus were recovered from 1 patient each. two of these patients died of pneumonia. outcome of lrti. of the 157 patients, 26 (17%) had lrti at the time of inclusion in the study (12 had only lrti and 14 had both urti and lrti). twelve additional patients who initially had only a urti later developed lrti (9% of the 131 cases of urti). thus, overall, 38 patients (24%) developed lrti during the study period. as specified above, 5 of these patients had a copathogen identified, 21 had only a respiratory virus identified as the possible etiologic agent, and there was no etiologic diagnosis for 12. seven (27%) of 26 patients with lrti in whom a respiratory virus was found died of pneumonia, as opposed to 2 (17%) of 12 for whom no respiratory virus was identified ( ). p p .1 risk factors for progression to lrti. twenty-six (18%) of the 145 patients who presented with symptoms of a urti either had pneumonia or had progression to pneumonia during the follow-up period. age, sex, underlying disease, receipt of an allogeneic hsct, neutropenia, use of corticosteroids, and infection with influenza virus were not predictive of progression to lrti. inpatients had a higher risk of having progression to pneumonia on univariate analysis ( . subanalyses of allogeneic hsct recipients and of patients with any respiratory viruses isolated during the episode revealed no significant variables, but the same trend was ob-served for rsv infection (data not shown). among the 97 episodes in hsct recipients, we found no correlation between the time after transplantation (analyzed a continuous variable) and the risk of progression to pneumonia. univariate and multivariate analyses were performed for all 157 episodes to analyze the characteristics of patients who had pneumonia any time during the study ( ). two variables n p 38 were associated with pneumonia on univariate and multivariate analyses: lymphocytopenia (!10 9 lymphocytes/l; on p p .05 multivariate analysis) and rsv infection ( ). lympho-p p .02 cytopenia was present in 24 (63%) of 38 episodes in patients with pneumonia and in 47 (39%) of 119 episodes in patients without pneumonia, whereas rsv infection was present in 8 episodes (21%) in patients with pneumonia and 7 (6%) in patients without pneumonia. we identified a respiratory virus in 48% of adult patients who had a hematologic malignancy present and signs and symptoms 0.5 ϫ 10 c lymphocyte count, lymphocytes/l. 9 1 ϫ 10 d includes patients with initial urti who later developed lrti and those with both urti and lrti at baseline. e presented with initial lrti. of urti. influenza a virus was by far the most common virus found (56 episodes), followed by rsv and parainfluenza virus. these respiratory viruses are the most commonly found viruses in studies that involve hsct recipients [1] [2] [3] [4] [5] [6] [7] , although their relative proportions vary between studies, probably because of the epidemic situation in the population during the study period. a large epidemic of influenza a virus infection occurred during our study period, and the isolates recovered from patients closely reflected the respiratory viruses isolated in the community, as shown in figure 1. the 7 enteroviruses found in our study confirm that these viruses also cause lrti and urti in these immunocompromised hosts [7, 11, 12] . however, we found no cases of infection with rhinoviruses or coronaviruses. the viral culture technique used in the study can detect infections with rhinoviruses but not coronavirus infections [6, 7, 10] . because rhinoviruses are commonly implicated in the common cold, we have no explanation for not having isolated any such virus in our study. on the other hand, a significant proportion of our patients (52%) with clear signs of urti or lrti had no respiratory viruses identified. many of these cases may be of noninfectious origin or nonviral origin, or they may have been caused by other undetected viral pathogens, whereas other cases may represent technical flaws in sample processing or lack of sensitivity to detect involved respiratory viruses. in a recent study, pcrbased techniques were found to increase the rate of respiratory virus identification in a similar patient population to 35% (compared with 19% for culture) [12] . thus, had we used such an approach, we might have found even more cases of respiratory virus infections. with respect to the clinical applicability of the microbiological methods used in our study for clinical decision making, findings for the direct immunofluorescence technique were available within 6-12 h after the sample arrived in the laboratory, but viral cultures usually took 7-10 days for results. an interesting finding in our study was the isolation of 11 respiratory virus in 19 episodes (12%). two or more respiratory viruses were found in the initial sample for 6 patients, whereas, in 13 cases, a new respiratory virus was later isolated during the same clinical episode, either in addition to ( ) or in n p 4 the absence of ( ) the initial virus. these observations n p 9 show that a patient can be superinfected with a new respiratory virus, even if they have not recovered from a prior infection. among the 145 patients with urti at the time of inclusion in the study, 26 (18%) developed lrti at some time during follow-up of the episode. an lrti occurred in 16 (21%) of 75 episodes in which a respiratory virus was identified and in 10 (14%) of 70 in which no respiratory virus was found, but the presence of any respiratory virus was not found to carry a higher risk of progression to pneumonia on multivariate analysis. of the specific respiratory viruses found, influenza a virus did not carry a higher risk of progression of infection to lrti (23%) on univariate and multivariate analyses. rsv infection, however, appeared to have a higher risk of lrti (46% among rsv-positive patients and 15% among the rest; ). this p p .01 finding is not surprising, because both rsv and parainfluenza virus have been found to lead to lrti in 30%-60% of immunosuppressed hsct recipients with urti [3] [4] [5] [13] [14] [15] . parainfluenza viruses 1 and 3 have also been described as carrying a high risk of producing severe lrti [2] , but we had too few cases of infections with these viruses for meaningful analysis. of note, all 4 patients with parainfluenza virus 3 infection developed pneumonia, whereas none of those infected with parainfluenza virus 1 had an lrti. we found a trend of lymphocytopenia being a risk factor for progression to lrti, as was previously found in a european multicenter study [5] . in all 157 episodes, this variable was also more frequently found among patients with pneumonia than among those with an isolated urti. this finding, however, requires confirmation in other series of patients. allogeneic hsct recipients are usually found to be at the highest risk for developing severe respiratory virus infections [1] [2] [3] [4] [5] [6] [7] . however, in our study, which included 97 hsct recipients (52 of whom received allogeneic transplants), we did not find that receipt of an allogeneic hsct was a risk factor for progression to pneumonia (20% vs. 17% for progression to lrti). in summary, our prospective study of both ambulatory and hospitalized adults who received treatment for a hematologic malignancy and who had signs and symptoms of urti and/ or lrti shows that a respiratory virus can be implicated in at least one-half the cases. the isolation of rsv and the presence of lymphocytopenia appear to increase the risk of developing pneumonia, the most dreaded complication of these infections. therefore, we suggest that screening for these viruses should be incorporated into the routine diagnostic study of patients with hematologic malignancies, both for epidemiological reasons (i.e., strict isolation of hospitalized patients with a respiratory virus infection) and for the use of currently available or emergent treatments in the most debilitated hosts. respiratory virus infections in bone marrow transplant recipients: the european perspective community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience community respiratory virus infections among hospitalized adult bone marrow transplant recipients community respiratory virus infections in immunocompromised patients with cancer respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation conventional respiratory viruses recovered from immunocompromised patients: clinical considerations community respiratory virus infections in patients with hematologic malignancies adenovirus infections in adult recipients of blood and marrow transplants rapid virus diagnosis-application of immunofluorescence cumulative techniques and procedures in clinical microbiology pulmonary enterovirus infections in stem cell transplant recipients polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience respiratory viral infections in immunocompetent and immunocompromised persons current concepts and challenges in the prevention and treatment of viral infections in immunocompromised cancer patients key: cord-276995-b003vcdc authors: wiese, andrew d; everson, jordan; grijalva, carlos g title: social distancing measures: evidence of interruption of seasonal influenza activity and early lessons of the sars-cov-2 pandemic date: 2020-06-20 journal: clin infect dis doi: 10.1093/cid/ciaa834 sha: doc_id: 276995 cord_uid: b003vcdc nan m a n u s c r i p t the unprecedented enactment of social distancing measures in response to the sars-cov-2 pandemic -including city lockdowns, school closures, stay-at-home orders, case isolation, quarantine of contacts, etc.was aimed at reducing the transmission of the virus and its burden on healthcare systems. since influenza and other common respiratory pathogens share a similar mode of transmission with sars-cov-2, their transmission could be indirectly impacted by those measures. and while novel surveillance systems have been implemented to monitor sars-cov-2 activity, pre-existing surveillance systems have the advantage of allowing comparison to trends in prior years to assess the impact of social distancing measures on the activity of influenza and other respiratory pathogens. syndromic surveillance is an important public health tool for the early detection, monitoring and characterization of outbreaks, and supports targeted investigations that may include detailed clinical and laboratory assessments. syndromic surveillance is especially informative when diagnostic tests are not available, applied selectively, or difficult to implement on a broad scale. globally, syndromic surveillance systems of influenza-like illness (ili) are maintained to characterize the onset, circulation, and characteristics of influenza activity. these activities are typically complemented with surveillance of laboratory-confirmed detection of influenza infections. maintained over multiple years, these surveillance systems help identify syndromic and viral activity that deviates from previous years' patterns. in this issue of the journal, hyunju lee and colleagues describe the use of national influenza surveillance data to assess the impact of social distancing measures, implemented in response to the sars-cov-2 pandemic, on seasonal influenza activity in korea. previous reports have demonstrated that the implementation of social distancing measures was associated with reductions in sars-cov-2 infections. [1] [2] [3] in this study, investigators compared the a c c e p t e d m a n u s c r i p t while surveillance data are helpful to identify abnormal activity of certain diseases of public health interest, and to demonstrate the impact of major interventions, such as implementation of social distancing measures, it is important to understand the limitations and strengths of specific surveillance systems. monitoring a new disease that rapidly reaches pandemic proportion is difficult for surveillance systems that rely on patients presenting to healthcare settings and getting tested. due to fear or other reasons, patients may avoid or delay healthcare encounters unless strictly necessary, and changes in testing practices could also affect the data captured by surveillance systems. complementing laboratory-based surveillance with syndromic surveillance data is helpful, but additional research is necessary to clarify and characterize the general healthcare utilization patterns during the pandemic. in korea, the us and most other settings, several social distancing measures were implemented in rapid sequence, making it difficult to isolate the impact of specific measures due to their temporal and regional aggregation. although the described comparisons are ecologic in nature, the population level assessment of the impact of these broad recommendations may be more relevant than individual level evaluations. finally, it is important to understand that there may be substantive surveillance variation over time and across regions ( figure) , and thus national figures may not represent the local or regional situation. looking beyond influenza, these surveillance systems will be important in different regions and countries as they initiate or continue relaxing social distancing requirements. in the us, a two-week downward trend in ili and cli activity was recommended as one indicator for reopening. 10 despite declines following social distancing, cli activity levels remained substantially elevated in certain regions through mid-may 2020, suggesting that high activity levels could rapidly resume once distancing measures are removed. in contrast with the abrupt quarantine alone or in combination with other public health measures to control covid-19: a rapid review. the cochrane database of systematic reviews effect of non-pharmaceutical interventions to contain covid-19 in china evaluating the effectiveness of social distancing interventions to delay or flatten the epidemic curve of coronavirus disease. emerging infectious diseases decreased influenza incidence under covid-19 control measures, singapore. emerging infectious disease journal seasonal influenza activity during the sars-cov-2 outbreak in japan collateral benefit of covid-19 control measures on influenza activity non-pharmaceutical interventions used for covid-19 had a major impact on reducing influenza in china in 2020 impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study us outpatient influenza-like illness surveillance network (ilinet); (3) us national syndromic surveillance program. cdc. cdc influenza tracker and syndromic surveillance web site a c c e p t e d m a n u s c r i p t key: cord-266232-2ctfmjb8 authors: trubiano, jason a; vogrin, sara; kwong, jason c; holmes, natasha e title: alterations in smell or taste – classic covid-19? date: 2020-05-28 journal: clin infect dis doi: 10.1093/cid/ciaa655 sha: doc_id: 266232 cord_uid: 2ctfmjb8 nan a c c e p t e d m a n u s c r i p t dear editor, there are increased reports of loss of smell (anosmia) and taste (ageusia) in patients with confirmed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection causing coronavirus disease 2019 , in particular in the setting of mild disease. the data to date has been presented predominantly from post-diagnosis surveys or retrospective cohort series [1] [2] [3] [4] [5] . the pathogenesis is postulated to be due to invasion of the olfactory neuroepithilium and olfactory bulb, seen previously in other coronaviruses, due to the high expression of angiotension-converting enzyme (the receptor which allows virus cellular entry) present in the respiratory system [1, 6] . luers and colleagues described from a retrospective adult cohort of confirmed sars-cov-2 from germany (n = 72) that 74% of patients reported anosmia and 69% ageusia [7] . spinato et al. prior to this also described from a retrospective cohort study of covid-19 patients interviewed 5-6 days post diagnosis that 64.4% reported alternations in taste or smell [1] . however, both these studies suffer from the absence of a control group and significant limitation of recall and selection bias. testing (etable 1). the distribution of symptom prevalence over time is displayed in efigure 1. in those who underwent sars-cov-2 testing, anosmia or ageusia were more frequently reported in females and in those reporting more symptoms (etable1). of those who reported anosmia or ageusia, 9.3% tested positive for covid-19 (positive predictive value), while the negative predictive value was 98.5%. ansomia and/or ageusia were more common in covid-19 positive than negative (39.3% a c c e p t e d m a n u s c r i p t vs 8.9%, p <0.001), and were more common when examined in isolation: anosmia (25% vs 5%, p <0.001) or ageusia (25% vs 6%, p = 0.002) ( table 1) . after adjusting for confounders, both anosmia and ageusia were independently associated with sars-cov-2 infection, etable 2. whilst supporting the observations made by leuers [7] and spinato [1] , our data also highlights a significantly lower prevalence of symptoms in a comparative outpatient covid-19 population (39.3% [aus] versus 64.4% [us] versus 68% [germany]) when prospective data is used. also, we demonstrate similar olfactory symptoms in the control group (sars-cov-2 negative). it is important for clinicians to realize that ansomia and ageusia are likely to be commonly reported symptoms in other upper respiratory tract infections, when appropriately asked (8.9% of our covid-19 test negative group) [3, 8] . from data available, anosmia and/or ageusia whilst associated with covid-19 should not yet be considered pathognomonic for the disease. larger prospective population studies are required to validate these findings, as we collectively search for key clinical predictors of covid-19 that can aid clinical decision making. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection self-reported olfactory loss associates with outpatient clinical course in covid-19 acute-onset smell and taste disorders in the context of covid-19: a pilot multicenter pcr-based case-control study clinical features of covid-19 self-reported olfactory and taste disorders in sars-cov-2 patients: a cross-sectional study axonal transport enables neuron-to-neuron propagation of human coronavirus oc43 olfactory and gustatory dysfunction in coronavirus disease 19 (covid-19) characteristics of olfactory disorders in relation to major causes of olfactory loss spo2, median (iqr) 98 (97, 99) 98 (97, 99) 98 temperature tympanic, median (iqr) systolic blood pressure, median (iqr) diastolic blood pressure, median (iqr) respiratory rate, median (iqr) 18 pulse rate, median (iqr) a c c e p t e d m a n u s c r i p t key: cord-289598-t8upoq9a authors: yoon, jane c; montgomery, martha p; buff, ann m; boyd, andrew t; jamison, calla; hernandez, alfonso; schmit, kristine; shah, sarita; ajoku, sophia; holland, david p; prieto, juliana; smith, sasha; swancutt, mark a; turner, kim; andrews, tom; flowers, kevin; wells, alyssa; marchman, cathryn; laney, emaline; bixler, danae; cavanaugh, sean; flowers, nicole; gaffga, nicholas; ko, jean y; paulin, heather n; weng, mark k; mosites, emily; morris, sapna bamrah title: covid-19 prevalence among people experiencing homelessness and homelessness service staff during early community transmission in atlanta, georgia, april–may 2020 date: 2020-09-08 journal: clin infect dis doi: 10.1093/cid/ciaa1340 sha: doc_id: 289598 cord_uid: t8upoq9a background: in response to reported covid-19 outbreaks among people experiencing homelessness (peh) in other u.s. cities, we conducted multiple, proactive, facility-wide testing events for peh living sheltered and unsheltered and homelessness service staff in atlanta, georgia. we describe sars-cov-2 prevalence and associated symptoms and review shelter infection prevention and control (ipc) policies methods: peh and staff were tested for sars-cov-2 by reverse transcription polymerase chain reaction (rt-pcr) during april 7–may 6, 2020. a subset of peh and staff was screened for symptoms. shelter assessments were conducted concurrently at a convenience sample of shelters using a standardized questionnaire results: overall, 2,875 individuals at 24 shelters and nine unsheltered outreach events underwent sars-cov-2 testing and 2,860 (99.5%) had conclusive test results. sars-cov-2 prevalence was 2.1% (36/1,684) among peh living sheltered, 0.5% (3/628) among peh living unsheltered, and 1.3% (7/548) among staff. reporting fever, cough, or shortness of breath in the last week during symptom screening was 14% sensitive and 89% specific for identifying covid-19 cases compared with rt-pcr. prevalence by shelter ranged 0%–27.6%. repeat testing 3–4 weeks later at four shelters documented decreased sars-cov-2 prevalence (0%–3.9%). nine of 24 shelters completed shelter assessments and implemented ipc measures as part of the covid-19 response conclusions: peh living in shelters experienced higher sars-cov-2 prevalence compared with peh living unsheltered. facility-wide testing in congregate settings allowed for identification and isolation of covid-19 cases and is an important strategy to interrupt sars-cov-2 transmission in 2019, approximately 570,000 people experienced homelessness on any given night in the united states (u.s.), and 63% used congregate shelters [1] . in atlanta, georgia, an estimated 3, 200 people experienced homelessness on any given night in 2019, and approximately one-quarter were living unsheltered (i.e., living in a place not meant for human habitation) [2] . risk of sars-cov-2 infection, the virus that causes covid-19, may be higher among people experiencing homelessness (peh) because of challenges in preventing respiratory disease transmission in congregate shelter settings. peh might also be at increased risk of severe covid-19 if infected due to a high prevalence of untreated, chronic medical conditions and obstacles to accessing healthcare [3] [4] [5] [6] [7] [8] . fulton county, the largest county in georgia, which includes 90% of the city of atlanta, reported the first covid-19 case on march 2, 2020. a sharp increase in cases was recorded in mid-april 2020. a door-to-door household survey conducted in fulton and neighboring dekalb counties during april 28-may 3, 2020, found an estimated 2.5% seroprevalence of sars-cov-2 antibodies [9] . reports of high sars-cov-2 infection rates and outbreaks within shelters in other metropolitan areas, in parallel with increasing local case-rates, led to concerns for widespread transmission in atlanta shelters [10] [11] [12] . to understand sars-cov-2 prevalence and prevent transmission among peh in atlanta, homeless service agencies partnered with local and federal government agencies to: (1) determine sars-cov-2 prevalence among clients living sheltered and unsheltered and homelessness service staff through viral testing; (2) a c c e p t e d m a n u s c r i p t 6 methods participants included clients living in shelters, clients living unsheltered, and staff in atlanta, georgia, during april 7-may 6, 2020. testing at homeless shelters was offered facility-wide to all clients and staff. testing was offered to clients living unsheltered during homeless outreach service events (e.g., meal services). participation was voluntary but encouraged by service agencies. written consent was obtained from each adult (≥16 years of age) or parent or guardian (for children <16 years) for administration of a brief, standardized screening questionnaire and testing for sars-cov-2 (see supplemental material #1). all screening interviews and specimen collections were conducted on-site at shelters or community events serving peh. at shelters with more than five people with positive sars-cov-2 results upon initial testing, clients and staff were re-screened and re-tested 3-4 weeks later, and testing was also offered to any new clients or staff. all participants (including parents or guardians of children <16 years) self-reported their sex, race, and ethnicity based on fixed-response categories. race and ethnicity were combined into mutually exclusive categories and were considered missing when race was missing and ethnicity was either non-hispanic or missing. participants from a convenience sample of testing events were interviewed using the screening questionnaire to collect information on symptoms, underlying medical conditions, pregnancy status, and tobacco use. persons interviewed were asked if they had any medical conditions in the following categories: diabetes, cardiovascular disease, chronic lung, kidney, and liver disease, immunocompromising conditions (e.g., hiv, chronic steroid use), and neurological conditions (e.g., seizure disorder). nasopharyngeal, oropharyngeal, or nasal mid-turbinate specimens were collected by clinical providers or supervised self-collection and tested by contracted commercial laboratories for sars-cov-2 by reverse transcription polymerase chain reaction (rt-pcr). positive sars-cov-2 test results were provided directly to the person (or parent or guardian for children <16 years) or to the shelter via the county public health department's standard notification procedure. clients living in shelters who had positive sars-cov-2 were isolated immediately in a a c c e p t e d m a n u s c r i p t 7 separate housing unit at the shelter or isolated until transported to an isolation hotel. for clients living unsheltered, results were provided via a clinic hotline number or clinical outreach teams, which located the clients with positive sars-cov-2 and arranged transport to the isolation hotel. staff with positive sars-cov-2 isolated at home. clients were not allowed to return to shared spaces in shelters, and isolated staff did not return to work until they met symptom-or time-based criteria in accordance with the centers for disease control and prevention (cdc) guidelines at the time [13] . shelter assessments were conducted in conjunction with screening and testing at a convenience sample of shelters. these shelters were selected based on availability of testing staff on the day of testing. using a standardized assessment questionnaire (see supplemental materials #2), shelter management was interviewed to collect quantitative and qualitative data on shelter characteristics and services offered. shelter characteristics included number of clients and staff, type (e.g., daytime only, 24 hours per day, transitional housing), services provided, and sleeping space configurations. measures and policies implemented by the shelter to mitigate sars-cov-2 transmission, including standardized client and staff screening, isolation and quarantine protocols, and ipc, were also collected. shelter staff were counseled on best practices to prevent transmission in the shelter. descriptive statistics were used to characterize the population tested and the proportions with current and recent symptoms, underlying medical conditions, and positive sars-cov-2. continuous variables were compared using student's t-test, and categorical variables were compared using the chi-square test. shelter characteristics were described in aggregate but were not analyzed in relation to sars-cov-2 prevalence due to small numbers. cdc determined this project to be non-research as demographic characteristics for tested participants are included in table 1 a c c e p t e d m a n u s c r i p t 9 the same subset of 1,997 people was screened for symptoms; 12 with inconclusive results were subsequently excluded (table 3) in one shelter housing adult men, testing revealed a widespread, undetected outbreak. this shelter represented 1% (29/2,050) of people tested on the initial round but 20% (8/40) of those with positive sars-cov-2. client census had been decreased by approximately half to facilitate physical distancing, but the shelter did not enforce city or state shelter-in-place orders nor restrict client movement. the shelter only had congregate sleeping spaces; sleeping mats and beds were placed at least six feet apart, but head-to-toe alignment was inconsistently used. showers upon entry were encouraged, but face coverings and hand sanitizer were not provided due to insufficient supply. household cleaning solutions and environmental protection agency-registered disinfectants appropriate for cleaning high-touch surfaces or items were also not available [15, 16] . 70% of all peh in atlanta based on the 2019 point-in-time count in atlanta [2] . overall sars-cov-2 prevalence among peh and staff tested in atlanta from april to may 2020 was low compared with reports among peh in other large, urban settings [10] [11] [12] . although sars-cov-2 prevalence among clients living in shelters was only 2.1%, it was four times higher than the 0.5% prevalence among a c c e p t e d m a n u s c r i p t 11 peh living unsheltered at the time. to our knowledge, this is the first report of sars-cov-2 prevalence in a population of peh living unsheltered. although the risk of sars-cov-2 was lower for peh living unsheltered, unsheltered living situations pose an increased risk of morbidity and mortality compared with living sheltered, so linkages to permanent supportive housing should remain a priority [17] . testing at shelters in other large, urban settings in the united states has primarily occurred in response to covid-19 clusters or outbreaks [10] [11] [12] . in early april 2020, baggett et al. investigated an outbreak in a large homeless shelter in boston with a sars-cov-2 detection rate of 36% [10] . during an outbreak across three affiliated shelters in seattle from march-april 2020, 18% of clients and 21% of staff had positive sars-cov-2 [11] . in atlanta, universal screening and testing of peh and staff was a proactive strategy, rather than in response to known covid-19 cases. a recent study of long-term care facilities in fulton county, georgia, found 1.5% of residents had positive sars-cov-2 when testing was proactive, compared to 47.2% after a case was already diagnosed [18] . only one shelter we tested was found to have a widespread, undetected outbreak. on march 23, 2020, 3 weeks after the first covid-19 case was identified in atlanta, the city implemented a 14-day shelterin-place order, which was followed by a georgia state-wide shelter-in-place order during april 3-30, 2020. over half of shelters assessed stopped taking clients during these shelter-in-place orders, and all reported a lower-than-average number of clients, which helped facilitate physical distancing. the low sars-cov-2 prevalence among peh and staff in atlanta compared with other cities may reflect the impact of shelter-in-place orders coupled with low community prevalence at the time of testing and the proactive testing strategy. [19, 20] . although the specificity was higher (81%-89%), screening would produce a high proportion of false positive results in low prevalence settings. most shelters assessed were conducting standardized symptom screening for clients, but only one-third were screening staff. despite the limitations of symptom screening, cdc recommends that homeless service providers regularly assess both clients and staff for symptoms using a standardized protocol [21] . people with covid-19 symptoms should immediately be provided with a face covering, isolated, and tested for sars-cov-2. shelters, in coordination with local public health authorities and community coalitions, should have plans to isolate people with suspected or confirmed sars-cov-2 infection to prevent spread [21] . however, because an estimated 40-45% of people with sars-cov-2 are asymptomatic, shelters should reduce the number of people served or expand to alternative housing sites, increase physical distancing (i.e., >6 feet), and mandate use of face coverings by all staff and clients inside shelters except when in bed or individual rooms [21, 22] . our finding of decreased prevalence in four shelters during repeat testing is consistent with reports from skilled nursing facilities and correctional facilities, supporting the use of universal (facility-wide) testing for early identification and isolation of those with positive sars-cov-2 as a strategy to interrupt transmission in congregate settings [23] [24] [25] . a potentially important factor in the observed decreased prevalence in these shelters was the ability to move all peh identified with sars-cov-2 infections to separate housing units at the shelter or to offsite locations. in homeless shelters and encampments located in areas where sars-cov-2 community transmission is substantial, cdc recommends that initial (baseline) and regular testing be considered, regardless of whether an initial covid-19 case has been identified [26] . in areas where community transmission is minimal to moderate, cdc provides examples of testing strategies that can be used to identify asymptomatic cases among both clients and staff, such as sentinel surveillance, positive symptom screening thresholds, or random testing (e.g., every third person) on a regular basis [26] . if a covid-19 case is identified, cdc recommends repeat testing of all previously negative or untested individuals until testing identifies no new covid-19 cases for at least 14 days [26] . a c c e p t e d m a n u s c r i p t 13 our findings are subject to several limitations. sars-cov-2 transmission dynamics are complex, and these results represent a single point in time early in the covid-19 pandemic. we did not screen or test all peh or shelters in atlanta; therefore, the results might not be representative of all peh or shelters; they are not generalizable to other large metropolitan areas. we cannot compare this prevalence to the general population of fulton county during this time period because representative testing had not been conducted among the general population. additionally, misclassification of sheltered and unsheltered housing status might have resulted from movement between settings and the difficulty of verifying unsheltered status. due to shortages of nasopharyngeal swabs, three specimen collection methods were used. specimen collection methods have different sars-cov-2 detection sensitivities; however, nasal and nasal midturbinate specimens were shown to have >90% sensitivity compared with nasopharyngeal samples [27] . symptom and medical condition screenings were only conducted at a subset of testing events, which oversampled the larger shelters and may not be generalizable. shelter clients might not have disclosed symptoms due to fears that they would be removed from shelters. thus, the proportion of people who reported symptoms might be underestimated. only nine shelters of 24 underwent facility assessments, including two shelters at which assessments were conducted after initial testing. these findings may reflect improved or modified ipc policies as a result of knowledge of positive cases at these two shelters. the findings provide an early view into the effects of the covid-19 pandemic on peh and homelessness service staff in atlanta. as evidence grows and guidance evolves during the covid-19 pandemic, shelters should prioritize mitigation strategies and best practices for congregate and highrisk settings to prevent sars-cov-2 transmission while continuing to provide essential services for a c c e p t e d m a n u s c r i p t 14 m a n u s c r i p t m a n u s c r i p t 19 a c c e p t e d m a n u s c r i p t 21 a c c e p t e d m a n u s c r i p t 22 the 2019 annual homeless assessment report (ahar) to congress. part 1: point-in-time estimates of homelessness state of homelessness: atlanta continuum of care america/homelessness-statistics/state-of-homelessness-dashboards/?state=georgia. accessed 29 preventing and controlling emerging and reemerging transmissible diseases in the homeless morbidity and mortality in homeless individuals, prisoners, sex workers, and individuals with substance use disorders in high-income countries: a systematic review and meta-analysis chronic disease burden of the homeless: a descriptive study of student-run free clinics in tampa, florida cardiovascular disease and homelessness out-of-hospital and emergency department utilization by adult homeless patients barriers to homeless persons acquiring health insurance through the affordable care act estimated community seroprevalence of sars-cov-2 antibodies -two georgia counties prevalence of sars-cov-2 infection in residents of a large homeless shelter in boston covid-19 outbreak among three affiliated homeless service sites assessment of sars-cov-2 infection prevalence in homeless shelters -four u.s. cities discontinuation of isolation for persons with covid -19 not in healthcare settings outbreak of drug-resistant mycobacterium tuberculosis among homeless people in list n: disinfectants for use against sars-cov-2 (covid-19) unsheltered status, and risk factors for mortality mass screening for sars-cov-2 infection among residents and staff in twenty-eight long-term care facilities in fulton county, georgia. medrxiv symptom screening at illness onset of health care personnel with sars-cov-2 infection in king county estimated effectiveness of symptom and risk screening to prevent the spread of covid-19 interim guidance for homeless service providers to plan and respond to coronavirus disease 2019 (covid-19) universal and serial laboratory testing for sars-cov-2 at a long-term care skilled nursing facility for veterans initial and repeated point prevalence surveys to inform sars-cov-2 infection prevention in 26 skilled nursing facilities -detroit serial laboratory testing for sars-cov-2 infection among incarcerated and detained persons in a correctional and detention facility -louisiana interim considerations for health departments for sars-cov-2 testing in homeless shelters and encampments swabs collected by patients or health care workers for sars-cov-2 testing we would like to thank anitra walker (mercy care), catherine christie (mercy key: cord-292372-kn27ghlv authors: de chaisemartin, clément; de chaisemartin, luc title: bcg vaccination in infancy does not protect against covid-19. evidence from a natural experiment in sweden date: 2020-08-23 journal: clin infect dis doi: 10.1093/cid/ciaa1223 sha: doc_id: 292372 cord_uid: kn27ghlv background: the bacille calmette-guérin (bcg) tuberculosis vaccine has immunity benefits against respiratory infections. accordingly, it has been hypothesized to have a protective effect against covid-19. recent research found that countries with universal bcg childhood vaccination policies tend to be less affected by the covid-19 pandemic. however, such ecological studies are biased by numerous confounders. instead, this paper takes advantage of a rare nationwide natural experiment that took place in sweden in 1975, where discontinuation of newborns bcg vaccination led to a dramatic fall of the bcg coverage rate, thus allowing us to estimate the bcg’s effect without the biases associated with cross-country comparisons. methods: numbers of covid-19 cases and hospitalizations were recorded for birth cohorts born just before and just after 1975, representing 1,026,304 and 1,018,544 individuals, respectively. we used regression discontinuity to assess the effect of bcg vaccination on covid-19 related outcomes. this method used on such a large population allows for a high precision that would be hard to achieve using a randomized controlled trial. results: the odds ratio for covid-19 cases and covid-19 related hospitalizations were 1·0005 (ci95: [0·8130-1·1881]) and 1·2046 (ci95: [0·7532-1·6560]), allowing us to reject fairly modest effects of universal bcg vaccination. we can reject with 95% confidence that universal bcg vaccination reduces the number of cases by 19% and the number of hospitalizations by 25%. conclusions: while the effect of a recent vaccination must be evaluated, we provide strong evidence that receiving the bcg vaccine at birth does not have a protective effect against covid-19 among middle-aged individuals. the bacille calmette-guérin (bcg) vaccine is an attenuated live vaccine that has been proven effective against tuberculosis, in particular its severe manifestations like meningeal and miliary tuberculosis [1] . besides its specific effects, the vaccine has immunity benefits against non-targeted pathogens [2] , and in particular against respiratory infections caused by rna viruses like influenza [3] . these effects are thought to be mediated mostly by "trained immunity", a recently described mechanism of epigenetic reprogramming of innate immune cells [4] . while this mechanism is still under investigation, most studies show that these effects tend to wane after 15 to 20 years [5, 6] . since sars-cov-2 is also a single-stranded rna virus, it has been hypothesized that differences in bcg vaccination coverage could explain the wide differences in disease burden observed between countries. a pioneering preprint paper by miller et al. found that countries with universal bacillus calmette-guérin (bcg) childhood vaccination policies tend to be less affected by the covid-19 pandemic, in terms of their number of cases and deaths [7] . while unpublished, this study had a great impact and gave rise to many comments and follow-up studies (reviewed in [8] ). some published studies were able to replicate this result [9] [10] [11] , but several authors underlined the important statistical flaws inherent to such ecological studies and concluded that randomized controlled trials (rct) were necessary to address the question [8, 12] . as of june 5th 2020, no less than 12 randomized controlled trials (rcts) studying the protective effect of the bcg against covid-19 are already registered on https://clinicaltrials.gov/. however, none has a primary completion date earlier than october 1 st 2020, so these rcts' first results will not be known until at least five or six months. with the epidemic still on the rise worldwide, and in the absence of a sars-cov-2 vaccine, there is an urgent need to know if bcg non-specific effects could be harnessed as a substitute prophylactic treatment. regression discontinuity (rd) is a method designed by social scientists to assess the effect of an exposure on an outcome. it is deemed as reliable as rcts to tease out causality from correlation [13] , a c c e p t e d m a n u s c r i p t 6 and typically yields results similar to those obtained in rcts [14, 15] . in this paper, we applied this method to a rare natural experiment that took place in sweden. sweden currently has the 5th highest ratio of covid-19 deaths per capita in the world. in april 1975, it stopped its newborns bcg vaccination program, leading to a dramatic drop of the bcg vaccination rate from 92% to 2% for cohorts born just before and just after the change [16] . we compared the number of covid-19 cases and hospitalizations per capita, for cohorts born just before and just after april 1975, representing 1,026,304 and 1,018,544 individuals, respectively. using rd, we were able to show that those cohorts do not have different numbers of covid-19 cases and hospitalizations per capita, with a high precision that would hardly be possible to reach with a rct design. regression discontinuity (rd) is a commonly-used method to measure the effect of a treatment on an outcome [18] . it is applicable when only individuals that satisfy a strict criterion are eligible for a policy. then, rd amounts to comparing the outcome of interest among individuals just above and just below the eligibility threshold. in this study, rd will amount to comparing the number of covid-19 cases, hospitalizations, and deaths among individuals born just before and just after april 1st 1975. the effect of universal bcg vaccination for individuals born around april 1st, 1975 was estimated using the state-of-the-art estimator for rd [19] . the estimator amounts to comparing treated and control units, in a narrow window around april 1st 1975. it uses linear regressions of the outcome on birth cohort to the left and to the right of the threshold, to predict the outcome of treated and untreated units at the threshold. then, the estimator is the difference between these two predicted values. the estimator and 95% confidence interval were computed using the rdrobust stata command, see [20] . the rd estimator in [19] assumes that the variable determining exposure is continuous, but it has also been used when that variable is discrete but takes a large number of values. our analysis is at the quarter-of-birth level, so the variable determining exposure to bcg vaccination is not continuous, but it takes a large number of values. for instance, we observe cases/1000 inhabitants for 288 qbcs. the validity of the rd estimator is usually assessed by testing if observations to the left and to the right of the threshold have similar characteristics [13] . in the supplementary table 3 a c c e p t e d m a n u s c r i p t 8 finally, because we observe deaths for groups of 3 consecutive ybcs till 1980, there are only two data points after 1975 for that variable (1975-1977 and 1978-1980) , so the rd method cannot be used. in the supplemental materials, we merely use a t-test to compare the death rates in the 1972-1974 and 1975-1977 ybcs. this method is less robust than the rd method we use for cases and hospitalizations, as it does not account for the fact that the average age of the treated and control groups differs by 3 years. this study uses the number of covid-19 cases per 1000 inhabitants for quarterly birth cohorts born table 1 for details. in an rd design, the presence or absence of a treatment effect can be assessed visually, by drawing a scatter-plot with the variable determining eligibility on the x-axis, and the outcome variable on the y-axis. if one observes that the relationship between these two variables jumps discontinuously at the eligibility threshold, this is indicative of a treatment effect. accordingly, figure 1 shows no discontinuity in the numbers of covid-19 cases per 1000 inhabitants for cohorts born just before and just after april 1975. this suggests that universal bcg vaccination has no effect on the number the effects in table 1 are intention-to-treat effects [21] , as the vaccination coverage pre-1975 was around 92%, and after the 1975 change in policy, about 2% of the population was still vaccinated. taking into account foreign-born residents (27·2% of the swedish population born in 1975), which were not affected by the policy, the policy led to a drop of the vaccination rate of 65.5%. to convert the intention-to-treat effects in table 1 into the effect of being vaccinated at birth, one needs to divide the intention-to-treat effects and their confidence intervals by 0·655 [22] . in odds ratios, the in this study, we took advantage of a change in vaccination policy in sweden to investigate the link between bcg vaccination in infancy and covid-19 cases, hospitalizations and deaths, using a regression discontinuity approach. contrarily to most studies on the question, we compared covid-19 cases between two very similar groups of people from the same country. this allows us to get rid of all confounders linked to crosscountries comparisons, and of confounders like sex or socio-economics status that are often present in observational studies that do not rely on a quasi-experimental design, unlike ours. another strength of this study is its statistical precision. since we could gather nationwide data in a country where covid-19 rates are high, we are able to reject fairly small effects of the bcg vaccine. achieving a comparable statistical precision in an rct would require an unrealistically large sample. even with a covid-19 hospitalization rate of 0·5%, as among the elderly swedish population, a randomized controlled trial that could reject bcg effects larger than 25% of the baseline hospitalization rate, as in our study, would require including around 15,000 participants. while previous studies mostly addressed differences in bcg vaccination policies but did not account for differences in actual bcg coverage, we work with two populations with well documented and very different vaccination rates. the termination of the universal bcg vaccination program in sweden had dramatic effects on the bcg coverage rate. based on nationwide reports on the vaccination status of children below 7 sent to the national bacteriological laboratory in 1981, 92% of children born in 1974 got vaccinated, against 26% of those born in 1975, and less than 2% of those born between 1976 and 1980 [16] . among children born in 1975, most of the vaccinated children were born in the first quarter of the year, when the universal vaccination policy was still in place [16] . prior to that, sweden had already eliminated its revaccination program at 7 years of age in 1965. sweden also stopped its revaccination program at 15 years of age in 1986, four years before the 1975 cohort turned 15 years old. finally, sweden stopped its vaccination program for conscripts m a n u s c r i p t 11 in 1979, long before the 1975 cohort would do its military service [23] . overall, children born before april 1st 1975 benefited from a bcg vaccination policy at birth, while children born after that did not benefit from any bcg vaccination policy. this being said, there is a number of limitations to this work that one has to bear in mind. as in many other countries, folkhälsomyndigheten's cases count probably underestimates the true number of cases, because it only includes cases confirmed by a laboratory test. its deaths count probably underestimates the true number of covid-19 deaths as well, as it only includes deaths where a covid-19 diagnosis has been confirmed during the past 30 days. however, these limitations are common to all studies on this question and are unlikely to affect much the results. moreover, cases confirmed by a laboratory test are arguably the more severe ones, given that our data covers a time period where testing resources were scarce in sweden, as in many other countries. our computation of the change of the vaccination rate induced by the 1975 policy relies on the assumption that the bcg vaccination rate of foreign-born residents is the same just before and just after april 1975. however, this is a reasonable assumption, since no other european country changed its bcg vaccination policy in 1975. furthermore, rd estimates only apply to units close to the eligibility threshold. for instance, this study estimates the effect of universal bcg vaccination for individuals born around april 1st 1975, who are in their mid-forties during the covid-19 pandemic, and cannot be generalized to the entire population. it seems reasonable, however, to speculate that our findings hold true for older people since most studies assessing the long-term heterologous effects of the bcg show they tend to fade rather than increase over time [5, 6] . two studies argue that bcg vaccination at birth could have larger effects against covid-19 on older individuals, those most at risk during the pandemic, than on middle-aged individuals [7, 11] . based on the literature, we estimate that this is very unlikely to be the case. a c c e p t e d m a n u s c r i p t 12 moreover, this study does not measure the covid-19 immunity benefit conferred by a recent bcg vaccination, as individuals born just before q2-1975 were vaccinated 45 years ago. the rcts currently underway will tell if the protective effect of a recent bcg vaccination differs from the effect measured in this study. overall, this study shows bcg vaccination at birth does not have a strong protective effect against covid-19, at least in middle-aged individuals. thus, it seems that bcg childhood vaccination policies cannot account for the differences in the severity of the pandemic across countries, as had been hypothesized by prior studies [7, 9, 10] . this advocates for a strict adherence to who's recommendation of the vaccine to infants outside of clinical trials [25] , and for restraint from starting new clinical trials on this question. the former point is of particular importance for a vaccine whose lengthy production process regularly leads to worldwide shortages with dire consequences on children from country with high prevalence of tuberculosis [26] . while rcts will complement this study by measuring the effect of a recent vaccination, this study comes much before results of the rcts will be made available, and with a greater precision. it exemplifies the potential of leveraging past medical policies and statistical techniques designed in the social sciences to answer current medical questions. (2), and its 95% confidence interval is shown in column (3). the effects and their 95% confidence interval were computed using the rdrobust stata command, using linear polynomials on both sides of the threshold, and the robust confidence intervals reported by the command. the rdrobust command first selects observations within a narrow bandwidth around the threshold, using the bandwidth proposed in [19] . then, linear regressions of the outcome on birth cohort are run to the left and to the right of the threshold, to predict the outcome of treated and untreated units at the threshold. finally, the estimator is the difference between these two predicted values. the number of quarters of birth used in the last two steps of the estimation is shown in column (4). protection by bcg vaccine against tuberculosis: a systematic review of randomized controlled trials the non-specific and sex-differential effects of vaccines non-specific effects of bcg vaccine on viral infections targeting innate immunity for tuberculosis vaccination the duration of protection of school-aged bcg vaccination in england: a population-based case-control study duration of bcg protection against tuberculosis and change in effectiveness with time since vaccination in norway: a retrospective population-based cohort study correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study stop playing with data: there is no sound evidence that bacille calmette-guérin may avoid sars-cov-2 infection (for now) relation between bcg coverage rate and covid-19 infection worldwide is bcg vaccination affecting the spread and severity of covid-19 bcg vaccine protection from severe coronavirus disease 2019 (covid-19) demystifying bcg vaccine and covid-19 relationship regression discontinuity designs in economics empirical tests of the validity of the regression discontinuity design. annales d'économie et de when does regression discontinuity design work? evidence from random election outcomes the impact of changing bcg coverage on tuberculosis incidence in swedish-born children between 1969 and 1989 regression discontinuity designs: a guide to practice robust nonparametric confidence intervals for regression-discontinuity designs robust data-driven inference in the regression-discontinuity design what is meant by intention to treat analysis? survey of published randomised controlled trials identification and estimation of local average treatment effects the bcg world atlas: a database of global bcg vaccination policies and practices some evidence of the efficacy of mass bcg vaccination bacille calmette-guérin (bcg) vaccination and covid-19 global shortages of bcg vaccine and tuberculous meningitis in children key: cord-267152-m9m0aunk authors: yang, philip; esper, annette m title: investigating the sex differences in covid-19: another step forward, but many unanswered questions date: 2020-06-28 journal: clin infect dis doi: 10.1093/cid/ciaa776 sha: doc_id: 267152 cord_uid: m9m0aunk nan scientific community has still devoted heroic efforts not only to control the spread of sars-cov-2, but also to understand the characteristics of covid-19 and test the efficacy of various treatments. within a few months of the beginning of the pandemic, several studies from various parts of the world have already reported the characteristics of patients affected by covid-19 [1] [2] [3] . while these studies provided valuable information about the risk factors and outcomes from covid-19, an important issue that has not been addressed in these studies is the sex differences in covid-19. other than simply reporting the proportion of male and female patients in their study populations, prior studies have not explored the gender differences in detail. as a result, little is currently known about how men and women may be affected differently by the disease. there are undoubtedly important differences between men and women with regard to their susceptibility and response to many diseases. the higher female susceptibility to autoimmune diseases is probably a widely known example, but such differences exist in infectious diseases as well. for example, it has been reported that women are more prone to human immunodeficiency virus (hiv) infections, whereas tuberculosis and parasitic infections are more prevalent in men 4 . the reasons for such differences are complex. there may be biologic factors such as chromosomal and hormonal differences between men and women that can impact their susceptibility to infections, immunologic responses to infections, and progression of disease. in addition, there are behavioral, cultural, and socioeconomic factors that cause health disparities between men and women, leading to different risks of exposure and a c c e p t e d m a n u s c r i p t transmission of infections 4, 5 . despite the knowledge of such differences, there is a scarcity of literature that investigates the sex differences in infectious diseases, and the underlying mechanisms are just starting to be understood. this is the knowledge gap that motivated qian and colleagues to explore the sex differences in a large number of patients affected by covid-19 in mainland china. in this study, the investigators utilized the data from a national registry of over 80,000 reported cases of covid-19 in mainland china, as of late april 2020. the main finding from the study was that while female patients were more likely to be affected by covid-19, female patients had a lower proportion of severe or critical cases (pscc) and a lower case fatality rate (cfr) compared to male patients. the study also showed higher overall attack rates, higher severity of illness, and higher cfr with increasing age, a trend that was consistent with the findings from earlier reports 1, 2, 6 . performing subgroup analyses by age groups showed that the attack rate was higher in women 50-69 years of age, but the pscc and the cfr was lower in women in most of the age groups. it appeared that the sex differences in the attack rate, pscc, and cfr in hubei province, which contained the vast majority of covid-19 cases in mainland china, likely shaped the overall trends seen nationwide; the trends seen in other regions were more variable. the lower pscc and cfr for female patients remained significant in multivariate logistic regression analysis after controlling for potential confounders. this study provides valuable insight into the epidemiology of covid-19 and adds to the increasing body of knowledge about this new disease. the results of this study are similar to those of a smaller prior study, which also reported more severe illness and higher mortality from covid-19 in men compared to women 7 . these findings may be one of the potential reasons for the predominance of male patients (82%) in the italian cohort of covid-19 patients admitted to a c c e p t e d m a n u s c r i p t the intensive care units 2 , suggesting that male patients were more likely to suffer severe disease and require higher levels of care. it is also worth noting that a similar coronavirus outbreak, the severe acute respiratory syndrome (sars) epidemic in 2002-2004, also showed a higher case fatality rate in men compared to women 7, 8 . these findings suggest that these viral illnesses can affect men and women differently, and indicate the need for a better understanding of various factors that may be contributing to these differences. the authors of this study propose several potential reasons for the sex differences found in covid-19. these include biologic factors such as the location of angiotensin converting enzyme-2 (which is involved in sars-cov-2 entry into cells) and immune-related genes on the x chromosome, as well as development of higher levels of igg against sars-cov-2 in females. they also suggest social and behavioral differences, such as the fact that women more commonly assume caregiver roles and that men have higher smoking rates. the higher proportion of females among healthcare workers, who tend to have less severe disease from sars-cov-2 than the general population 9 , may have contributed as well. unfortunately, we are still left with many unanswered questions, some of which arise from the limitations of the study itself. first, the criteria used for diagnosing sars-cov-2 infection, classifying the severity of illness, and reporting the cases to the national surveillance database may have been inconsistent, especially early on in the outbreak. the database also did not include detailed information regarding the patients' medical comorbidities or the treatments received. as the authors admit, there was likely a wide variation in how these patients were treated, especially early on in the outbreak when there was no clear guidance on how to best treat covid-19 and many patients were being given unproven therapies. it is impossible to know whether any of these treatments or the underlying comorbidities of the patients may have a c c e p t e d m a n u s c r i p t confounded the results of this study in any way. moreover, the vast majority of patients in this study were from hubei province, and the sex differences noted in other regions of china appeared more variable. it is not yet clear whether the findings of this study will be generalizable to other parts of the world. as discussed previously, currently available studies describing the characteristics of covid-19 patients in other countries do not provide a deep exploration of the sex differences. with the disease still continuing to spread all throughout the world, our understanding of the sex differences in covid-19 will likely continue to evolve in the coming months. overall, this study of a large number of covid-19 patients in china points out the different impact of covid-19 based on sex, and pushes us another step forward in understanding this novel disease. it is too early to determine whether sex differences identified in this study would have practical implications for management, especially because the numerous potential reasons for such differences still need to be further elucidated. nonetheless, the study highlights the importance of recognizing specific populations at increased risk for an infection and better understanding the differences in outcomes. it raises awareness of this important issue and provides a valuable foundation on which to conduct future studies investigating the sex differences in infectious diseases. we hope that understanding the sex differences will someday contribute to formulating individualized treatments and determining better interventions, not only for covid-19 but also for other infectious diseases. neither author has any potential conflicts of interest. clinical characteristics of coronavirus disease 2019 in china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region covid-19 in critically ill patients in the seattle region -case series sex differences in infectious diseases-common but neglected gender differences in emerging infectious diseases case-fatality rate and characteristics of patients dying in relation to covid-19 in italy gender differences in patients with covid-19: focus on severity and mortality. front public health do men have a higher case fatality rate of severe acute respiratory syndrome than women do? characteristics of health care personnel with covid-19 -united states a c c e p t e d m a n u s c r i p t key: cord-292546-un0blb3w authors: dandachi, dima; geiger, grant; montgomery, mary w; karmen-tuohy, savannah; golzy, mojgan; antar, annukka a r; llibre, josep m; camazine, maraya; díaz-de santiago, alberto; carlucci, philip m; zacharioudakis, ioannis m; rahimian, joseph; wanjalla, celestine n; slim, jihad; arinze, folasade; kratz, ann marie porreca; jones, joyce l; patel, shital m; kitchell, ellen; francis, adero; ray, manoj; koren, david e; baddley, john w; hill, brannon; sax, paul e; chow, jeremy title: characteristics, comorbidities, and outcomes in a multicenter registry of patients with hiv and coronavirus disease-19 date: 2020-09-09 journal: clin infect dis doi: 10.1093/cid/ciaa1339 sha: doc_id: 292546 cord_uid: un0blb3w background: people with hiv (pwh) may have numerous risk factors for acquiring coronavirus disease-19 (covid-19) and developing severe outcomes, but current data are conflicting. methods: healthcare providers enrolled consecutively by non-random sampling pwh with lab-confirmed covid-19, diagnosed at their facilities between april 1st and july 1st, 2020. de-identified data were entered into an electronic research electronic data capture (redcap). the primary endpoint was severe outcome, defined as a composite endpoint of intensive care unit (icu) admission, mechanical ventilation, or death. the secondary outcome was the need for hospitalization. results: 286 patients were included; the mean age was 51.4 years (sd, 14.4), 25.9% were female, and 75.4% were african-american or hispanic. most patients (94.3%) were on antiretroviral therapy (art), 88.7% had hiv virologic suppression, and 80.8% had comorbidities. within 30 days of positive sars-cov-2 testing, 164 (57.3%) patients were hospitalized, and 47 (16.5%) required icu admission. mortality rates were 9.4% (27/286) overall, 16.5% (27/164) among those hospitalized, and 51.5% (24/47) among those admitted to an icu. the primary composite endpoint occurred in 17.5% (50/286) of all patients and 30.5% (50/164) of hospitalized patients. older age, chronic lung disease, and hypertension were associated with severe outcomes. a lower cd4 count (<200 cells/mm³) was associated with the primary and secondary endpoints. there was no association between the antiretroviral regimen or lack of viral suppression and predefined outcomes. conclusion: severe clinical outcomes occurred commonly in pwh and covid-19. the risk for poor outcomes was higher in those with comorbidities and lower cd4 cell counts, despite hiv viral suppression. patients with severe coronavirus disease-19 requiring hospital admission are more likely to be older, male, and have underlying comorbidities. [1] [2] [3] additionally, immunocompromising conditions such as malignancy and solid organ transplantation may increase patients' risk for severe covid-19 and death. [4] [5] [6] [7] [8] data are conflicting whether people with hiv (pwh) are also at increased risk. pwh may have numerous factors that could increase their risk of exposure to and acquisition of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). first, pwh are aging and have high rates of smoking, chronic cardiovascular and lung disease, and obesity. 9 in addition, racial and ethnic minorities are disproportionately affected by hiv disease and covid-19. ongoing research aims to determine the impact of structural racism in covid-19 outcomes. while the causes of health disparities are multifaceted, lack of access to healthcare, differences in socioeconomic, and prevalence of chronic diseases are potential contributors. 10, 11 several case series have described clinical characteristics of pwh and covid19 . many have been either limited to single-center studies, hospitalized patients, or included patients with suspected but not confirmed covid-19. most were also limited by small numbers of patients or lacked important hiv-specific variables such as cd4 cell counts, antiretroviral therapy (art), and plasma hiv viral load. [12] [13] [14] [15] some of these studies reported that pwh with covid-19 had similar clinical characteristics and comparable risk of severe disease to the general population. [16] [17] [18] [19] [20] [21] however, other studies found higher rates of sars-cov-2 infection and worse outcomes among pwh. [22] [23] [24] because current data are conflicting and limited, further investigation in hiv and covid-19 is warranted. we aim to describe the clinical characteristics and outcomes of covid-19 in pwh and to characterize pwh at the highest risk for severe covid-19-associated outcomes through an extensive multicenter registry. a c c e p t e d m a n u s c r i p t the covid-19 in pwh registry was sponsored by the university of missouri, columbia. the study was reviewed by the university of missouri institutional review board and considered to be exempt. anonymized patient data were collected without the need for informed consent. this multicenter registry was for pwh and covid-19, who received care between april 1 st and july in both inpatient and outpatient settings were eligible for study inclusion. healthcare providers collected study data by chart review of pwh and covid-19 diagnosed at their facilities and entered anonymous information into a secure electronic research electronic data capture (redcap). 25, 26 patients were enrolled consecutively by non-random sampling. study variables included patient demographics, hiv-associated variables, underlying medical problems, covid-19 clinical presentation as reported by patients, laboratory values, treatment, and clinical outcomes. providers certified that the information submitted was accurate to the best of their knowledge. two reviewers cross-validated the data for duplicity by age, gender, race, location, and hiv-1 rna (viral load). a c c e p t e d m a n u s c r i p t laboratory-confirmed covid-19 was defined as positive reverse-transcriptase-polymerase-chainreaction (rt-pcr) in respiratory samples or serum sars-cov-2 specific igg/igm. the us geographical region of residence was based on the cdc's national hiv surveillance system region distribution. 27 chronic lung disease included asthma and copd. cardiovascular disease included coronary artery disease and congestive heart failure. chronic liver disease included cirrhosis, chronic hepatitis b, and chronic untreated hepatitis c. active malignancy excluded non-melanoma skin cancer. we defined obesity as body mass index (bmi) of ≥ 30. 28 we defined virologic suppression as hiv-1 rna (viral load) < 200 copies/ml. 29 we collected the most recent hiv viral load measured before or at the time of covid-19 presentation. art categorizations were mutually exclusive. the primary endpoint was severe clinical outcome, defined as a composite endpoint of icu admission, use of mechanical ventilation, or death. 1 outcomes for survival analyses were time from positive sars-cov-2 test to icu admission and death. the secondary outcome of interest was hospitalization. we used descriptive statistics to summarize patient data. for categorical variables, we used frequency and calculated proportions using the number of patients with data available as the denominator. for continuous variables, we used the mean with standard deviation (sd) or the median with interquartile range (iqr). we analyzed the association between baseline variables with the defined outcomes by univariate analysis using the chi-square test, fisher's exact test, or t-tests as indicated. a multivariable logistic regression model was used to assess the association between each outcome of interest, severe outcome a c c e p t e d m a n u s c r i p t and hospitalization, and independent variables. to obtain the parsimonious model for each multivariable analysis, we identified the significant independent variables using the backward selection method. variables with a p-value ≤ 0.2 were included in the model in addition to clinically relevant variables. to adjust for within-region differences, we fitted a generalized estimating equation (gee) logistic regression model, assuming an exchangeable correlation structure and regions as clusters, for each outcome. to test whether there was a differential effect of cd4 count on icu-free survival and overall survival, we used the kaplan-meier method to analyze survival outcomes and log-rank statistics to compare the survival distribution of the cd4 groups (< 200, 200 -500, > 500 cells/mm³) with respect to time from positive sars-cov-2 test to icu admission and death. in a post-hoc sub-analysis, we included patients from the us to examine the clinical presentation and study outcomes, excluding international locations. user-defined missing values were treated as missing and not imputed. all tests were two-sided, with a level of significance defined as ≤ 0.05. sas ® software was used for statistical analysis of data. between april 1 st and july 1 st , 2020, we identified 286 unique pwh and laboratory-confirmed covid-19. thirty-six institutions from 21 states and three international locations contributed to the data; five duplicate cases were removed. a c c e p t e d m a n u s c r i p t art regimen was an integrase inhibitor with two nucleoside reverse transcriptase inhibitors (61.3%; 171/279). hypertension (46.5%), obesity (32.3%), and diabetes (21.3%) were the most common underlying medical problems. when stratified by hospitalization status, older age, lower cd4 counts, number of years living with hiv, not being on art or virally suppressed, and high comorbidity burden were associated with higher hospitalization rates (table 1) . a sars-cov-2 rt-pcr test was used to diagnose all but one patient, who was diagnosed based on serologic testing. the most frequently reported symptoms within 72 hours of diagnostic testing were cough (76.2%; 205/269), fever (70.7%; 198/280), and fatigue (66.0%; 140/212). pwh hospitalized with covid-19 were significantly more likely to have fever, fatigue, dyspnea, gastrointestinal symptoms, and altered mental status. whereas, pwh who were not hospitalized were more likely to have upper respiratory symptoms such as sore throat and nasal congestion, and headache. at presentation, all patients with a peripheral oxygen saturation of < 94% on room air (30.6%; 72/235) and a quick sequential organ failure assessment (q-sofa) score of ≥ 2 (8.3%;17/206) were hospitalized. a chest x-ray was obtained in 194 (67.8%) patients, of whom 77.8% had abnormal findings, and a computerized tomography (ct) scan was performed for 44 (15.4%) patients of whom 86.4% had abnormal findings. patients who were hospitalized were more likely to have imaging done and to have abnormal findings (table 2) . within 30 days of positive sars-cov-2 testing, 164 (57.3%) patients were hospitalized. among hospitalized pwh, potential sars-cov-2 targeted treatments, excluding placebo-controlled clinical trials, were given to 99 (60.4%) patients, the most common medication being hydroxychloroquine (40.9%) ( table 3) . c c e p t e d m a n u s c r i p t testing. when stratified by age groups (<40, 40 -60, >60 yrs.), the relative risk for all poor outcomes was more than one for older age groups compared to less than 40. patients who are older than 60 were significantly more likely to require respiratory support, develop acute kidney injury, and die, compared to patients who were less than 40 years old (table 3) . multivariable analysis identified higher age, lower cd4 counts, chronic kidney disease, and chronic lung disease as independent predictors of hospitalization. phw with three or more comorbidities, compared to having only hiv disease, were also more likely to be hospitalized. (table 4) . chronic lung disease (32.0 vs. 14.0%; p<0.01), and ckd (30.0 vs. 14.0%; p<0.01). in a multivariable analysis, older age, lower cd4 count, chronic lung disease, hypertension, and high comorbidity burden were significantly associated with severe outcomes (table 4) . based on 47 patients admitted to an icu and 27 deceased patients, cd4 cell count had a significant effect on survival. the pairwise comparison showed a significant difference between the cd4 count < 200 and cd4 count > 500 cells/mm3 for both icu-free survival (p=0.04) and overall survival (p=0.05) (figure 1 ). in post-hoc analysis, we included pwh diagnosed with covid-19 from the us (n=265) and excluded those from international locations (n=21). there were no significant differences in presentation and predictors of outcomes, except for hypertension. hypertension was not significantly associated with severe outcome, after adjusting for other variables. analysis presented in supplementary material (tables 1-4 a c c e p t e d m a n u s c r i p t in this multicenter analysis, severe clinical outcomes occurred commonly in pwh and covid-19. as reported in multiple other studies in people without hiv, we found that age, chronic lung disease, and comorbidity burden were associated with increased rates of severe outcomes. in addition, among hiv-specific factors, we found that a lower cd4 count (< 200 cells/mm3) was associated with poor outcomes, including higher hospitalization rates, lower icu-free survival, and overall survival. our study is the first to characterize outcomes in a large number of geographically diverse pwh with laboratory-confirmed covid-19. the clinical and radiologic presentations of the pwh in our study were similar to those reported in other studies of patients with covid-19, with or without hiv co-infection. 1, 17, 22, 30 our study confirms the unequal racial and gender distribution of pwh and covid-19. it mirrors the demographics of pwh in the us with a higher proportion of men, african american, and hispanic patients. 31 however, we did not find that race and gender were associated with worse outcomes in this cohort. our findings demonstrate a high prevalence of comorbidities among pwh and covid-19. consistent with other studies, 22, 32 we found that underlying comorbidities constitute a significant risk factor for hospitalization and poor outcomes in pwh. available data indicate considerable variability in mortality rates, icu admission rates and need for invasive mechanical ventilation among pwh diagnosed with covid-19. 16, [18] [19] [20] 22 based on our analyses, rates of icu admission, mechanical ventilation use, and death among pwh and covid-19 were consistent with the general us data. 30 we did not identify hiv viremia (a proxy for not taking art) as a risk factor for severe covid-19, but the proportion of study participants with hiv viremia was small and more than 90% of our study enrollees were receiving art. hence our ability to compare the outcomes between those with and without hiv control was limited. a c c e p t e d m a n u s c r i p t it has been postulated that patients with advanced hiv, low cd4 counts, and severe immunosuppression cannot mount the robust inflammatory response responsible for covid-19associated complications. 15, 16, 18, 21 our study does not support this hypothesis for those who are virologically suppressed but have low cd4 cell counts. although we did not collect information about nadir cd4 cell count or duration of art, this population (low cd4 but virally suppressed) usually has a history of severe immunosuppression, recent art initiation, or both. our findings show a significant association between low cd4 counts and poor outcomes contrasting recently published cases series. the study by karmen-tuohy et al. showed that 6 patients out of 19 had cd4 < 200 cells/mm3 and the cd4 count was not associated with mortality in hiv-positive patients. 19 a recent study from spain suggested that pwh receiving tenofovir disoproxil fumarate (tdf) in combination with emtricitabine as part of their art regimen have a lower risk for sars-cov-2 acquisition and related hospitalizations than those on other art regimens. 34 although the data we gathered were not specific to address the differences between those on tdf and other nucleoside reverse transcriptase inhibitors, in our study, we did not find an association between the class of art or the use of darunavir-containing regimens and predefined outcomes. this study has several limitations. first, this study cannot comment on the prevalence of sars-cov-2 infection among pwh. second, covid-19 testing, treatment, and hospitalization were all done at the discretion of individual healthcare providers and may have varied widely between sites as well as between different time points during the pandemic, reflective of local test availability and policies. there may also be selection bias, as contributors entered cases voluntarily and may not have entered every case from their institutions or clinics. however, 17 out of 36 institutions (accounting for 246 patients out of 286), have included systematically all hiv patients with covid identified through the search performed in their corresponding centers during the study period. we could not control for a c c e p t e d m a n u s c r i p t covid-19 therapies because we did not collect data on steroid use and clinical trial participation, as well as the small number of patients in each treatment group. we also did not collect data on social determinants of health, which may have impacted the clinical course of covid-19. future studies should assess whether specific socioeconomic factors impart a greater risk related to covid-19 for pwh. finally, death is counted as all-cause mortality; we did not verify the exact cause of death. despite these limitations, evaluating outcomes for pwh with covid-19 who were hospitalized or non-hospitalized from multiple sites and settings (community hospitals, clinics, and large academic centers) make these results more generalizable. in conclusion, our study adds to existing reports of the clinical characteristics of covid-19 among as principal investigator, dr. dandachi had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. analysis and interpretation of data: all authors. critical revision of the manuscript for important intellectual content: all authors. m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t the model for hospitalization outcome is adjusted for age, sex, race/ethnicity, years with hiv, cd4 count, hiv viral load suppression, antiretroviral regimen, hypertension, diabetes, chronic lung disease, chronic kidney disease, cardiovascular disease, active malignancy, and chronic liver disease. the model for severe outcome is adjusted for age, sex, race/ethnicity, cd4 count, hiv viral load suppression, hypertension, diabetes, chronic lung disease, chronic kidney disease, and chronic liver disease. the model for the association between hospitalization, severe outcome, and comorbidity burden is adjusted for age, sex, and race/ethnicity. † severe outcome, defined as a composite outcome of intensive care admission, invasive mechanical ventilation, or death. m a n u s c r i p t a c c e p t e d m a n u s c r i p t figure 1 comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region obesity in patients younger than 60 years is a risk factor for covid-19 hospital admission covid-19 in immunocompromised hosts: what we know so far patients with cancer appear more vulnerable to sars-cov-2: a multicenter study during the covid-19 outbreak covid-19 in persons with haematological cancers covid-19 and kidney transplantation cdc. hiv public health partners. policy, planning, and strategic communication. 2020. 10. price-haywood eg covid-19 and african americans co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area a survey for covid-19 among hiv/aids patients in two districts of wuhan, china (3/4/2020) covid-19 in people living with human immunodeficiency virus: a case series of 33 patients. infection clinical features and outcomes of hiv patients with coronavirus disease 2019 outcomes among hiv-positive patients hospitalized with covid-19 covid-19 and people with hiv infection: outcomes for hospitalized patients clinical characteristics, comorbidities and outcomes among persons with hiv hospitalized with coronavirus disease description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort. lancet hiv risk of covid-19 death: a population cohort study from the western cape province characterization and clinical course of 1000 patients with coronavirus disease 2019 in new york: retrospective case series research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners defining treatment failure in resource-rich settings interim analysis of risk factors for severe outcomes among a cohort of hospitalized adults identified through the u.s. coronavirus disease 2019 (covid-19)-associated hospitalization surveillance network (covid-net) disproportionate burden of covid-19 among racial minorities and those in congregate settings among a large cohort of people with hiv updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-300411-ppcdkfxq authors: xu, xiao-ke; liu, xiao-fan; wu, ye; ali, sheikh taslim; du, zhanwei; bosetti, paolo; lau, eric h y; cowling, benjamin j; wang, lin title: reconstruction of transmission pairs for novel coronavirus disease 2019 (covid-19) in mainland china: estimation of super-spreading events, serial interval, and hazard of infection date: 2020-06-18 journal: clin infect dis doi: 10.1093/cid/ciaa790 sha: doc_id: 300411 cord_uid: ppcdkfxq background: knowledge on the epidemiological features and transmission patterns of covid-19 is accumulating. detailed line-list data with household settings can advance the understanding of covid-19 transmission dynamics. methods: a unique database with detailed demographic characteristics, travel history, social relationships, and epidemiological timelines for 1,407 transmission pairs that formed 643 transmission clusters in mainland china was reconstructed from 9,120 covid-19 confirmed cases reported during january 15 february 29, 2020. statistical model fittings were used to identify the super-spreaders and estimate serial interval distributions. age and gender-stratified hazard of infection were estimated for household versus non-household transmissions. results: there were 34 primary cases identified as super-spreaders, with 5 super-spreading events occurred within households. mean and standard deviation of serial intervals were estimated as 5.0 (95% cri: 4.4, 5.5) and 5.2 (95% cri: 4.9, 5.7) days for household transmissions and 5.2 (95% cri: 4.6, 5.8) and 5.3 (95% cri: 4.9, 5.7) days for non-household transmissions, respectively. hazard of being infected outside of households is higher for age between 18 and 64 years, whereas hazard of being infected within households is higher for young and old people. conclusions: non-negligible frequency of super-spreading events, short serial intervals, and a higher risk of being infected outside of households for male people of working age indicate a significant barrier to the identification and management of covid-19 cases, which requires enhanced non-pharmaceutical interventions to mitigate this pandemic. in december 2019, a novel coronavirus disease have also been reported in several recent studies [5] [6] [7] [8] . starting from the last week of january 2020, more than 260 chinese cities have implemented intensive social distancing and confinement policies, which brought the epidemic under control [7] [8] [9] [10] . however, the epidemic has still caused more than 10,000 confirmed cases in china outside hubei province within a month. to enhance public health preparedness and awareness, chinese health authorities have publicly reported detailed records of confirmed covid-19 cases since mid-january. this provides a unique resource for studying the transmission patterns, routes, and risk factors of a c c e p t e d m a n u s c r i p t 5 in mainland china, 27 provincial and 264 urban health commissions have publicly posted 9,120 confirmed case reports online during january 15 -february 29, 2020, which accounts for 72% of all cases confirmed in mainland china outside hubei province. we compiled a unique line-list database using these reports, which contains detailed information about the demographic feature, social relationship, travel history and key epidemiological timelines (e.g., dates of symptom onset, confirmation, and hospitalization). in contrast to several published covid-19 data repositories [11] [12] [13] [14] [15] [16] which focus on describing information about individual cases, our database allows to reconstruct transmission pairs and clusters by inferring potential causal associations among different cases. the entire dataset of transmission pairs is available at our github (https://github.com/linwangidd/covid19_transmissionpairs_china). see supplementary materials for more details. we reconstructed 1,407 transmission pairs using the epidemiological evidence among reported cases. the section "reconstruction of transmission pairs" in supplementary materials specifies how we identified a pair or a group of confirmed cases using information about their close contacts, stratified transmission pairs into household and non-household settings using information about familial relationships, and determined the direction of transmission between infector and infectee using information about travel histories. for each transmission pair, we term the infector the primary case and the infectee the secondary case. we also consider connected chains of confirmed cases, in which we term the original case the index and the entire chain of cases, including the index, the transmission cluster ( figure 1a ). we categorized each transmission pair by the social relationship between primary and secondary cases (e.g., familial members of the same household, non-household relatives, colleagues, classmates, friends, and other face-to-face contacts). considering that during the spring festival travel season (january 10 -february 18, 2020) several billion human movements can occur because of the tradition of chinese new year (to visit and a c c e p t e d m a n u s c r i p t 6 live with their original families), we considered any transmission pair with immediate familial relationships (e.g., a person's spouse, parents, and children) as a household transmission pair, and with other familial relationships (e.g., a person's siblings with age older than 17) or close contacts with no familial information (e.g., classmates, colleagues) as a non-household transmission pair. the numbers of household (662) and nonhousehold (745) transmission pairs are almost even. a c c e p t e d m a n u s c r i p t 7 we estimated the age-stratified hazard of infection for household versus non-household transmissions by the ratio between the probability that a secondary case of age group b was infected by a primary case of age group a within the same household and the probability that a secondary case of age group b was infected by a primary case of age group a outside of households, i.e., . if , then the infection within households has a higher risk than the infection outside of households for secondary cases of age group b being infected by primary cases of age group a. we estimated the gender-specific hazard of infection for household versus non-household transmissions by the ratio between the probability that a secondary case of gender b was infected by a primary case of gender a within the same household and the probability that a secondary case of gender b was infected by a primary case of gender a via non-household transmission. the we in total reconstructed 643 transmission clusters formed by 1,407 transmission pairs (figure 1a) hazard of being infected within households was higher for the age groups of young (<18) and elderly (>65) people, whereas the hazard of being infected outside of households was higher for age groups between 18 and 64 years (table 1 ). primary cases of elderly (>65) people were more prone to cause household infections. hazard of infection between different genders was higher for households than non-household transmission (table 2) . a c c e p t e d m a n u s c r i p t 9 we have built a line-list database with detailed demographic information, travel history, epidemiological timelines, and social relationships for 1,407 transmission pairs that formed 643 transmission clusters in mainland china outside hubei province. we identified 34 primary cases as super-spreaders. majority of superspreading events were observed for non-household transmissions, which is consistent with a recent study 21 on transmission settings of covid-19 (e.g., hospitals, residential care, prisons, boarding schools, cruise ships). this indicates the importance of non-pharmaceutical interventions (e.g. isolation, quarantine, social distancing, and confinement 7,22-24 ) in mitigating the covid-19 epidemic. household studies are helpful to identify risk factors for certain demographic groups 25, 26 . the analysis of the age-stratified and gender-specific hazard of infection suggests a higher risk of infection within households for age groups of young (<18), elderly (>65) and female people. the higher risk of being infected outside of households for male people of age between 18 and 64 years may indicate their role in driving household secondary infections, perhaps because these were travelers from wuhan of working age. we identified 50 transmission pairs (~3.5%) with secondary case reported symptom onset earlier than primary case (i.e., negative-valued serial intervals), which is consistent with recent clinical reports 27,28 and epidemiological studies 29, 30 . we estimated that the mean serial interval is around 5 days for both household and non-household infections, which is considerably shorter than the mean serial interval estimated for sars (e.g., 8 .4 days 31 ) and mers (e.g. 7.6 days 32 ). our findings have several limitations. first, the household sizes and primary cases with no secondary infections were not provided from the original public case reports. this may give rise to biased estimates if we estimate the household reproduction number and secondary attack rate from the raw data. field surveys will be helpful to adjust such biases. second, the information on nosocomial infections and public gathering settings was not available from original case reports, so that the observation of super-spreading events may be less common from our dataset. third, caution is needed when attempting to generalize the age-stratified hazard of infection to other a c c e p t e d m a n u s c r i p t 10 demographic settings. for example, in our study (table 1) a c c e p t e d m a n u s c r i p t 16 a c c e p t e d m a n u s c r i p t 18 figure 1 detail/30-01-2020-statement-on-the-second-meeting-of-theinternational-health-regulations world health organization. coronavirus disease 2019 (covid-19), situation report -133 risk for transportation of 2019 novel coronavirus disease from wuhan to other cities in china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak an investigation of transmission control measures during the first 50 days of the covid-19 epidemic in china the effect of human mobility and control measures on the covid-19 epidemic in china early dynamics of transmission and control of covid-19: a mathematical modelling study effect of non-pharmaceutical interventions for containing the covid-19 outbreak in china. medrxiv open access epidemiological data from the covid-19 outbreak epidemiological data from the covid-19 outbreak, realtime case information evolving epidemiology and transmission dynamics of coronavirus disease 2019 outside hubei province, china: a descriptive and modelling study an interactive web-based dashboard to track covid-19 in real time early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population-level observational study the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application superspreading and the effect of individual variation on disease emergence serial intervals of respiratory infectious diseases: a systematic review and analysis secondary attack rate and superspreading events for sars-cov-2 what settings have been linked to sars-cov-2 transmission clusters? impact assessment of non-pharmaceutical interventions against covid-19 and influenza in hong kong: an observational study. medrxiv interventions to mitigate early spread of sars-cov-2 in singapore: a modelling study the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-ncov) outbreak covid-19 -studies needed presumed asymptomatic carrier transmission of covid-19 asymptomatic cases in a family cluster with sars-cov-2 infection serial interval of covid-19 among publicly reported confirmed cases temporal dynamics in viral shedding and transmissibility of covid-19 transmission dynamics and control of severe acute respiratory syndrome hospital outbreak of middle east respiratory syndrome coronavirus household size and composition around the world a c c e p t e d m a n u s c r i p t 12 a c c e p t e d m a n u s c r i p t 13 key: cord-291286-diwigcy9 authors: de schutter, iris; de wachter, elke; crokaert, françoise; verhaegen, jan; soetens, oriane; piérard, denis; malfroot, anne title: microbiology of bronchoalveolar lavage fluid in children with acute nonresponding or recurrent community-acquired pneumonia: identification of nontypeable haemophilus influenzae as a major pathogen date: 2011-06-15 journal: clin infect dis doi: 10.1093/cid/cir235 sha: doc_id: 291286 cord_uid: diwigcy9 background. precise etiologic diagnosis in pediatric community-acquired pneumonia (cap) remains challenging. methods. we conducted a retrospective study of cap etiology in 2 groups of pediatric patients who underwent flexible bronchoscopy (fob) with bronchoalveolar lavage (bal); children with acute nonresponsive cap (nr-cap; n = 127) or recurrent cap (rec-cap; n = 123). procedural measures were taken to limit contamination risk and quantitative bacterial culture of bal fluid (significance cutoff point, ≥10(4) colony-forming units/ml) was used. blood culture results, serological test results, nasopharyngeal secretion findings, and pleural fluid culture results were also assessed, where available. results. an infectious agent was detected in 76.0% of cases. in 51.2% of infections, aerobic bacteria were isolated, of which 75.0%, 28.9%, and 13.3% were haemophilus influenzae, moraxella catarrhalis, and streptococcus pneumoniae, respectively. most (97.9%) of the h. influenzae strains were nontypeable (nthi). h. influenzae was detected in 26.0% of nr-cap cases and 51.2% of rec-cap cases, whereas mycoplasma pneumoniae was the predominant pathogen in the nr-cap group (accounting for 34.9% of cases) but not in the rec-cap group (19.3%). viruses were found in 30.4% of cases, with respiratory syncytial virus, parainfluenzaviruses, and influenzaviruses detected most frequently. mixed infections were found in 18.9% of nr-cap cases and 30.1% of rec-cap cases. conclusions. a variety of microorganisms were isolated with frequent mixed infection. nthi was one of the major pathogens found, especially in association with recurrent cap, possibly because of improved detection with the fob with bal procedure. this suggests that the burden of pediatric cap could be reduced by addressing nthi as a major causative pathogen. direct sampling from the lung and lower airways can only be achieved with invasive methods, such as transthoracic needle aspiration (tna) and puncture of pleural effusion [7] . in adults, sputum samples are considered to be a valuable, noninvasive alternative for bacterial detection, but it is often not feasible to obtain a sputum sample from children. therefore, most pediatric studies are based on samples obtained from the upper respiratory tract (urt) and blood samples. however, the urt is frequently colonized by bacterial pathogens [3, 7] , making it difficult to discriminate commensal organisms and acute pathogens. moreover, although blood is normally sterile and blood cultures are highly specific, the sensitivity of blood cultures in childhood cap is low (4%-18%) [6, 7] . serological tests for the detection of viruses and atypical microorganisms are of limited use in clinical practice because of the need for convalescent-phase serum samples. flexible bronchoscopy (fob) with bronchoalveolar lavage (bal) is an option if noninvasive samples from the lower respiratory tract (lrt) are impossible to obtain or for selected patients with nonresponse to antibiotic treatment [8, 9] . bal fluid has the advantage of being suitable for multiple detection methods, including microscopy, aerobic culture, viral culture, and polymerase chain reaction (pcr). protected brushes cannot pass the pediatric flexible bronchoscope, but the risk of oropharyngeal contamination can be reduced considerably with careful technique [10] . interpretation of bal results is more reliable with the use of quantitative bacterial culture [1, 7, 8, 11, 12] . the reliability of fob plus bal used with these precautions has been demonstrated, with some authors reporting specificities of 97%-100% for potential pathogens in relation to actual pneumonia [7, 13] . moreover, fob plus bal has been proven to be safe, even in critically ill children [8, 14] . in this retrospective study, we assessed the etiology of cap in 2 groups of pediatric patients who underwent fob plus bal: children with acute nonresponsive cap and children with recurrent cap. among children, these cap features account for a significant proportion of physician and emergency department visits, hospitalizations, and antibiotic prescribing in the community. in addition to bal fluid culture, blood cultures, serological testing, culture and pcr results of nasopharyngeal aspirates of nasopharyngeal secretions (npa), and pleural fluid culture were assessed, where available. this was a retrospective analysis of pediatric patients who underwent fob plus bal from january 2005 through december 2007. the study was approved by the ethics committee of universitair ziekenhuis brussel (uz brussels, belgium). patients selected for analysis had acute nonresponsive community-acquired (broncho)pneumonia (nr-cap group) or recurrent communityacquired (broncho)pneumonia (rec-cap group). nr-cap case patients were defined according to current pediatric cap guidelines [15] as patients with persistent fever (.38.5°c) and persistent elevated infection parameters in the peripheral blood, associated in some cases with worsening consolidation visible on chest radiographs after at least 48 h of antibiotic treatment. the rec-cap group had an episode of cap with a history of at least 2 other cap episodes. exclusion criteria included the presence of severe and chronic conditions (such as cystic fibrosis, primary ciliary disease, immune deficiency, bronchopulmonary dysplasia, cardiopathy, neuromuscular diseases, asplenia, and tuberculosis) and nosocomial respiratory tract infection. retrospective analyses were conducted of demographic data, antibiotic use before fob plus bal, and the results of a broad microbiological investigation. fob and bal were performed according to european respiratory society recommendations [8] . a pediatric flexible fiberoptic bronchoscope (bf3c20 olympus) was inserted orally to avoid nasal contamination. aliquots of saline solution (0.9%) were instilled and reaspirated in the diseased lobar or segmental bronchus (maximum volume, 3 ml/kg body weight). the first bronchial lavage fraction was discarded. all subjects were sedated with intravenous midazolam, atropinesulphate, and tramadol hydrochloride; were transcutaneously monitored for oxygen saturation and heart rate; and received supplementary oxygen as needed during the procedure. blood cultures and serological testing were performed according to routine procedures when cap was diagnosed, before starting antibiotic treatment. npa was performed routinely in children ,4 years of age with cap with use of a flexible sterile catheter (vaginal catheter ch.8; medinorm) introduced into a nostril up to the nasopharynx. at this level, saline solution (2 ml; 0.9%) was instilled and immediately reaspirated. pleural fluid was obtained from patients with a significant amount of pleural fluid for safe puncture, using routine disinfection and puncture methods. samples were processed using standard techniques and interpreted as listed in table 1 . for haemophilus influenzae biotyping and capsular typing using a pcr and agglutination technique were performed at centre hospitalier universitaire st pierre, brussels. nontypeable h. influenzae (nthi) strains were distinguished from nonhemolytic haemophilus haemolyticus [16, 17] . b-lactamase activity was assessed using a nitrocephin-based test, and antibiotic susceptibility was determined by the etest technique (biomérieux). serotyping and antibiotic susceptibility testing of streptococcus pneumoniae was performed as described elsewhere [18] . viral culture and an in-house multiplex pcr (mpcr) were used on bal fluid and npa to detect respiratory viruses and atypical microorganisms (mycoplasma pneumoniae and chlamydophila pneumoniae). serological tests were performed by the complement fixation technique. the v 2 test with yate's correction or, when one of the subjected figures was ,5, the fisher's exact test were used to compare positivity rates between the 2 groups. statistically significant differences were defined as those for which the probability of occurrence was %5%. of the 700 children who had fob plus bal, 250 children-127 with nr-cap and 123 with rec-cap-met the inclusion criteria. the median age and sex ratio in both groups were similar ( table 2 ). most patients with nr-cap (77%) had received antibiotic treatment within 48 h before fob plus bal, whereas most patients with rec-cap (70%) had not been treated ( table 2) . a pathogen was detected in approximately three-quarters of children in both groups (table 3) . exclusively bacterial pathogens were found in 20.5% of the nr-cap group and 38.2% of the rec-cap group, and mixed infection was found in 18.9% and 30.1%, respectively. all bacterial pathogens except 3 s. pneumoniae were isolated from bal specimens, whereas only 2 blood cultures and 2 pleural fluid cultures were positive. in both patients with positive blood cultures, s. pneumoniae was isolated from the blood cultures, whereas h. influenzae and m. catharralis were isolated from bal specimens from the same patients. in 1 patient, staphylococcus aureus was isolated from pleural fluid culture, whereas bal yielded the same organism in combination with enterobacter aerogenes. in the latter case, s. pneumoniae was isolated from the pleural fluid specimen, but bal and blood cultures remained negative. overall, aerobic bacteria were isolated from 128 (51.2%) of 250 patients with infection; 75.0% of the aerobic bacteria isolated were h. influenzae. bacterial pathogens were found significantly more often in patients with rec-cap than in patients with nr-cap ( (table 4 ). in the remaining cases, h. influenzae was found with another bacterial pathogen (moraxella catarrhalis in 15 cases, s. pneumoniae in 6 cases, and s. aureus in 3 cases). h. influenzae was found in 21 mixed bacterial-viral infections, 11 mixed bacterial-atypical infections, and 5 mixed infections with mycoplasma species and a respiratory virus (table 5) . of the 96 h. influenzae isolates, 94 (97.9%) were nthi, and 2 were lost for typing. three isolates were initially misidentified by agglutination technique as being encapsulated strains, 2 type e and 1 type b. however, pcr results were negative in all 3 cases, resulting in a definitive identification as nthi. the majority of h. influenzae isolates (77.1%) were b-lactamase negative, including 20 of 33 isolates from nr-cap cases and 54 of 63 isolates from rec-cap cases. two b-lactamase-negative isolates were intermediate susceptible to ampicillin (minimum inhibitory concentration [mic], 2 mg/l), but no b-lactamasenegative, fully ampicillin-resistant strain was detected. after h. influenzae, m. catarrhalis and s. pneumoniae were the next most common bacterial pathogens, accounting for 28.9% and 13.4% of cases, respectively, in which aerobic bacteria were isolated. m. catarrhalis was isolated in 8.7% of nr-cap cases and 21.1% of rec-cap cases, whereas s. pneumoniae was isolated in 6.3% of nr-cap cases and 7.3% of rec-cap cases ( table 4 ). although s. pneumoniae was isolated in only 17 cases, a variety of pneumococcal serogroups-serotypes (sgt) was found, with a predominance of sgt 19, 23, and 6, which were isolated in 4, 2, and 2 cases, respectively. all 17 s. pneumoniae isolates were fully susceptible to parenterally administrated penicillin (mic, %2 lg/ml), whereas 3 isolates were intermediate susceptible to orally administrated penicillin (mic, 0.12-1 lg/ml). m. catarrhalis and s. pneumoniae were mostly found with another bacterial pathogen in 26 (70.3%) of 37 and 12 (70.6%) of 17 cases, respectively ( atypical microorganisms alone were detected significantly more often in patients with nr-cap than in patients with rec-cap (table 3) . m. pneumoniae was detected in 34.9% of the samples from patients with nr-cap that were tested and in 19.3% of the samples from patients with rec-cap that were tested, and c. pneumoniae was detected in 5.6% of rec-cap cases (table 4 ). viruses were found in 30.4% of cases overall. exclusive viral infection was reported in 16.5% of nr-cap cases and 7.3% of rec-cap cases (table 3) ; overall, respiratory syncytial virus (rsv), parainfluenzaviruses, influenzaviruses, adenovirus, and human metapneumovirus were detected most frequently (table 4 ). viral coinfection was found in 11 (8.7%) of 127 nr-cap and 5 (4.1%) of 123 rec-cap cases. we assessed 250 otherwise healthy children with either nr-cap or rec-cap to determine the etiology of the infection. a heterogeneous group of patients was included, which reflected pediatric cap cases that often require hospitalization in developed countries. an infectious agent was detected in 76% of cases, compared with detection in 17%-91% of cases in previous studies of pediatric cap [5, 14, 19, 20] . however, most of these studies involved urt samples, and thus had limited specificity for detecting bacterial pathogens, with few studies involving samples of the lrt or lung aspirates [3, 21, 22] . among the aerobic bacteria, which were isolated in approximately half of infections, we found a predominance of nthi in both groups of children; approximately three-quarters were h. influenzae, of which 97.9% were nthi. to our knowledge, this is the first such reported finding since those of shann et al [22] and weinberg et al [23] in the 1980s. since then, studies involving children with cap or lrt infection (using serological testing or tna) have suggested a possible role for nthi in pediatric pneumonia but have failed to define it as a significant pathogen [24] . for example, in a recent study involving 34 children with cap, 16 cases were attributed to s. pneumoniae and no case of h. influenzae infection was found using tna [25] . however, the study included a small number of patients and investigated lobar cap only. it is also possible that nthi infection was missed in previous studies of pediatric cap because the methodology was not appropriate for nthi detection. nthi does not easily invade the bloodstream or pleura [22, 26] , and misclassification of nthi strains as hib strains using the slide agglutination technique has been reported [27] . also, in many tna studies, the culture media used were not appropriate for detecting h. influenzae and, if h. influenzae was found, strains were not serotyped [22] . the reliability of our finding that nthi is frequently involved as a pathogen in childhood cap is supported by the sterility of the lrt in healthy subjects and the finding that, in children with unilateral cap, bal culture of the contralateral side has negative results [10, 12] , as well as by the established pathogenicity of nthi in another normally sterile site, the middle ear. nthi is associated with persistent, nonresponsive acute otitis media (aom) [28, 29] , possibly attributable to the choice of treatment (amoxicillin is used as first-line treatment), leading to the selection of b-lactamase-positive h. influenzae [29] . this was not the case in our study, because 77% of nthi isolates were b-lactamase negative. in aom, nthi has a clear association with recurrence .1 month after the completion of antibiotic therapy, with an incidence of 32% in third and subsequent episodes [29] . nthi infection contributes to complications in aom and is clinically indistinguishable from aom caused by s. pneumoniae [29] . it is therefore possible chlamydophila pneumoniae 0/58 tested (0) 3/54 tested (5.6) ns note. nr-cap, nonresponding community-acquired pneumonia; rec-cap, recurrent community-acquired pneumonia; coinfection: cases in which at least 2 infective agents of the same microbiological group were isolated; ns, not significant. a n 5 122 for viruses. that, as in aom, nthi is not an innocent bystander in the lrt. indeed, it is now increasingly recognized that nthi is capable of causing invasive disease, particularly neonatal sepsis, and is also a cause of cap, bacteremia and, to a lesser extent, meningitis [30, 31] . moreover, we are confident that the risk of contamination in bal samples was limited considerably, because various technical measures were taken to avoid contamination by urt secretions and a significance cutoff value was used for bacterial bal culture [1, 8, [10] [11] [12] . although a significance cutoff of r10 3 colonyforming units (cfu)/ml has been shown to be reliable in adults [7, 32] , we have chosen to use a higher cutoff value of r10 4 cfu/ml, because colonization of the urt is more common in children. the finding that only 2.8% of the haemophilus strains detected in our study were nonhemolytic h. haemolyticus, whereas previous studies showed that the prevalence of nonhemolytic h. haemolyticus in throat swab samples obtained from healthy children or adults was 16%-21% [16, 33] , also supports the reliability of our results. other typical bacterial pathogens for pneumonia, such as m. catarrhalis and s. pneumoniae, were detected in 28.9% and 13.3% of cases, respectively, in which aerobic bacteria were isolated. bacterial coinfections were reported in several earlier studies, including studies that used tna [3, 21, 22] ; most such coinfections were due to s. pneumoniae and h. influenzae, although coinfections due to h. influenzae and m. catarrhalis, as recorded in our study, were also reported [22] . although the role of each isolated pathogen cannot be defined, previous findings of coinfection and recent insights into interspecies quorum signaling suggest an individual role for each pathogen and/or possible interaction between them [34] . in belgium, universal childhood vaccination against h. influenzae type b (hib) was introduced in the 1990s and might explain the absence of any case of infection due to hib in our study. however, because universal childhood pneumococcal vaccination using pcv7 was only started in 2007, it is very unlikely that it may have influenced our results. the proportion of cases with an identified etiology was comparable between both groups. bacterial etiology was found more often in rec-cap cases than in nr-cap cases, probably because only approximately a quarter of patients with rec-cap received antibiotics before fob plus bal [35] . in antibiotic-pretreated patients, the diagnostic yield might have been increased with the use of bacterial serological testing [36] or quantitative pcr [37] . the relatively low detection rates for m. pneumoniae and c. pneumoniae may be related to the young age of the patients, because atypical infections are most common in children .5 years of age [20, 38, 39] . m. pneumoniae was a predominant pathogen in the nr-cap group but not in the rec-cap group. most of the patients with nr-cap had been treated with penicillin or aminopenicillins, the first-line antibiotics for treatment of cap, and the predominance of m. pneumoniae gives a possible reason for nonresponsiveness. viruses were found in 30.4% of cases, with rsv, parainfluenzaviruses, and influenzaviruses being detected most frequently. this incidence is relatively low, compared with the findings of other studies [5, 20] , and may have been due to omission of rhinovirus in the pcr assay. virus detection was slightly more frequent in the nr-cap group. viruses were the sole pathogen more than twice as often in the nr-cap group than in the rec-cap group, possibly providing another explanation for nonresponsiveness to initial antibiotic therapy. viral coinfections, which were found in 6.4% of cases overall, are rarely reported in most studies of cap, although one study reported an incidence of 14% for all cap cases and 22% for viral cap cases [5] . mixed infections were found in 24.4% of cases, which was comparable to previous findings [19, 20, 40] . our findings are compatible with current guidelines that recommend antibiotic treatment for all cap cases, because an established viral or atypical etiology does not rule out a concomitant bacterial infection [15, 39] . the predominance of nthi in rec-cap cases in our study suggests the use of aminopenicillins with or without b-lactamase inhibitors for the initial treatment in recurrent cases. in case of nonresponsiveness, the addition of macrolides to the initial treatment regimen is supported by the relatively high incidence of m. pneumoniae infection in nr-cap cases. in conclusion, as in previous studies, we found that a variety of microorganisms were involved in pediatric nonresponsive and recurrent cap with frequent mixed infection. in contrast to other reports, we found nthi to be a major bacterial pathogen, possibly because of improved detection with fob plus bal. this finding might explain cases of nonresponsiveness to antibiotic treatment or recurrence in pediatric cap, similar to observations in aom, which is frequently associated with nthi infection. additional research is needed to elucidate the pathogenic mechanisms of nthi in pediatric cap. however, our findings suggest that the burden of pediatric cap could be reduced by addressing nthi as a major causative pathogen in treatment and prevention strategies. the role of bronchoalveolar lavage in diagnosing nonopportunistic bacterial pneumonia community-acquired pneumonia in children diagnosis of acute bacterial pneumonia in nigerian children. value of needle aspiration of lung of countercurrent immunoelectrophoresis pneumonia after implementation of the pneumococcal conjugate microbiology of cap in children d cid vaccine program in the province of quebec etiology of community-acquired pneumonia in 254 hospitalized children etiology of childhood community acquired pneumonia and its implications for vaccination optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections bronchoalveolar lavage in children. ers task force on bronchoalveolar lavage in children infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults bilateral fiberoptic bronchoalveolar lavage in acute unilateral lobar pneumonia usefulness of quantitative cultures of bal fluid for diagnosing nosocomial pneumonia in ventilated patients quantitative bacterial cultures of bronchoalveolar lavage fluids and protected brush catheter specimens from normal subjects quantitative culture of bronchoalveolar lavage fluid in community-acquired lower respiratory tract infections flexible bronchoscopy and bronchoalveolar lavage in pediatric patients with lung disease pneumonia in childhood guideline development group of british thoracic society standards of care committee. british thoracic society guidelines for the management of community acquired pneumonia in childhood haemophilus haemolyticus: a human respiratory tract commensal to be distinguished from haemophilus influenzae detection of cryptic genospecies misidentified as haemophilus influenzae in routine clinical samples by assessment of marker genes fuck, hap, and sodc pneumococcal serogroups and serotypes in severe pneumococcal pneumonia in belgian children: theoretical coverage of the 7-valent and 9-valent pneumococcal conjugate vaccines epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections acute pneumonia in zimbabwe: bacterial isolates by lung aspiration aetiology of pneumonia in children in goroka hospital clonal analysis of hemophilus influenzae isolated from children from pakistan with lower respiratory tract infections serotypes and pathogens in paediatric pneumonia etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods role of nontypeable haemophilus influenzae in pediatric respiratory tract infections haemophilus influenzae infections in adults: characterization of strains by serotypes, biotypes, and beta-lactamase production microbiology of the middle ear fluid in costa rican children between haemophilus influenzae: a significant pathogen in acute otitis media non-type b haemophilus influenzae disease: clinical and epidemiologic characteristics in the haemophilus influenzae type b vaccine era invasive haemophilus influenzae disease quantitative culture of bronchoalveolar lavage fluid for the diagnosis of bacterial pneumonia pharyngeal colonization dynamics of haemophilus influenzae and haemophilus haemolyticus in healthy adult carriers indirect pathogenicity of haemophilus influenzae and moraxella catarrhalis in polymicrobial otitis media occurs via interspecies quorum signaling effects of antibiotics on protected specimen brush sampling in ventilator-associated pneumonia aetiology of community-acquired pneumonia: serological results of a paediatric survey evaluation of a multiplex pcr for bacterial pathogens applied to bronchoalveolar lavage etiology of childhood pneumonia: serologic results of a prospective, population-based study diagnosis and management of pneumonia in children mixed microbial aetiology of community-acquired pneumonia in children we thank dr. joanne knowles, for her outstanding editorial assistance, and dr. jos bellens, of the department of medical informatics, universitair ziekenhuis brussel (uz brussel), for his help with database production.financial support. glaxosmithkline biologicals provided an unrestricted scientific grant.potential conflicts of interest. a. m. has participated in advisory boards on pneumococcal diseases and vaccines for wyeth-pfizer and glax-osmithkline; is a member of a steering committee of a project sponsored by wyeth-pfizer; received payment for the development of educational presentations from glaxosmithkline; is coordinating a steering committee of a research project sponsored by wyeth-pfizer; has been a speaker for wyeth-pfizer and/or glaxosmithkline on pneumococcal diseases and epidemiology; and has received travel expenses from glaxosmithkline, novartis, abbott, sanofi-pasteur, and pfizer (weyth). i. d. s. has participated in advisory boards on nthi for glaxosmithkline and advisory boards on pneumococcal diseases and vaccines for wyeth-pfizer and glaxosmithkline; is a member of a steering committee of a project sponsored by wyeth-pfizer; and has received travel expenses from glaxosmithkline and sanofi-pasteur. j. v. is a member of a steering committee of a research project sponsored by wyeth-pfizer and is heading the national reference laboratory for s. pneumoniae that received grants from wyeth-pfizer and glaxosmithkline for serotyping isolates of ipd studies. d. p. has received grants from janssen-cilag, astrazeneca, wyeth, and pfizer and travel expenses from pfizer.all other authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed in the acknowledgements section. key: cord-269455-pkjov371 authors: faust, jeremy samuel title: towards a better case fatality estimate for sars-cov-2 during the early phase of the united states outbreak date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa639 sha: doc_id: 269455 cord_uid: pkjov371 nan given the above considerations, data from the model as proposed by kou et al can be harnessed for calculating a denominator statement with acceptable face validity for symptomatic disease at three time points, ranging from march 7 to march 21. we can further posit an alternative denominator that takes asymptomatic infection into account-which their model does not. while some estimates state that only 11 percent of cases are ultimately asymptomatic, other estimates are closer to 18 percent. but presymptomatic disease comprises a substantial fraction of infections at any given time and should therefore also be considered. universal screening among one healthy population detected that the rate of asymptomatic or presymptomatic disease was as high as 88 percent. 3 another study of older patients who were sicker at baseline found that 56 percent of patients with a positive sars-cov-2 swab were asymptomatic at the time of testing, and only developed symptoms later (median time from test to symptoms = 4 days). such patients would not be picked up in the final data point in use. taken together, a reasonably conservative a c c e p t e d m a n u s c r i p t attempt to add symptom-free cases to numbers proposed by kou et al could include a 50 percent addition to their estimates. 4 5 numerator statements, meanwhile, can reasonably be assumed to be sufficiently close to the running cumulative total number of counted covid-19 deaths as recorded at least two weeks after the day used to estimate the denominator. these counts are relatively reliable because covid-19 is currently a reportable cause of death in all us states and territories. while excess deaths may ultimately offer an attractive alternative for use as the numerator, expected lags in all-cause mortality reporting renders these numbers incomplete for several weeks. 6 once those numbers are available, they may serve as a partial measure of quality for numerator statements based on counted covid-19 deaths, which are prone to some degree of error. thus, the use of excess mortality may at some point provide another lens through which to verify the accuracy of these counts, as excess all-cause mortality figures does not rely on the subjective judgement of those filling out death certificates. march 21 st appears to be the best available date upon which to estimate a denominator for the cfr of sars-sov-2 using the model provided by kou et al. this date has the advantage both of being the peak of ili reporting to the cdc while being directly prior to the time when the effects of many of the mitigation strategies and changes in public behavior mentioned above began to become noticeable on a systemic level. as of april 5 th , public covid-19 trackers reported a crude cfr of 3.5 percent worldwide. using the kou model as a source for the denominator (cases as of march 21 st ) and all deaths through april 4 th as the numerator (including all deaths that occurred on us soil prior to march 21), the calculated cfr appears to have been approximately 22 percent of estimates on public-facing covid19 trackers-and this only accounts for symptomatic cases ( table 1 , column 1). further, allowing for the addition of pre-or asymptomatic cases into the denominator reveals a cfr of just 12 percent of the figures published on covid-19 trackers ( table 1 , column 2). these figures mirror estimates obtained in closed systems where universal testing was achieved, such as the diamond princess cruise. while the crude cfr on the diamond princess appears to have settled at around 1.8 percent, passengers aged 70 or older were over-represented as compared to other cohorts by a factor of approximately four. 7 8 this implies an age-adjusted cfr for the diamond princess of 0.45, which is remarkably similar to implied rates we calculate here using the denominator based on kou et al with adjustment for symptom-free infection ( table 1 , column 2). these numbers are higher, though not astronomically, than estimates given in the increasingly controversial santa clara county serology study. 9 if we instead use some of the higher reported numbers of pre-or asymptomatic cases found in the emerging literature, the estimated cfr we might calculate would indeed approach the 0.17 percent figure proposed by the authors of the santa clara study. together, these data imply that a more accurate cfr for sars-cov-2 may rest between 0.5 and 0.8 percent for symptomatic cases, and 0.2 and 0.4 percent for all cases including pre-and asymptomatic infections. however, this would also appear to imply that sars-cov-2 has a cfr that is between one and eight times greater than reported figures for seasonal flu. based upon recent ground conditions during the covid-19 outbreak compared to the peak of the worst flu seasons from recent years (as well as the 2009 h1n1 pandemic), no credible case can be covid-19 fatality is likely overestimated fact sheet medicare telemedicine health care provider fact sheet universal screening for sars-cov-2 in women admitted for delivery: nejm department of medicine. asymptomatic transmission, the achilles' heel of current strategies to control covid-19: nejm. 2020. available at estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship excess deaths associated with covid-19 field briefing: diamond princess covid-19 cases. available at covid-19 antibody seroprevalence a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-259471-lsdodl0a authors: pagliano, pasquale; piazza, ornella; de caro, francesco; ascione, tiziana; filippelli, amelia title: is hydroxychloroquine a possible postexposure prophylaxis drug to limit the transmission to healthcare workers exposed to coronavirus disease 2019? date: 2020-03-24 journal: clin infect dis doi: 10.1093/cid/ciaa320 sha: doc_id: 259471 cord_uid: lsdodl0a nan to the editor-we read with great interest the study by yao et al [1] reporting that hydroxychloroquine has better antiviral activity than chloroquine against severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which is associated with a high lung-to-plasma concentration ratio and immunomodulatory effects. moreover, the authors highlighted that hydroxychloroquine half maximal effective concentration values "tended to decrease with longer incubation times" and that, in the simulation model, the lung trough concentrations were still above the target concentration on day 10 after a 5-day course of hydroxychloroquine. besides household contacts, sars-cov-2 can be acquired in hospital settings, with healthcare workers (hcws) attending at invasive procedures on the respiratory tract (ie, intubation, endotracheal tube aspiration, or bronchoscopy) at the highest risk. accidently infected hcws represent an important source of infection during the period that they are asymptomatic or presymptomatic, as they can transmit the virus to household contacts and to patients seeking medical care for reasons unrelated to sars-cov-2 infection. for these reasons, we suggest preventive strategies for those accidentally exposed to sars-cov-2 such as preexposure or postexposure prophylaxis to avoid viral transmission, as largely accepted for those exposed to other viral agents such as human immunodeficiency virus (hiv) [2] . chloroquine and hydroxychloroquine are able to inhibit replication at early stages of viral infection, increasing endosomal ph at the time of virus/cell fusion, as well as impairing the glycosylation of cellular receptors of many viruses including coronavirus [3] . in contrast, no similar effect on early phases of coronavirus infection has been reported for other drugs proposed for sars-cov-2 treatment, which are able to interfere only after cell infection, affecting protease cleavage (protease inhibitors) or viral genome replication (remdesivir or ribavirin). these effects of chloroquine on early phases of viral replication permit the attenuation of vertical transmission in an animal model of zika virus infection, significantly reducing brain viral load, and make cells refractory to sars-cov infection in an in vitro model [4, 5] . hydroxychloroquine, the hiv protease inhibitors (particularly lopinavir), ribavirin, and remdesivir are the most promising drugs proposed for coronavirus disease 2019 (covid-19) treatment, but currently no drug has been proposed for postexposure or preexposure prophylaxis for those accidently exposed to sars-cov-2 [6] . on the basis of these investigations, we believe that hydroxychloroquine can be effective in preventing respiratory tract invasion in hcws exposed to sars-cov-2 and that hydroxychloroquine administration as a prophylactic agent could be particularly useful for hcws attending to high-risk procedures on the respiratory tract in covid-19 patients. hydroxychloroquine's effectiveness profile, its ability to inhibit lung viral replication for a 10-day period after only a 5-day cycle of therapy, and the large amounts of knowledge in term of safety deriving from its use for malaria prophylaxis and rheumatologic diseases lead us to recommend its preexposure or postexposure use for those performing procedures at high risk of viral diffusion in patients with covid-19 pneumonia. in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) protecting health care workers during the covid-19 coronavirus outbreak: lessons from taiwan's sars response effects of chloroquine on viral infections: an old drug against today's diseases? repurposing of the anti-malaria drug chloroquine for zika virus treatment and prophylaxis chloroquine is a potent inhibitor of sars coronavirus infection and spread clinical trial analysis of 2019-ncov therapy registered in china key: cord-300013-6m1f4q5r authors: brahier, thomas; meuwly, jean-yves; pantet, olivier; brochu vez, marie-josée; gerhard donnet, hélène; hartley, mary-anne; hugli, olivier; boillat-blanco, noémie title: lung ultrasonography for risk stratification in patients with covid-19: a prospective observational cohort study date: 2020-09-17 journal: clin infect dis doi: 10.1093/cid/ciaa1408 sha: doc_id: 300013 cord_uid: 6m1f4q5r background: point-of-care lung ultrasound (lus) is a promising pragmatic risk stratification tool in covid-19. this study describes and compares lus characteristics between patients with different clinical outcomes methods: prospective observational study of pcr-confirmed covid-19 adults with symptoms of lower respiratory tract infection in the emergency department (ed) of lausanne university hospital. a trained physician recorded lus images using a standardized protocol. two experts reviewed images blinded to patient outcome. we describe and compare early lus findings (acquired within 24hours of presentation to the ed) between patient groups based on their outcome at 7 days after inclusion: 1) outpatients, 2) hospitalised and 3) intubated/death. normalized lus score was used to discriminate between groups results: between march 6 and april 3 2020, we included 80 patients (17 outpatients, 42 hospitalized and 21 intubated/dead). 73 patients (91%) had abnormal lus (70% outpatients, 95% hospitalised and 100% intubated/death; p=0.003). the proportion of involved zones was lower in outpatients compared with other groups (median 30% [iqr 0-40%], 44% [31-70%] and 70% [50-88%], p<0.001). predominant abnormal patterns were bilateral and multifocal spread thickening of the pleura with pleural line irregularities (70%), confluent b lines (60%) and pathologic b lines (50%). posterior inferior zones were more often affected. median normalized lus score had a good level of discrimination between outpatients and others with area under the roc of 0.80 (95% ci 0.68-0.92) conclusions: systematic lus has potential as a reliable, cheap and easy-to-use triage tool for the early risk stratification in covid-19 patients presenting in eds the coronavirus disease pandemic has overwhelmed the health systems in several high-income settings (1) , and is now spreading in the low-income countries. there is a critical need for accessible and low cost methods to stratify risk for evidence-based resource allocation (2). while the majority of covid-19 patients have a paucisymptomatic or asymptomatic course, some may rapidly deteriorate leading to hospitalisation and the need for respiratory support. it has been suggested that early identification of patients at high risk of respiratory compromise is associated with lower mortality (3) . several studies have shown the predictive value of ct imaging, where the extent and patterns of lung involvement correlated well with severity of covid-19 on admission to hospital. other studies have described a progression of lung anomalies on consecutive chest cts during the course of the disease, with rapid evolution from focal unilateral to diffuse bilateral ground-glass opacities and finally, consolidation (7) . however, ct imaging has important limitations in triaging patients during the context of covid-19, not only due to their limited availability, high cost and exposure to radiation, but more critically, due to their immobile nature, thus necessitating the movement of infectious patients (8) . point-of-care ultrasound applied to lung is a promising alternative diagnostic tool, which can shorten time-to-diagnosis for the aetiology of acute dyspnoea, as well as stratify severity in the emergency department (ed) (9) . it is widely used in routine practice of swiss eds, can be performed at the bedside without radiation exposure and is easy-to-use in patients requiring protective isolation. so far, the use of lung ultrasonography (lus) in covid-19 has only been described in cohorts of severe hospitalised patients (10) (11) (12) (13) . however, it has already shown excellent performance to detect non-covid-19 pneumonia, compared to ct as a reference standard (14) , and matches the discriminative power of ct in patients with acute respiratory distress syndrome (ards) (15) . a c c e p t e d m a n u s c r i p t lus has potential in the pragmatic triage of covid-19 patients especially in low-resource settings. this study aims to describe lus characteristics in a prospective cohort of patients with covid-19 and explore their predictive capacity for risk stratification. this study is nested in a prospective cohort study of patients with lower respiratory tract infections, which started on february 6 th , 2020 in the ed of the lausanne university hospital, switzerland. we prospectively screened consecutive adult patients (age ≥ 18 years) presenting in the ed with an acute lower respiratory tract infection (cough, sputum, dyspnoea or chest pain for <21 days) (16) . patients with covid-19 confirmed by real time polymerase chain reaction (rt-pcr) for sars-cov-2 in a nasopharyngeal swab were included in this study. patients were excluded if lus could not be performed within 24 hours of admission or if the patient was receiving therapeutic prone ventilation before the lus. the study team collected patient's data using a standardized electronic case report form in redcap® (research electronic data capture). we assessed day-7 outcome by checking the electronic health record and we classified patients in three groups: group 1 (outpatients: absence of admission within 7 days of inclusion); group 2 (hospital admission within 7 days of inclusion); group 3 (intubation and/or death within 7 days of inclusion). a c c e p t e d m a n u s c r i p t a trained physician (tb) in lung ultrasonography performed all lus at inclusion in the ed. acquisition was standardized according to the "10-zone method" (17, 18) . two images (sagittal and transverse) and 5 second videos were systematically recorded in every zone with a butterfly iq tm , using the lung preset. the study physician (tb) and an expert radiologist (jym) standardized the reporting of pathological lus features based on covid-19 patterns (figure 1 ) (efigure 1 , efigure 2 and video v1 in the online data supplement) (10, 19) . for every zone, the following patterns were reported: (1) normal appearance (a lines, < 3 b lines), (2) pathologic b lines (≥3 b lines), (3) confluent b lines, (4) thickening of the pleura with pleural line irregularities (subpleural consolidation < 1 cm) or (5) consolidation (≥ 1 cm). the presence of pleural effusion was also recorded. the lus score, used as a correlate of loss of lung tissue aeration, as well as a normalized lus score (nlus score) corrected for the number of examined zone, were calculated in every patient (15, 19, 20) . blinded to patient outcome, both physicians independently filled the standardized report. discordance between the two readers was resolved by a third expert (op). supplementary table 1 shows the potential correlation of visible features between ct and lus images based on physical explanations behind their generation in several retrospective human studies (11, (21) (22) (23) (24) an animal study (25) and biomedical analysis (26) . differences between the three groups was evaluated by one-way anova, kruskal-wallis or chi-squared test, as appropriate. a bilateral p value <0.05 was considered indicative of a c c e p t e d m a n u s c r i p t statistical significance. the kappa coefficient was calculated to measure the inter-rater agreement between the two lus readers. the prognostic accuracy of the lus score, the nlus score and the proportion of lus affected zones to predict outcome was assessed by calculating the area under the receiver operating characteristic curve (auroc). we determined the optimal nlus score cut-off by choosing the value with the best sensitivity and a specificity superior to 50%. ethical approval was granted by the swiss ethics committee of the canton of vaud (cer-vd 2019-02283). table 1 shows demographics, clinical characteristics and laboratory results of the study population by group. overall, the mean age was 62 years (sd 17 years), and 34 (42%) patients were female. outpatients were significantly younger than patients in the other two groups (mean of 51 years, p=0.002). at inclusion, the median duration of symptoms was 7 days (iqr 6-11) and not different between groups. the most common symptoms were cough (91%), fever (83%) and dyspnoea (75%). dyspnoea occurred with increasing frequency across severity groups (p=0.014). heart and respiratory rates were lower in outpatients compared with patients in the other two groups (median 78/min vs 91/min, p=0.002 and 20/min vs 24/min, p=0.002, respectively). leukocyte count and c-reactive protein were significantly and gradually higher with increasing severity. overall, eight patients (10%) had a ct scan and 95% had a chest x-ray. x-rays were abnormal in 76% and outpatients had fewer abnormal x-rays than patients in the other two groups (38.5% versus 84%, p<0.001). among 10 patients with a normal chest x-ray (9 in the hospitalized group, one in the intubated/death group), 9 had lus abnormalities. at ed inclusion, 73 patients (91%) had an abnormal lus, the proportion of which increased progressively across severity groups to reach 100% in intubated/death patients (p=0.001) ( table 2) . a total of 735 lung zones were explored with lus in all patients. a median of 10 zones were recorded for each patient (iqr 9, 10); 10 zones (iqr 10, 10) in outpatients, 10 zones (iqr 9, with increased severity, lung anomalies affected both apical and basal lung regions (efigure 4 in the online data supplement) and were more bilaterally distributed. figure 3 ). in terms of the predominant abnormal lus pattern, outpatients mostly had a "non-confluent b lines" pattern, while the other two groups more frequently presented with "thickening of the pleura with pleural line irregularities" pattern (efigure 5 in the online data supplement). while the patterns of "pathologic b lines" and "confluent b lines" were more commonly the median lus score was 10 (iqr 5, 15) and the median nlus score was 1.1 (iqr 0.5the optimal nlus score cut-off to differentiate between outpatients and admitted patients including those who were intubated or died was 0.6 (sensitivity 81%, specificity 59%, positive predictive value 88%, negative predictive value 45%, positive likelihood ratio 1.97, negative likelihood ratio 0.32). if this nlus score had been used at the first ed visit, it would have correctly recommended primary hospitalisation for the three patients who were initially discharged (later returning for secondary hospitalization). the lus score, the nlus score and the proportion of affected zones had a poor level of discrimination between patients who died or were intubated and the other two groups. the two observers showed good reproducibility for all explored zones with a kappa of 0.74 based on the standardized us report. the reproducibility was excellent to differentiate normal and abnormal zones with a kappa of 0.90. despite the potential of lus as a cheap, portable and accessible point-of-care triage tool in acute respiratory disease (especially in low resource settings), a multinational consensus recently stated that the lack of studies limited specific recommendations for the management of covid-19 patients (27) . using a standardized approach in a prospective ed cohort of 80 patients, we described the characteristics of lus findings in covid-19 pneumonia. most a c c e p t e d m a n u s c r i p t patients presented abnormal lus with bilateral and multifocal involvement as previously shown in a large ct scan study (28) . the most common patterns seen on lus in decreasing frequency were thickening of the pleura with pleural line irregularities, confluent b lines, pathologic b lines and rarely, consolidations and minor pleural effusions. abnormalities affected all lung regions but were more frequent in posterior and inferior zones. lus findings also evolved with increasing disease severity, both in anatomic scope (progressing from unilateral to bilateral and pan-lung involvement), and pathological type (progressing from the "non-confluent b lines" pattern to "irregular pleural thickening"). we also describe a risk gradient in lus findings that can be summarised in a simple ordinal scoring system (lus score) which was able to discriminate between outcome groups in ed a c c e p t e d m a n u s c r i p t triage. the lus score can be used to quantify the loss of lung aeration and is thus useful for monitoring patients with ards. this simple lus scoring method may help in assessing covid-19 disease severity and support ed triage to decide on admission or close monitoring. previous studies have evaluated the lus score in covid-19 patients. in the intensive care unit, the lus score was higher in patients with refractory respiratory failure compared with others (31) . a good correlation existed between the lus score and a ct scan severity score. both scores correlated with clinical severity (21, 24). in our study, lus score also increased progressively according to clinical severity. however, we did not have the power to predict intubation and/or death with a good accuracy. to our knowledge, our study is the first including the complete range of disease severity, i.e.outpatients and patients who were intubated or died. our findings provide additional evidence that the lus score could be used as a triage tool to decide on admission. the role of lus to evaluate several respiratory diseases such as pneumonia and ards has been widely documented (14, 15) . lus has several advantages over chest ct such as its ease-of-use at point-of-care, low cost, absence of radiation, reproducibility and a reduced risk of nosocomial infection through its portability (reducing patient transport to imaging suites and lengthy disinfection protocol for the ct suite) (32, 33). lus allows a rapid assessment of severity at presentation in the ed. this study also shows that physicians with basic training in us (1-day theoretical course and 20 supervised acquisitions) are able to identify pathology with excellent concordance compared with experts: a critical proof of concept for its rapid deployment in covid-19 and for its general use in low resource settings. this study does not correlate lus with chest ct imaging. however, current recommendations specify that ct imaging should not be used for screening and is rather reserved for hospitalised, symptomatic patients, with specific indications (34). interestingly, two studies showed that the lus and ct scan scores have good agreement in the assessment a c c e p t e d m a n u s c r i p t of clinical severity (21, 24). excluding chest ct from the inclusion criteria eliminates a potential selection bias. on the other hand, we cannot propose a direct correlation between ct imaging and lus. acquisition of lus is dependent on the accessibility of anatomic sites, which is sometimes challenging in respiratory patients unable to mobilise. indeed, this study reported approximately 15% of missing values in posterior lung regions, which were mostly in severely ill patients. we mitigated this bias by normalising our score according to the number of available zones. regardless the discriminatory power of the score reveals that the predictive capacity of accessible zones is already highly informative. work is underway to identify the most informative zones and devise personalised imputations for such missing values. lus image interpretation is operator dependant, which is a potential disadvantage of this technique. however, in our study, we found a good agreement between the two observers. furthermore, using a standardized procedure and a pre-defined scoring method could minimize this limitation. in conclusion, lus is a promising tool for early risk stratification in covid-19. lung involvement visualized with us correlates with disease severity and summarising this into a simple ordinal scoring system has potential to discriminate patients requiring hospitalisation in the ed and thus better allocate scarce resources. work is ongoing to confirm these findings in a larger outpatient cohort. glasgow coma scale < 15; n (%) 2 (2.6) 0 (0) 0 (0) 2 (10) 0.044 leukocyte count, g/l; median (iqr) 6.2 (4.9, 8.5) 5 (4.3, 6.0) 6.3 (5.0, 7.3) 8.9 (6.2, 10) <0.001 :m1090. 2. who. home care for patients with covid-19 presenting with mild symptoms and management of their contact. the world health organisation lower mortality of covid-19 by early recognition and intervention: experience from jiangsu province clinical characteristics of coronavirus disease 2019 in china. the new england journal of medicine admission chest ct score predicts 5-day outcome in patients with covid-19 coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study. the lancet infectious diseases clinical diagnostic value of ct imaging in covid-19 with multiple negative rt-pcr testing point-of-care ultrasonography for evaluation of acute dyspnea in the ed chinese critical care ultrasound study g. findings of lung ultrasonography of novel corona virus pneumonia during the 2019-2020 epidemic. intensive care medicine a clinical study of noninvasive assessment of lung lesions in patients with coronavirus disease-19 (covid-19) by bedside ultrasound can lung us help critical care clinicians in the early diagnosis of novel coronavirus (covid-19) pneumonia? radiology our italian experience using lung ultrasound for identification, grading and serial follow-up of severity of lung involvement for management of patients with covid-19 lung ultrasound in diagnosing pneumonia in the emergency department: a systematic review and meta-analysis thoracic ultrasonography: a narrative review guidelines for the management of adult lower respiratory tract infections--full version. clinical microbiology and infection : the official publication of the european society of clinical microbiology and infectious diseases thoracic ultrasound: technique, applications, and interpretation international evidence-based recommendations for point-of-care lung ultrasound proposal for international standardization of the use of lung ultrasound for patients with covid-19: a simple, quantitative, reproducible method the value of lung ultrasound score on evaluating clinical severity and prognosis in patients with acute respiratory distress syndrome semiquantitative lung ultrasound scores in the evaluation and follow-up of critically ill patients with covid-19: a single-center study covid-19 pneumonia manifestations at the admission on chest ultrasound, radiographs, and ct: single-center study and comprehensive radiologic literature review chest ct manifestations of new coronavirus disease 2019 (covid-19): a pictorial review comparative study of lung ultrasound and chest computed tomography scan in the assessment of severity of confirmed covid-19 pneumonia b-lines quantify the lung water content: a lung ultrasound versus lung gravimetry study in acute lung injury on the physical basis of pulmonary sonographic interstitial syndrome the role of chest imaging in patient management during the covid-19 pandemic: a multinational consensus statement from the fleischner society ct imaging features of 4121 patients with covid-19: a meta-analysis frequency of abnormalities detected by point-of-care lung ultrasound in symptomatic covid-19 patients: systematic review and meta-analysis. the american journal of tropical medicine and hygiene early recognition of the 2009 pandemic influenza a (h1n1) pneumonia by chest ultrasound lung ultrasound score in evaluating the severity of coronavirus disease 2019 (covid-19) pneumonia how to face the novel coronavirus infection during the 2019-2020 epidemic: the experience of sichuan provincial people's hospital. intensive care medicine iqr) 72 (30, 147) we thank all the patients who accepted to participate and make this study possible. we thank professor carron, head of the emergency department, who supported the study. we thank all healthcare workers of the emergency department, internal medicine ward, infectious disease service and intensive care unit of the university hospital of lausanne, who managed covida c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-007321-7gi6xrci authors: chow, anthony w.; hall, caroline b.; klein, jerome o.; kammer, robert b.; meyer, richard d.; remington, jack s. title: evaluation of new anti-infective drugs for the treatment of respiratory tract infections date: 1992-11-17 journal: clin infect dis doi: 10.1093/clind/15.supplement_1.s62 sha: doc_id: 7321 cord_uid: 7gi6xrci these guidelines deal with the evaluation of anti-infective drugs for the treatment of respiratory tract infections. five clinical entities are described: streptococcal pharyngitis and tonsillitis, otitis media, sinusitis, bronchitis, and pneumonia. a wide variety of microorganisms are potentially pathogenetic in these diseases; these guidelines focus on the bacterial infections. inclusion of a patient in a trial of a new drug is based on the clinical entity, with the requirement that a reasonable attempt will be made to establish a specific microbial etiology. microbiologic evaluation of efficacy requires isolation of the pathogen and testing for in vitro susceptibility. alternatively, surrogate markers may be used to identify the etiologic agent. the efficacy of new drugs is evaluated with reference to anticipated response rates. establishment of the microbial etiology of respiratory tract infections is hampered by the presence of “normal flora” of the nose, mouth, and pharynx, which may include asymptomatic carriage of potential pathogens. this issue is addressed for each category of infection described. for example, it is suggested that for initial phase 2 trials of acute otitis media and acute sinusitis tympanocentesis or direct sinus puncture be used to collect exudate for culture. acute exacerbations of chronic bronchitis also present difficulties in the establishment of microbial etiology. these guidelines suggest that clinical trials employ an active control drug but leave open the possibility of a placebo-controlled trial. for pneumonia, the guidelines suggest the identification and enrollment of patients by the clinical type of pneumonia, e.g., atypical pneumonia or acute bacterial pneumonia, rather than by etiologic organism or according to whether it was community or hospital acquired. for each respiratory infection, the clinical response is judged as cure, failure, or indeterminate. clinical improvement is not acceptable unless quantitative response measures can be applied. these guidelines deal with the evaluation of anti-infective drugs for the treatment of respiratory tract infections. five clinical entities are described: streptococcal pharyngitis and tonsillitis, otitis media, sinusitis, bronchitis, and pneumonia. a wide variety of microorganisms are potentially pathogenetic in these diseases; these guidelines focus on the bacterial infections. inclusion of a patient in a trial of a new drug is based on the clinical entity, with the requirement that a reasonable attempt will be made to establish a specific microbial etiology. microbiologicevaluation of efficacy requires isolation of the pathogen and testing for in vitro susceptibility. alternatively, surrogate markers may be used to identify the etiologic agent. the efficacy of new drugs is evaluated with reference to anticipated response rates. establishment of the microbial etiology of respiratory tract infections is hampered by the presence of "normal flora" of the nose, mouth, and pharynx, which may include asymptomatic carriage of potential pathogens. this issue is addressed for each category of infection described. for example, it is suggested that for initial phase 2 trials of acute otitis media and acute sinusitis tympanocentesis or direct sinus puncture be used to collectexudate for culture. acute exacerbationsof chronic bronchitis also present difficulties in the establishment of microbial etiology. these guidelines suggest that clinical trials employ an active control drug but leave open the possibility of a placebo-controlled trial. for pneumonia, the guidelines suggest the identification and enrollment of patients by the clinical type of pneumonia, e.g., atypical pneumonia or acute bacterial pneumonia, rather than by etiologic organism or according to whether it was community or hospital acquired. for each respiratory infection, the clinical response is judged as cure, failure, or indeterminate. clinical improvement is not acceptable unless quantitative response measures can be applied. this is one of a series of disease-specific guidelines that have been prepared to assist sponsors and investigators in the development, conduct, and analysis of studies of new antiinfective drugs. these guidelines deal with the conduct of phase 1 through phase 4 clinical trials and are subsets of the general guidelines for the clinical evaluation of anti-infective drug products, which should be consulted for prerequisites to conducting studies in humans. these guidelines for the evaluation of drugs for the treatment of respiratory tract infections include acute streptococcal pharyngitis and tonsillitis, acute otitis media, acute and chronic sinusitis, acute exacerbations of chronic bronchitis, and acute infectious pneumonia (table 1). the focus is primarily on infections of bacterial etiology, especially those due to respiratory pathogens such as streptococcus pyogenes, streptococcus pneumoniae, haemophilus irfiuenzae, and moraxella (branhamella) catarrhalis and respiratory anaerobes (e.g., bacteroides species, fusobacterium nucleatum, and peptostreptococcus species). readers should consult the specific guidelines for the evaluation of new anti-infective drugs for mycobacterial and fungal infections. the guidelines for clinical microbiology provide important background information and should be used in concert with the current guidelines. the respiratory tract infections considered in these guidelines are among the most frequent disease entities encountered in both children and adults. they are associated with potentially serious morbidity if unattended or treated subop-timally. they also are infections in which evaluation of specific anti-infective therapy may be difficult. the reasons for the difficulties include: (1) routine noninvasive collection of specimens and culture techniques are often inadequate, and specimens are regularly contaminated by the indigenous microflora of the oropharynx and the upper airways; (2) the microbial etiology is often complex and polymicrobial; and (3) newly recognized etiologic agents continue to emerge (e.g. , legionella species, chlamydia pneumoniae, and coxiella burnettii). even with the use of sophisticated sampling and microbiologic techniques, the causative agents can be identified only for a small proportion (60%-80% at best) of patients. furthermore, good clinical practice requires empiric initiation of anti-infective therapy for these conditions (with the possible exception of group a streptococcal pharyngitis) on the basis of a presumptive initial diagnosis before confirmatory microbiologic data are available. frequently, the microbiologic response to therapy cannot be definitively evaluated, even when the etiologic agent has been identified. this is often the case in otitis media, sinusitis, and pneumonia, when the use of invasive procedures such as tympanocentesis, sinus puncture, or transtracheal aspiration to confirm microbial eradication in the patient who is improving clinically generally is considered unjustified. thus, whereas microbiologic failure can be documented by repeat cultures, microbiologic eradication can only be assessed presumptively on the basis of clinical response. the current standards of anti-infective therapy for the respiratory tract infections encompassed in these guidelines are summarized in table 1. in addition to the changing trends in microbial etiology, several controversial areas exist: (1) the clinical significance of ,b-iactamaseproduction among respiratory pathogens and the respiratory tract infections under study should be categorized according to the ageof the patient, chronicity of the disease, and anyunderlying or concomitant disease(s) in the patient. theinvestigational drugshould have in vitroactivity against the specific respiratory tract pathogen to be evaluated in a pathogen-specific studyandactivity against the vastmajority ofstrains ofthemost likely encountered pathogens in a diseasespecific study. evaluation oftheinfluence ofcombination therapy is desirable, as is assessment of the emergence of resistance in vitro. information obtained from studies in animals maybe of assistancein identifying preliminary dosage schedules for humans. evaluations of efficacy in standardized animalmodels of infection may be performed. determination of drug levels in respiratorytract secretions (suchas sinus, middle-ear, or endotracheal aspirates) or in tissue (such as pulmonary parenchyma) is not required because at present the clinical significance of these concentrations is uncertain. institutions should be capable of performing the following studies relevant to the management of respiratorytract infections when appropriate to a specific protocol: nucleic acid probeanalysis foridentification ofselected respiratory pathogens, radiography andcomputerized tomography (ct) and/or magnetic resonance imaging of the head and neck, sinuses, and chest; arterialblood gasdeterminations; tympanocente-sis; sinus puncture; thoracentesis; and bronchoscopy. these studies should be done in addition to routine diagnostic microbiologic testing. alternatively, special studies may be performed at a reference laboratory skilled in these procedures and approved by the appropriate authorities. the preferred design is the randomassignment of patients to the investigational-drug and active-control-drug groups. therandomization schedule shouldbe maintained bya study monitor. patients should be stratified according to age, severity of infection, presence of underlying disease, and concomitant non-antibacterial therapy. blinding of both subjects and investigators to treatment group(double-blind design) is encouraged whenever feasible. in all cases, the inclusion and exclusion criteria should be clearly identified prior to initiation of the study. all patients enrolled in the study should be assessed on the basis of "intention to treat." a uniform approach to clinical and microbiologic assessment duringand after therapy shouldbe implemented. endpointsforbothclinicaland microbiologic evaluation should be clearly stated, and whenever possible a quantitative scoring system should be devised. patient compliance shouldbe verified (e.g., by pill counts or by appropriate assays of drug concentrations in serum or other body fluids). the clinical entity addressed in this guideline is group a j3-hemolytic streptococcalpharyngitis and tonsillitis. not included are clinical cases of pharyngitis due to other agents or cases in which streptococcihavebeen isolated in cultures of throatspecimens but have notbeen documented to be group a j3-hemolytic streptococci. 28 groupa j3-hemolytic streptococcal pharyngitis remains one of the most frequentacute infections seen in ambulatory patients, especially school children between 5 and 8 years of age [1] [2] [3] [4] [5] [6] . antibiotic therapy for streptococcalpharyngitis is aimed not only at symptomatic improvement of the acute infection [7] [8] [9] [10] [11] [12] and the prevention of suppurative complications but also, and most importantly, at the prevention of the subsequent occurrenceof acute rheumatic fever [1, 13] . the incidence of acute rheumatic fever in the united states has declined dramatically overthe past severaldecades, suchthat by the 1980sit wasa rare sequelaof streptococcal pharyngitis [1, 5, 6] . however, between1984and 1986fourmajor outbreaks of acute rheumatic fever in three states resulted in a heightened concern for optimal treatment of streptococcal pharyngitis [14] [15] [16] . penicillin, given orally or intramuscularly, has generally beenconsidered the drug of choiceand the drugagainst which other regimens havemost often been judged. a full 10 days of oral therapy or a single injection of benzathine penicillin is required [3] [4] [5] [6] . shortening a course of penicillin by even a few days has been shown to resultin an appreciable increase in the rate of treatment failure. however, even withthe recommended 10 days of oral therapy, the failure rate may still be high [3] [4] [5] [6] . in recent studies, penicillintherapy, givenorally or intramuscularly, has been associated withratesof microbiologic failure as high as 20%-30%, in contrast to the rates of5%-10% seen 20 years ago. the reasons for this increase are not clear,although the presenceof j3-lactamase-producing organisms in the throat flora and an increasein the tolerance of streptococci to penicillin havebeen suggested as contributing causes. resistanceto erythromycin, a frequently used algroup a j3-hemolytic streptococcalpharyngitiscan not be diagnosed accuratelyon clinical grounds alone because it is frequently difficult to differentiate this entity from pharyngitis caused by other organisms. therefore, diagnosis requires a positiveculture for group a j3-hemolytic streptococcifrom a throatswab specimen in a patientwith symptomatic pharyngitis. alternatively, the diagnosismaybe made by use of one of therapiddiagnostic kitsthatcandetectgroupa j3-hemolytic streptococcal antigen directly from a throat swab specimen [19] [20] [21] [22] . for the purposeof the evaluation of newdrugs, however, the diagnosis shouldbe confirmedwith a throat culture, since the sensitivity (37%-100%, most 60%-95%) and specificity (70%-100%, most >90%) of the rapid detection kits are quite variable [19] [20] [21] [22] . drugsused for the treatmentof group a j3-hemolytic streptococcalpharyngitis shouldhavebeen shown to havebactericidal activity against group a j3-hemolytic streptococci and to haveundergone relevant phase 1 studiesprior to the initiation of clinical investigations. the drug under consideration shouldhave a lowindex of toxicity in bothchildrenandadults, sincea numberof otheragents existthat offer acceptabletherapyfor group a j3-hemolytic streptococciand since streptococcal pharyngitis is usually a minor disease. furthermore, the drug shouldresult in clinical improvement within 24-48 hoursoftheinitiation oftherapy, withresolution offever within 48 hours in uncomplicated streptococcalpharyngitis, as can be expected withpenicillin andother antimicrobial agents currentlyapproved for treatmentof streptococcalpharyngitis [3, [7] [8] [9] [10] [11] [12] . the drug under considerationalso should provide an acceptably low rate of microbiologic failure associated with recurrence or persistent carriage and a rate no greater than that associatedwith current standard therapy with penicillin (10 %-20 %) [3] [4] [5] [6] . the drug shouldbe capableof preventing the suppurative complications of group a streptococcal pharyngitis and, ideally, of preventing rheumatic fever. it would be desirable to have data indicating that the drug is capable ofpreventing rheumatic fever, but it is recognized that this goal may not be achievable. a current concern in the treatment of acute streptococcal pharyngitis is whether the failure rate for penicillin therapy will continue to climb and whether penicillin should still be considered the standard therapy. in addition, there is controversy about when antimicrobial therapy should be initiated. clinical differentiation of group a streptococcal pharyngitis from other causes of sore throat is not alwayspossible, a problem that raises the question of whether antibiotic therapy should be initiated before bacteriologic confirmation is available. furthermore, prompt treatment of group a (3-hemolytic streptococcal pharyngitis has been shown to interfere with the antibody response and possibly to result in a higher rate of recurrence than that seen in patients whose therapy is delayed for a few days [17] . last, controversy exists concerning whether post-treatment cultures should be obtained to detect bacteriologic failures and whether asymptomatic carriage necessitates treatment [18] . patients eligible for study entrance are children or adults with symptomatic pharyngitis or tonsillitis of acute onset clinically consistent with infection with group a i3-hemolytic streptococci and from whom group a (3-hemolytic streptococci have been isolated in cultures of throat -swab specimen or for whom a rapid screening test has indicated the presence of streptococci. to be evaluable for efficacy, the screening test results must be confirmed by culture. the guideline generally applies to ambulatory patients. signs and symptoms of acute pharyngitis or tonsillitis of acute onset include sore throat and evidence on physical examination of inflammation of the uvula and pharynx or tonsils, including erythema, often with edema of the tissues, with or without exudate. fever mayor may not be present. a single culture specimen should be obtained from the posterior pharynx prior to initiation of anti-infective therapy. at least 10 colonies of group a i3-hemolytic streptococci should be present on the culture plate. a throat specimen for culture is obtained with use of a throat swab that is passed over both sides of the posterior pharynx and the uvula [3] . the preferred culture medium is sheep's-blood agar. all cultures negative at 24 hours should be reincubated for another 24 hours. reduced oxygen tension may enhance identification of group a (3-hemolytic streptococci. such a reduction may be achieved in a simple manner by stabbing the agar after the sample is streaked or by using a coverglass pressed onto the primary zone of inoculation [19] . group a streptococci are identified by the bacitracin method or by an-other method of at least equal sensitivity and specificity [3, 19, 21] . if a rapid diagnostic test is used for identification of group a streptococci, the findings must be confirmed by culture [20] [21] [22] . the streptococci obtained on culture should be saved for subsequent typing when possible. the drug under consideration should be active in vitro against group a (3-hemolytic streptococci. the institution or the investigator should have access to a clinical microbiology laboratory where the following tests can be performed: culture of throat swabs on sheep's-blood agar and identification of group a (3-hemolytic streptococci. alternatively, a single laboratory may process samples referred from participating centers. clinical studies should include patients of different age groups, since the clinical manifestations of group a streptococcal pharyngitis and tonsillitis may vary with age of the patient. streptococcal pharyngitis is uncommon in children <3 years of age. classic exudative pharyngitis is most frequently observed in school-aged children. group a streptococcal pharyngitis in teenagers and adults is often atypical. children, adolescents, and adults of both sexes should be included. for other considerations, see general guidelines, section ix. it is not considered ethical to use a placebo control. an active control drug should be used. the control agent should be selected on the basis of previous experience demonstrating that it is among the most effective agents for the treatment of group a (3-hemolytic streptococcal pharyngitis at standardized and well-tolerated doses. the study should compare the trial drug with the active control drug. the treatment regimens should be randomized and of a double-blind design whenever possible. phase 1 studies shouldprovide adequateinformation concerning dose, dosage interval, andotherpharmacokinetic characteristics. the usefulness of monitoring concentrations in serumor other bodyfluids or tissuesshouldhave been determined. the form of the drug (liquid, tablet, capsule)should be acceptable for patients of any age included in the study and should be an accurate dose (e.g., no cutting of tablets required). theusualtreatment coursewithstandard regimens (e.g., penicillin or erythromycin) is 10days. the optimalduration of therapy with the study drug maybe determined by additional studies. the initiation of therapy should be standardized, i.e., at the time of clinical diagnosis or at the time of culture confirmation. ifit proves necessary to add a seconddrug or to substitute a newantimicrobial drug, treatment is considered to have failed clinically. in the eventof allergy to or failure of either drug being evaluated, the patient shouldbe treated with an alternative, standard active drug. response should be evaluated by both clinical and bacteriologic assessment. clinical assessment should include history andphysical examination. documentation of the clinical response with regard to symptoms and signs, including fever, shouldbe obtainedat 3-5 days after initiation of therapy and at weekly intervals (±2 days) thereafteruntil the patient is asymptomatic. the 3-to 5-day assessment may take the form of a telephone call. patients should be observed posttherapy for a sufficient time to permit detection ofrelapse of disease and/orpost-streptococcal nephritisor carditis. the periodof post-treatment evaluation will varywithknowledge of the durationof anti-infective activitysubsequent to terminationof administration of the test drugs. asa generalguide, patients shouldbe followed-up for 2-4 weeks after termination of therapy. evaluation of thebacteriologic response requires a repeated throatcultureat the firstfollow-up visit, within4-7 days after the end of therapy, and at any time clinical symptoms recur. additional posttreatment throatculturesmaybe necessary for patients treatedwith drugs known to remain in serum or tissue for intervals beyond the initial4-to 7-day evaluation. all organisms recovered should be saved for typing if possible. groupa streptococci recovered duringtherapy or at the time of the follow-up visit should be evaluated for their in vitro susceptibility to the study drug. theserologic response to groupa~hemolytic streptococci may be evaluated in acute-and convalescent-phase sera for titersofantibody to streptolysin-o (aso) or otherstreptococcal antigens. serologic evaluation, however, is not required for evaluation of drug efficacy. compliance shouldbe evaluated by the return of all medicationcontainers andof anyremaining drugat the endoftherapy. documentation of drug in the urine or blood may also be used to assess compliance. (1) definition ofclincial response. clinical cureis defined as complete disappearance of signs and symptoms without recurrence; clinical cure with recurrence is defined as the development of symptomatic pharyngitis documented to be causedbygroupa~hemolytic streptococci beforeor during follow-up in patients who were asymptomatic at the initial follow-up assessment; and clinical failure is defined as lack of any response to therapy. (2) definition of microbiologic response. microbiologic eradication is defined as eradication of group a ,8-hemolytic streptococci at the initialand subsequent follow-up examinations; microbiologicpersistence is defined as failure to eradicate group a ,8-hemolytic streptococci at the time of initial follow-up; andmicrobiologic relapse is defined as initialsuppression of groupa~hemolytic streptococci withsubsequent positive cultures for group a ,8-hemolytic streptococci. thefinal assessment of efficacy maybe categorized according to both clinical and microbiologic criteria as in table 3. otitis media is the most frequent diagnosis recorded for infants and children who visit physicians because of illness [23] . before 3 years of age more than two-thirds of children have had one or more episodes of acute otitis media (aom) and more than one-third have had three or more episodes [24] . the highest incidence of aom is in children 6-24 months of age. the incidence declines with age except for a limited reversal of the downward trend at the time of entry into day care or school. although middle-ear infection is considered uncommon in adults, a recent survey identified almost 4 million visits to physicians by adults each year for this problem [25] . males have a significantly increased risk for aom, and native americans and canadian and alaskan eskimos have high rates and severe disease. incomplete data suggest that american blacks have fewer episodes of ear infection than do members of other racial groups in the united states. early occurrence of the first episode of aom, sibling history of recurrent aom, not being breast fed, and attendance in day care are all associated with increased risk for recurrent aom [24, 26] . since aom and secretory otitis media (som) are defined by the presence of middle-ear effusion (mee), techniques to determine the presence of air or fluid in the middle ear are critical to diagnosis. three methods are available: the standard technique of pneumatic otoscopy, typanometry, and acoustic reflectrometry. tympanometry uses an electroacoustic impedance bridge to record compliance of the tympanic membrane (tm) and provides objective evidence of the status of the middle ear and the presence or absence of fluid. technical difficulties limit the use of tympanometry in children during the first 6 months of life. the acoustic otoscope or reflectometer is a hand-held instrument that utilizes principles of reflected sound waves to diagnose the presence of air or fluid in the middle ear. the microbiology of aom has been documented by appropriate cultures of mee obtained by needle aspiration. many studies have been performed in the united states, scandinavia, and japan. the bacteriologic results are consistent in demonstrating the importance of s. pneumoniae, h. influenzae (90 % nontypable, 10% type b), and m. catarrhalis [27] . s. pneumoniae is the most important bacterial cause of otitis media and is defined in mee of about one-third of children with aom. otitis media due to h. irfiuenzae has been associated with 20%-30% of cases of aom, and rv20%-30% ofthese strains produce j3-lactamase. m. catarrhalis has been isolated from mee in 7%-20% of cases of aom, and a majority of these strains produce j3-lactamase. virologic and epidemiologic data suggest that viral infection frequently is associated with aom. mycoplasma pneumoniae does not appear to playa role in aom, although some patients with lower respiratory tract disease due to m. pneumoniae may have con-comitantaom. c. trachomatis is a cause of aom but almost exclusively in infants <6 months of age. the microbiologic diagnosis of aom can be made only by aspiration of mee. this procedure should be done only by persons skilled in the technique. cultures of throat and nasopharyngeal swab specimens are of no value because they are neither sensitive nor specific when compared with culturs of isolates from the middle ear. the results of cultures of middle-ear fluids from the two ears are disparate in rv20 % of cases of aom (e.g., effusion from one ear may be sterile while the effusion from the other yields a bacterial pathogen, or different bacterial pathogens are isolated from the two ears). therefore, for evaluation of new drugs or vaccines, it is important that each diseased ear be aspirated for a complete microbiologic assessment and that outcome for each ear be evaluated separately [28] . suppurative sequelae such as mastoiditis and other infratemporal and intracranial complications occur but are uncommon in developed countries. hearing loss is the most important complication of aom and mee. patients with mee suffer from hearing loss of variable severity. on average, a patient with fluid in the middle ear has a 25-decibel hearing loss. since intellectual development is dynamic during infancy, when the incidence of aom is highest, there is concern that any impediment to reception or interpretation of auditory stimuli might have an adverse effecton development of speech, language, and cognitive abilities. some studies suggest that children with histories of recurrent aom have lower scores in tests of linguistic and cognitive abilities than do their diseasefree peers [29] . the clinical entity discussed in this guideline is limited to aom (synonyms include acute suppurative om and acute purulent om). the microorganisms considered are s. pneumoniae, h. infiuenzae, and m. catarrhalis. not included in this guideline are secretory otitis media and chronic suppurative otitis media. som is defined as the presence of mee behind an intact tm without acute signs or symptoms (synonyms include chronic om with effusion, persistent mee, om with effusion, and serous om). chronic suppurative om is defined as chronic discharge from the middle ear through a perforation of the tm (synonym includes chronic om). tympanocentesis and culture of mee is required for microbiologic diagnosis of aom. nose and throat cultures are of no value. tympanocentesis is a safe procedure when performed by skilled and experienced persons. the procedure provides not only specific microbiologic diagnosis but also symptomatic relief of acute pain by decompressing the em 1992;15 (suppl 1) middle-earabscess. there is transientpain during the few seconds of the procedure. rare untoward events may occur, including bleeding, tearing of the tympanic membrane, and ossicular dislocation. approximately one-third of children with aom caused by a bacterial pathogen improve without treatment with antibacterial drugs. clinical resolution may occur because the contents of the middle ear are spontaneously discharged, either through the eustachian tube or by means of a spontaneous perforation of the tm. with appropriate antimicrobial therapy, however, signs and symptoms of aomimprovewithin48-72 hours. mee maypersist (even though sterile) for weeks to months after onset of aom. the goals of antimicrobial therapy for aom are the rapid resolution of signs and symptoms of disease; sterilization of the mee; prevention of suppurative sequelae; reduction of the occurrence of relapse and recurrences; and decrease in time spent with mee. the preferredantimicrobialagent for the patient with aom must be active againsts. pneumoniae, h. irfluenzae, and m. catarrhalis. group a streptococci, staphylococcus aureus, gram-negative enteric bacilli, and anaerobic bacteria are infrequent causes of aomand need not be considered in initial therapeutic decisions. amoxicillin or an equivalenthas been the standard regimenfor aomsinceit is effective againstmost strains of the three major pathogens and is well tolerated, producing limited adverse effects. however, since at present 20%-30% of h. irfluenzae strainsand 50%-70% of m. catarrhalis strains in the united states produce~-lactamase, ã -lactamase-stable agent (such as amoxicillinplus a~-lacta mase inhibitor, a second-or third-generation cephalosporin) or a combination such as trimethoprim-sulfamethoxazole or erythromycin/sulfisoxazole may also be used. clinical trials with these agents indicate that all regimens are of approximately equal clinical efficacy when the bacterial pathogens are susceptible [30] . the control drug chosen for a clinical trial should be among the most effective and safe agentsavailable for treatment. it is expected that an effective agent will sterilize the middle-ear fluid of bacterial pathogens in >80% of infected ears within 72 hours. a second aspiration of middle-earfluidshouldbe consideredfor anypatientfor whom the outcome at 72 hours is clinical failure. chemoprophylaxis has been shown to be of value in the prevention of acute illness in children who have had recurrent aom [31] . more than 10 studies in which a penicillin, a sulfonamide, or erythromycinwas used haveidentifiedprotective efficacy against new episodes of aom in 60%-90% of cases in comparisons with a placebo control group. the changing susceptibilitypatterns of bacterial pathogens associatedwith aomwarrant considerationof new and effectivedrugs with activityagainstall major pathogens. newdrugs should have advantages over currently available agents, including (1) ease of administration to ensure compliance and greater conveniencefor the patient (e.g., once-a-day dosing, drug stabilityat room temperature, prolongeddrug shelf-life); (2) reduced incidence of relapse and recurrence; and (3) reduced duration of mee after resolution of acute signs and symptoms. newdiagnostic instrumentswith improvedcapacity for examinationof the middle ear (of most importance is diagnosis of the presence of fluid in the middle ear) also are needed. even the most experienced otoscopists are accurate in diagnosing the presence of mee in only rv80% of cases. tympanometryand acoustic reflectometry are of value in assisting the otoscopist but are insufficiently sensitive and specific to assure accuracy of diagnosis for all children enrolled in clinical trials. a noninvasive technique for determining the organisms present in mee is needed for the facilitation of appropriate microbiologic diagnosis and optimal use of approved drugs. currently, only needle aspiration of the fluid from both middle ears assures definition of the etiologicagentsof aom. patients eligible for inclusion in studies will be children or adults with symptomsand signs clinicallycompatiblewith aom. (1) clinical criteria. aom is defined as inflammation of the middle ear evidencedby the presence of fluid and accompanied by specific signs or symptoms such as ear pain, ear drainage, hearing loss, or nonspecific findings such as fever, lethargy, irritability, anorexia, vomiting, or diarrhea. thepresence ofmee is definedby pneumatic otoscopy with or without use of tympanometry or acoustic reflectometry. (2) microbiologic criteria. specific microbiologic diagnoses of aom can be determinedonly by aspirationof mee. both ears should be aspirated when the patient has bilateral aom. tympanocentesis is a standard procedure and is described in various texts on otolaryngology [32] . the procedure should be performed only by qualified personnel with previous experience. nose and throat cultures are of no value in the microbiologic diagnosis of aom since they are neither sensitive nor specific for predicting bacteria present in mee. specimens for such cultures may be obtained from selected patients for monitoring change in susceptibility patterns of nasopharyngeal or oropharyngeal isolates during the course of antimicrobial therapy. the drug under consideration should have proven in vitro activity against s. pneumoniae, h. injluenzae, and m. catarrhalis. group a streptococci, s. aureus, gram-negative enteric bacilli, and anaerobic bacteria are infrequent causes of adm and need not be considered in initial therapeutic decisions. in vivo evidence of sterilization of bacterial pathogens should be obtained with use of an appropriate dosage schedule in an animal model of adm. the chinchilla has been used most frequently in assessments of pathogenesis and therapy and should be considered for such in vivo studies. the investigator or the institution should have access to a clinical microbiologic laboratory where personnel can perform the following tests: culture of mee for the isolation and identification of common pathogens in adm and in vitro susceptibility testing, including tests for ,b-iactamase production. the institution should have appropriate facilities and investigators experienced in middle-ear examination and aspiration of mee. clinical studies should be conducted with patients of different age groups and racial backgrounds. in newborns and infants up to 6 weeks of age, the bacterial pathogens in adm differ from those in older children and include organisms acquired during delivery. in addition, pharmacologic considerations are different for older infants and children. the incidence of adm is highest between the ages of 6 and 24 months. the risk for adm is significantly increased in males, native americans, and canadian and alaskan eskimos, and the risk may be lower for black americans than for white americans. children, adolescents, and adults of both sexes should be included in studies. phase 1 evaluations may include singledose administration before tympanocentesis to assess the penetration of drug into middle-ear fluids. initial clinical studies should not include children with focal anatomic, physiologic, or systemic immune defects; children who had received a systemic antimicrobial agent within the past 7 days for treatment of an illness other than adm; and neonates or infants <12 weeks of age. the control agent should be selected on the basis of expected patterns of in vitro susceptibility of the most common pathogens (s. pneumoniae, h. injluenzae, and m. catarrhalis) in the community. because of the difficulties in obtaining reliable cultural information in adm even under protocol conditions, it may be appropriate to adopt a sequential study strategy: (1) a small (r'-ji00 patients) phase 2 trial can be conducted in which mee aspiration and culture is performed for all patients to document the unique microbiology of the population to be studied. in vitro antimicrobial susceptibility testing should be performed for all mee isolates, and both clinical and presumed microbiologic outcome should be assessed (see definitions below). repeat aspiration ofmee is required only if there is evidence of clinical failure. in the phase 2 trial, an "open" uncontrolled study may be conducted. because the number of centers that perform tympanocenteses is presently limited and a second aspiration of mee cannot be recommended for children who are clinically cured or improved, the microbiologic response is correctly termed presumptive eradication. clinical and presumed microbiologic efficacyfor a minimum of 60 patients with documented adm, with 20 cases each due to the three major bacterial pathogens (s. pneumoniae, h. injiuenzae, and m. catarrhalis, respectively) should be sufficient to determine whether the drug is effective on an organism-specific basis. both organism-specific and disease-specific responses should be evaluated. for the purpose of organism-specific evaluation, a minimum of 20 isolates from~20 patients is required for evaluation. (2) if the preliminary assessment is favorable (i.e., a clinical and presumed microbiologic response rate of~80 %), a larger, comparative phase 3 trial with an active control should be conducted. a double-blind study design is desirable whenever feasible; in any event, the evaluator should be blinded. aspiration of mee for microbiologic diagnosis before treatment is desirable but not required, but aspirates from those patients who fail to respond clinically are required. all drugs will be provided to the patient by the investigator or his or her designee. for young children unable to swallow tablets or for those with a small body mass, the use of a suspension or other acceptable formulation is necessary for accurate dosing. it is not anticipated that addition of a new antimicrobial agent will be required. if there is clinical failure (see definition below) after 72 hours of therapy, tympanocentesis should be performed; modification of antimicrobial therapy will be based on the data obtained from culture and from susceptibility testing. both clinical and presumed microbiologic responses should be assessed. after enrollment, observations should be made 3-5 daysafter initiation of therapy and at least 2 and 4-6 weeks later. the precise period of posttreatment evaluation will vary according to knowledge of the anticipated duration of antiinfective activity subsequent to termination of administration of the test drugs. at each visit an interval medical history should be obtained and otoscopic examination, including tympanometry or acoustic reflectometry, should be performed to determine the status of the middle ear. during reexaminations, children should be assessed for other foci of infection and for adverse effects of the test drug. the treatment outcomes for the study and control groups should be compared according to the proportion of patients in the following outcome categories: (1) clinical cure with presumed microbiologic eradication; (2) clinical failure with microbiologic persistence; and (3) clinical relapse or recurrence. (1) definition of clinical response. clincal cureis defined as resolution of signs and symptoms (e.g., pain, fever, vomiting), exclusive of mee, within 72 hours in a child who remains well throughout the course of therapy and follow-up. clinical failure is defined as lack of resolution of signs and symptoms, exclusive of mee, within 72 hours of onset of therapy. relapse is defined as reappearance of signs and symptoms of adm after initial response during or within 4 days of conclusion of therapy. recurrence is defined as reappearance of signs and symptoms of adm~5 days after the conclusion of therapy. (2) definition ofmicrobiologic response. it is recognized that whereas the microbiologic response can be accurately assessedonly by repeat aspirationsof mee during or after com-pletion of antimicrobial therapy, repeat tympanocentesis in a patient who is clinically improving is generally not warranted. all patients for whom outcome is classified as clinical failure, relapse, or recurrence should undergo repeated aspiration of mee before their antimicrobial regimens are toms of acute sinusitis are often difficult to distinguish from those of the common cold or from allergic (vasomotor) rhinitis. the most common complaints are cough (80 %) and nasal discharge (75 %). parents often notice a malodorous breath among preschoolers (50% of cases) who have neither signs of pharyngitis nor poor dental hygiene [34, 35] . in adults, postnatal purulent discharge and facial pain over the affected sinus that worsens with movement or percussion are the cardinal symptoms [36, 37] . fever occurs in <50% of cases. hyposmia, jaw pain with mastication, nasal congestion, and a history of recent upper respiratory infection are other manifestations. in patients with nosocomial sinusitis secondary to prolonged nasotracheal intubation, the clinical features, except for unexplained fever, may be relatively silent. symptoms associated with chronic sinusitis are usually less intense but more protracted than those in acute sinusitis. fever is uncommon. fatigue, general malaise, and an ill-defined feeling of unwellness and irritability can be more prominent than local symptoms of nasal congestion, facial pain, or postnasal drip [38] . the precise microbial etiology of sinusitis can be determined only by direct aspiration of the sinus, since nasopharyngeal secretions are regularly contaminated by the indigenous flora and culture results correlate poorly with results for sinus aspirates [36, 39] . this difficulty may limit the ability to make a definitive assessment of the microbiologic response to anti-infective therapy. s. pneumoniae and unencapsulated h. influenzae are responsible for >50% of cases of acute sinusitis in adults, while m. catarrhalis in addition to s. pneumoniae and h. influenzae account for two-thirds of cases in children [34, 39, 40] (table 4 ). s. aureus is a common nasal contaminant and an infrequent cause of acute sinusitis. obligate anaerobes are uncommonly isolated in acute sinusitis. in contrast, the microbiology of chronic sinusitis is usually logic persistence, emergence of resistance, or superinfection and optional determinations of drug concentrations in mee for such patients; (3) repeated hematologic, hepatic, and renal function studies as appropriate; (4) monitoring of change in susceptibility patterns of bacterial isolates in nasoor oropharyngeal culture specimens for selected patients; and (5) recording of allergic or toxic reactions or important adverse effects, which will be grounds for terminating the use of either the study drug or the standard drug. patients should be followed up clinically and by otoscopy biweekly until mee has completely resolved. repeated aspiration of mee should be performed for patients with clinical relapse or recurrence. the time to resolution of mee should be recorded. laboratory studies to monitor resolution of infection and adverse reactions should be repeated according to the protocol. c. sinusitis sinusitis is a common disorder both in children and adults. approximately 0.5 % of upper respiratory infections in children are complicated by acute sinusitis, and 0.02 % of adults have chronic sinusitis. because of the location and rich vascular supply of the sinuses, these infections are potentially life-threatening in that intracranial suppurative complications may result, including epidural or subdural empyema, brain abscess, or cavernous sinus thrombosis. early diagnosis and effective antimicrobial therapy are critical for the prevention of such complications as well as chronic sequelae. the paranasal sinuses are lined with ciliated pseudo-columnar epithelium and are connected to each other through small tubular openings, the sinus ostia, which drain into various regions of the nasal cavity. the paranasal sinuses are generally considered to be sterile, although transient colonization by the resident upper respiratory flora does occur [33] . conditions that affect the patency of the sinus ostia, .the normal mucociliary function of the sinus epithelium, or immune defenses of the upper airways or events that facilitate direct introduction of microorganisms into the paranasal sinuses are the key predisposing factors to sinus infection [34] . such conditions include viral upper respiratory tract infections, respiratory allergies, alterations in mucus (e.g., cystic fibrosis), and selective deficiencies in immunoglobulins. dental extraction or periapical infections of the maxillary molar teeth are a particularly important cause of maxillary and chronic sinusitis. the clinical manifestations of sinusitis vary greatly depending on the duration of infection (i.e., acute or chronic) and the age of the patient (i.e., child or adult). in children, symp[41, 42] . viridans streptococci and nonencapsulated h. infiuenzae are the major aerobic isolates. nosocomially acquired sinusitis secondary to head trauma or prolonged nasotrachea1 intubation is commonly causedbypolymicrobial gram-negative bacilli and s. aureus as well as by anaerobes [43] . fungal sinusitis is rare, but aspergillus, mucor, candida, pseudoallescheria boydii (scedosporium spiospermum) and other saprophytic fungi can cause invasive diseaseusually in the debilitated host. although antecedent viral upper respiratory infection is an important cause of acute sinusitis, viruses (e.g., rhinovirus, influenza, parainfluenza, and adenovirus) are isolated only in 15%of antralaspirates [39] . the clinical entities included in this guideline are acute sinusitis (symptoms present for~4 weeks) and chronic sinusitis (symptoms present for~3 months). not included in this guideline are subacute cases (symptoms lasting 1-3 months), whichhave a variable naturalhistory and in which the bacterial etiology is poorly defined. the goals of antimicrobial therapy for acute sinusitis are (1) the eradication of the causative pathogens; (2) the provision of symptomatic relief; (3) the restoration and improvementof sinusfunction; and (4) the prevention of intracranial complications and chronic sequelae. although many management options are available, antimicrobial agents are the mainstay of therapy for acute sinusitis. the therapeutic efficacy of anti-infective agents for acute sinusitis hasbeenestablished by placebo-controlled clinical trials [44] and in studies that employed sinus aspiration before and after treatment [39, 45] . standardtherapy is usuallyselected on anempiricbasisanddirected against themost likely pathogens, including h. infiuenzae, s. pneumoniae, and m. catarrhalis (see table 4 ). oral therapy with a j3-lactam agent suchas ampicillin for 10-14 days is generally prescribed and is considered the standardregimenfor acute sinusitis in bothchildren and adults. a favorable rate ofclinical response of70%-80% canbe expected withthisregimen. in penicillinallergic patients, a second-generation cephalosporin (e.g., cefaclor), a macrolide/sulfonamide (e.g., erythromycin/sulfisoxazole) or trimethoprim/sulfonamide (such as trimethoprimsulfamethoxazole) combinations have yielded comparable results. penicillins (such as amoxicillin plus a j3-lactamase inhibitor) or cephalosporins thathavea more-extended spectrum have not yielded superior results in controlled trials [44] [45] [46] even though theprevalence ofj3-lactamase-producing strains among respiratory pathogens appears to be increasing(upto 20% ofh. influenzae strains, 50%-70% ofm. catarrhalis strains,and20%-30% of respiratoryanaerobes) [47] . in 'patients with chronic sinusitis, surgical procedures to facilitate sinus drainage through the creationof an artificial ostium and submucosal resection of diseased tissue appear to be the mainstays of treatment. the role of anti-infective agents in chronic sinusitis is not as clear as that in acute sinusitis. conservative therapy with anti-infective agents or sinusirrigationwithout surgical intervention is successful in onlyone-thirdof cases [36, 48] . withcombined medicaland surgical treatment, the curerate forchronicmaxillary sinusitis is >60% after 3 years of follow-up [48] . anti-infective agents useful forchronic sinusitis should have broad-spectrum activity against respiratory anaerobes as well as against viridansstreptococci, s. pneumoniae, h. influenzae, and m. catarrhalis. several issuesin the management of acute and chronic sinusitis remain controversial. these include: (1) the optimal duration of therapy for acute and chronic sinusitis; (2) the clinicalrelevance of the increasing prevalence of in vitro resistanceto j3-lactam agents among upper respiratorypathogens; (3)the roleofrespiratory allergy in recurrent or chronic sinusitis; (4) the value of adjunctive measures such as oral or topical decongestants, antihistamines, and intranasal steroids in the treatment of acuteand chronic sinusitis (such measures must be standardized in both study and control groups during initialassessment of new antibiotic regimens for both acute and chronic sinusitis); (5) the optimal mode of surgical management in chronic sinusitis (i.e., preservation of sinus epithelium vs. radical mucosal resection); (6) avoidance of the need for sinus puncture by the use of endoscopic sinoscopy for performing quantitative cultures. patients eligible for study will be children or adults with symptoms and signs clinically compatible with acute or chronic sinusitis. (i) clinical criteria. acute sinusitis is defined as inflammation of the sinuses associated with symptoms lasting~4 weeks. clinical findings suchas fever, headache, malartenderness, andnasal discharge (which are often nonspecific) should be supported by objective localizing studies such as radiography, ultrasonography, or ct. transillumination of the s75 sinuseshas a relatively low sensitively (74%) and specificity (47%) for acute sinusitis [39] and should not be used as the solediagnostic criterion. transillumination is alsoless informativein children<6 years of age (40% concordanceand 20% discordance compared with radiographic findings) because of either poor cooperationof the child in performingthe test or thedevelopmental variations of the sinuses in this agegroup [34] . anterior rhinoscopy mayrevealhyperemicand edematous nasal turbinates, often with purulent dischargefrom the middle meatus where the orifices of the maxillary, frontal, andanteriorethmoidal sinusesenterthe intranasal cavity [49] . imaging studies (roentgenography, ultrasonography, or ct) should be performed in all cases. other laboratory studies suchas neutrophil count, erythrocyte sedimentation rate, and c-reactive protein should also be performed. chronic sinusitis is definedas inflammation of the sinuses associated with symptoms lasting >3 months that are compatiblewithradiographic abnormalities (determined byroentgenography, ultrasonography, or ct). if possible, chronic sinusitis should be confirmedby endoscopic sinoscopy with direct visualization of the sinusmucosa, appropriatemicrobiologic sampling, and histopathologic evaluation [49, 50] . (2) microbiologic criteria. the precise microbial etiology of sinusitis can be determined only by direct aspiration or injection wash of the sinus cavity. cultures of the surface of the nasal vestibule or the nasopharynx are unreliable becauseof their regularcontamination bythe residentmicroflora and should not be used for assessment of microbiologic efficacy of study regimens. access to the maxillary sinus can be obtainedintranasally througha puncturebelow the inferior turbinate and to the frontal sinus through a puncture below the infraorbital rim oftheeye. thorough cleansing of thepuncture site with an appropriate antiseptic is important to minimize contamination of the specimen with surface bacteria. if no fluid is obtained, 1 ml of sterile normal saline without bactericidalpreservativeshouldbe instilledand the washings reaspirated. specimens should be sent to the laboratory for leukocytecounting, gram staining, and culture for aerobes, anaerobes, fungi, and mycobacteria. viral cultures are of investigational interest. withthe appropriate technique, >76% of such specimenswill yield positivecultures in acute maxillary sinusitis [40] . furthermore, if organisms are seen on gram-stained preparations of antral secretions, a presumptive diagnosis can be made by assessing the bacterial morphotype in up to 90% of cases [51] . quantitative cultures (~103 cfu/ml of aspirate) are usefulin distinguishing true infection from colonization or contamination [39, 52] , but such studies are labor-intensive and are not required for microbiologic diagnosis in clinical trials. in chronic sinusitis, microbiologic diagnosis can be confirmedby cultureof diseasedmucosaobtainedby biopsyduring endoscopicsinoscopy or surgery. in such cases, the culture results should be correlated with the histopathologic findings to exclude the possibility of specimen contamination. for a pathogen-specific evaluation, the drug under consideration should haveprovenin vitro activity against the specific bacteriaprevalent in sinusitis, and for a disease-specific evaluation (i.e., acute vs. chronic, pediatric vs. adult), the drug should havea broad range of activity against the most prevalent pathogens. the investigator or subinvestigator should have the necessary skillsto perform sinuspuncturefor microbiologic evaluationsof acuteandchronicsinusitis and endoscopic sinoscopy for studies of chronic sinusitis. the institution should have the facilities and personnel with expertise to perform and interpret radiographs, ultrasonography, or ct and microbiologic studies of the paranasal sinuses. clinical evaluation of new treatment regimens should be conducted with patients grouped by specified age, underlying disease, duration of symptoms, and presence or absence of respiratory allergy. since these factors appear important both in predictingthe microbial etiologyand in overallprognosis,their contribution to treatmentoutcomeshouldbe carefully controlledby appropriate randomizationduring patient enrollment or by stratification eitherprospectively or posthoc during analysis of results. children, adolescents, and adults of both sexesare eligible for inclusion. patientswho havereceivedother antimicrobial therapy within the preceding 2 weeks, patients with hypersensitivity reactions to drugs of a similar class, and patients with other concurrent, acute infectious illnesses should be excluded. in acute sinusitis, an active control regimen with proven efficacy againsts. pneumoniae, h. injluenzae, and m. catarrhalis shouldbe used. in chronicsinusitis, a placebo-controlled trial is consideredjustified since the role of antimicrobial therapy for this condition remains unclear at this time. because of the difficulties in obtaining reliable cultural information about sinusitis even under protocol conditions, it may be appropriate to adopt the following sequential study strategy. (1) conduct a small (rvloo patients) phase 2 trial in which sinus puncture and culture is performed for all patients to document the unique microbiology of the intended study population, with at least 20 cases of each of three major bacterial pathogens implicated (s. pneumoniae, h. irfiuenzae, m. catarrhalis). in vitro antimicrobial susceptibility testing of all sinus isolates should be performed. both clinical and presumed microbiologic outcomes are assessed (see definitions below). repeated aspiration of the sinus is required only if there is evidence of clinical failure. in the phase 2 trial, an "open" uncontrolled study may be conducted, although a randomized comparative double-blind trial with an active control is still desirable despite the clearly inadequate size of the sample for meaningful comparisons of clinical response rates. a conrolled comparison provides additional information regardmg the expected response rate in a particular community. both organism-specific and disease-specific responses should be evaluated. for purposes of organism-specific evaluation a minimum of 20 isolates from~20 patients is required for evaluation. (2) if the preliminary assessment is favorable (i.e., a clinial and presumed microbiologic response rate of~70 %), it is reasonable to conduct a larger, comparative phase 3 trial with at1 active control. sinus puncture for microbiologic diagnosis and sinus radiography before treatment are desirable but not required, but examination of aspirates and sinus radiographs is necessary for those patients who fail to respond clinically. in vitro antimicrobial susceptibility testing should be performed for all isolates from cultures. use of adjunctive medications such as oral or nasal decongestants, antihistamines, or intranasal steroids should be standardized such that hey~re used either in both the study and control groups or in neither of the groups. similarly, in studies of chronic sinusitis, the mode of concomitant surgical therapy (i.e., endoscopic sinuscopy with limited mucosal curettage vs. a more conventional approach of radial mucosal resections) should also be standardized or stratified. the projected sample size must include consideration of the expected difference in efficacy of the study and control regimens, the expected proportion of cases due to each of the major bacterial pathogens (and that one-fourth of all cases of acute sinusitis are due to nonbacterial causes that would not be affected by either antibacterial agent), and an anticipated rate of spontaneous clinical cure of rv30 %among children with acute sinusitis [34] . the treatment course is usually 10-14days for acute sinus-itis. si.nce the opt~mal duration of therapy has not been clearly estabhshed for either acute or chronic sinusitis, this could be the mainfocus of evaluationin phase 4 trials. patients should be assigned randomly to the test or "control" group, and if p~e~reatment cultures of the sinuses are not performed, the chmcal and presumed microbiologic response should be evaluated by a blinded observer. for children unable to swallow tablets or whose body mass is small, either a suspension or an acceptable alternative formulation of the study drug or the control drug is necessary for precise dosing. modification of the study by the addition of a new antimicrobial agent may be necessary if the clinical response after 3-5 days of therapy is suboptimal. in such instances sinus aspiration for documentation of the microbiologic response is required before the therapeutic regimen is modified. addition of a new antimicrobial agent constitutes a clinical failure of the initial treatment regimen. both clinical and presumed microbiologic responses should be~s~:ss.ed. clinical evaluation should be made 3-5 days after imuation of therapy and weekly or biweekly thereafter until the resolution of all symptoms and signs. use of a scoring system, pa:ticularly a binomial (yes/no) objective scoring system, for signs and symptoms such as fever, pain, headache, tenderness, nasal discharge, and purulence is strongly encouraged. imaging studies (roentgenography, ultrasonography, or ct) should be repeated at least at the completion of antimicrobial therapy. patients with chronic sinusitis should be further assessed by repeated endoscopic sinoscopy before or after completion of therapy. information about concentrations ?f dru~in sinus aspirates or mucosal biopsies may be of value iii studies of chronic sinusitis, but they are not critical to studies of efficacyin acute sinusitis and are not required for final evaluation. since a repeat of sinus puncture is generally not justified in patients who have responded clinically to therapy, the microbiologic response in such patients can only be judged presumptively. comparisons of treatment outcomes in the study and control groups should be made according to the proportion of patients in the following outcome categories: (1) clinical cure with presumed microbiologic eradication; (2) clinical failure with microbiologic persistence; (3) clinical and/or microbiologic relapse and recurrence; and (4) indeterminate. (1) definition ofclinical response. clinical cure is defined as complete resolution of signs and symptoms at the conclu-sio~of antimicrobial therapy and at follow-up. clinical failure is defined as lack of improvement in signs and symptoms within a defined period of therapy (72 hours for acute sinusi-sn tis and 2 weeksfor chronic sinusitis). earlyrelapse is defined as reappearane of signsand symptoms or newclinicalfindings of sinusitis within 14daysafter the conclusionof therapy. late relapse is definedas the reapearrance of signs and symptoms or new clinical findings of sinus infection after 14 days but within 1 month after the conclusion of therapy. (2) definition of microbiologic reaponse. presumed microbiologic eradication is definedas cases in whichpretreatment cultures of sinus aspirates were positive, clinical signs and symptoms resolvecompletely, and posttreatmentculturesare not performed because clinical response is complete. confirmed microbiologic response is defined as cases in which the causativeorganismcannot be isolated in cultures of sinus aspirates performed after 72 hours of antimicrobial therapy. such repeated cultures of sinus aspirates are likely to be performed only in the setting of clinical failure. a statement as to microbiologic eradication is not possible because of the influence ofconcomitantantimicrobial therapy. microbiologic persistence is defined as a positive culture of sinus aspirates after at least 72 hours of antimicrobial therapy. if a pretreatment culture of sinus aspirate was performed and was positive, the isolation of the same organism after~72 hours of therapy is considered confirmed microbiologic persistence. if no pretreatment culture of sinus aspirates was performed, isolation of a pathogen after~72 hours of treatment is considered presumptive microbiologic persistence. superinfectionis defined as the emergenceof new or resistant organisms in cultures of sinus aspirates after~72 hours of antimicrobial therapy. (1) initial clinical evaluation and imaging (roentgenography, ultrasonography, or ct) of the paranasal sinuses; (2) hematologic, hepatic, and renal function studies; (3) sinus puncture and microbiologicstudies for phase 2 trials and optionallyfor phase 3 trials; and (4) endoscopic sinoscopy, bacterial cultures,andoptionaltissuebiopsyfor studiesof chronic sinusitis. (b) assessment during the course of therapy should include: (1) clinical evaluation at 2-3 and 5-7 days after initiation of antimicrobial therapy, and weekly or biweekly thereafter until resolution of all symptoms and signs; (2) aspiration of sinuses for microbiologic studies for patients who fail to respond clinically after at least 72 hours of antimicrobial therapy to define microbiologic persistence, emergence of resistance, or superinfection; and (3) repeated imaging, hematologic. hepatic, and renal function studies as appropriate. patients should be followed up clinically and with imaging for at least 2 weeksafter completionof antimicrobial therapy to assess relapse or recurrence, clinical complications, and adverseeffects of the antimicrobialregimen. sinus aspiration should be performed for those patients with clinical relapse or recurrence. bronchitis is an inflammatory conditionof the tracheobronchial tree. it is both acute and chronic and is caused by a variety of irritants and infectiousagents. productive cough is the commondenominator of this condition, and the sputum produced ranges from mucoid to frankly purulent. acute bronchitis is generally an infectious process. it occurs in all age groups and is most common in the winter months, when acuterespiratory infections are prevalent. most cases are thought to be due to respiratory viruses, including those associatedwith the common cold and other respiratory viruses involved in infections of the lower respiratory tract (e.g., adenovirus, rhinovirus, coronavirus, influenza, parainfluenza, respiratory syncytial virus, coxsackievirus). m. pneumoniae, c. pneumoniae, and legionella specieshave also been implicated in some cases. the frequency of infection due to these pathogens is not certain. chronic bronchitis generally is defined as a condition characterized by cough and excessive secretion of mucus in patients who have coughed up sputum on most days during 3 consecutive months for >2 successive years. this disease is caused by prolonged exposure to pulmonary irritants, the mostprominentof whichis cigarette smoke.atmospheric pollution also playssome role, as do recurrent episodes of infection. chronic bronchitis results in widely ranging degrees of respiratoryembarrassment. in its most severe forms, obstructive pulmonary disease, emphysema, and respiratory failure occur. patientswith chronic bronchitis frequently experienceepisodes of acute disease superimposedon the chronic process. these exacerbationsare characterized by some combination of increasing cough, sputum volume and purulence, and respiratory distress. the role of infectionin these episodes has been difficult to define. the bacterial speciesmost oftenmentioned as potential etiologic pathogens include s. pneumoniae, typable(especially typeb)and nontypable h. irfiuenzae, and m. catarrhalis. however, the same organisms, particularly haemophilus species, can be isolated from the respiratory secretionsof patientswith chronic bronchitiswho do not present with evidence of acute exacerbation [53] . gump et al. did report an association between purulence of sputum and an increase in the number of pneumococciin the sputum em 1992;15 (suppl 1) of patients with acute exacerbations [54] . haemophilus parainfluenzae, viridans streptococci, and strains of enterobacteriaceae also are isolated from patients with acute exacerbations of bronchitis, but their pathogenic role is even less welldefined. viruses, m. pneumoniae, c. pneumoniae, andperhapslegionella speciesplayan etiologic role in some cases of acute exacerbations. the only clinical entity included in this guidelineis acute exacerbation of chronic bronchitis. considerable controversy surrounds the use of antibacterial agents for patientswith acute exacerbations of chronic bronchitis [55, 56] . tager and speizer [57] reviewed the existing studies in 1975 and concluded that the role of antimicrobial agentsin the management of these patients needed reassessment and that respiratory infections appeared to contribute to worsening of episodesof coughand productionof sputum. a recent double-blind randomizedplacebo-controlled study by anthonisen et al. [58] showed a significant clinicalbenefit in association withantibacterial therapy. the recovery of peak air flow wasmore rapid and the rate of clinicaldeteriorations requiring therapeutic intervention was lower in antibiotictreatedpatients. response to treatment wasevidenced by the trilogy of decreased dyspnea, sputum volume, and sputum purulence. treatment success wasdefined as resolution within 21 days of all symptoms that accompanied the exacerbation. no attempt at microbiologic confirmation wasperformed in this study. antibacterial agentsutilizedin thetreatmentgroup included trimethoprim-sulfamethoxazole, amoxicillin, and doxycycline. althoughcontroversy aboutpossiblemicrobialpathogenesis persists, most clinicianselect to treat the acute exacerbations as infectious events and direct that therapy at s. pneumoniae andh. influenzae and, morerecently, at m. catarrhalis. the duration of therapy is generally 7-10 days. it should be recognized that up to 25%of strains of h. influenzae and 50%-70% of m. catarrhalis strains produce (3-lactamase. the etiologicrole of viruses, m. pneumoniae, c. pneumoniae, and legionella in acute exacerbations of chronic bronchitis needs clarification. determination of their role will be facilitated by the application of more sensitive and specific microbiologic diagnostic techniques (e.g., nucleic acidprobes, polymerase chain reactions, antigen detection). patients eligible for study will primarily be adults with symptoms and signscompatible with acute exacerbations of chronic bronchitis. (1) clinical criteria. patients must (a) havehad a chronic cough and sputumproduction for >2 consecutive years and on mostdaysfor 3 consecutive monthsand (b) haveevidence of acute exacerbation as indicated by some combination of increasedcough and/or dyspnea, increased sputumvolume, or increased sputum purulence. (2) microbiologic and other laboratory criteria. these criteria include (a) negative chest roentgenogram to rule out pneumonia; (b) productionof purulent sputumas defined by the presence on a gram-stainedpreparation of >25 polymorphonuclear leukocytes and <10 squamous epithelial cells per low-power magnification (x 10) field(thepresenceofpredominant bacterialmorphology maybe noted); (c) documentation of the presence or absence of potential bacterial pathogens and monitoringof emergenceof resistant isolates during antimicrobial therapyby sputumcultureand susceptibility tests. the drug under consideration should have provenin vitro activityagainsts. pneumoniae, h. influenzae, and m. catarrhalis. dosage ofthe studydrugmustbe determined by means of pharmacokinetic and in vitro studies. the investigator or subinvestigator shouldhavethe necessary skills to assesspulmonary functionand interpret radiographicstudies. the institution shouldhave adequate facilities for performance of laboratorystudies, including hematologic, hepatic, andrenalfunction testsandstudies ofpulmonary function, especially arterial blood gas analysis, forced vital capacity, fev! (forcedexpiratory volumein 1 sec), total lung capacity, and peak flow spirometry. 5 . design and implementation of phase 1, 2, and 3 clinical trials onlyadultpatients(~18 years)with stablechronicpulmonary disease should be included. patients who are experiencing an acute exacerbation of chronic bronchitis are eligible. patients with cystic fibrosis, patients unable to give informed consent, and patients with a known history of hypersensitivity to the study or control drug should be excluded. steroid use is not necessarily a criterion for exclusion. even though the use of antibacterial agents for treatment of acute exacerbations of chronic bronchitis is controversial, the presence of s. pneumoniaeand other potential pathogens in some patients and the concomitant need for corticosteroids in some patients suggest the need for an active control drug. both control and study drugs should be active in vitro against s. pneumoniae, h. influenzae type b, and m. catarrhalis. placebo-controlled trials may be conducted. in phase 1 studies, human pharmacologic and pharmacokinetic studies should demonstrate sufficient absorption and achievement of peak serum concentrations that exceed the mic90 for the major respiratory pathogens. in phase 2 and 3 trials, study patients should be stratified according to major host factors (e.g., history and duration of smoking). whenever feasible, studies should be prospective, randomized, and of double-blind design. no additional antimicrobial agent is permitted. concurrent medications (e.g., bronchodilators) should be administered in the same manner to study and control groups. the study patients may be stratified according to the use of concomitant steroid therapy. another strategy might be to design a four-arm randomized comparison: (1) study drug with steroids; (2) control drug with steroids; (3) study drug with no steroids; and (4) control drug with no steroids. in projecting a sample size, consideration must be given to the expected difference in efficacy of the study and control regimens and the desirability of undertaking poststudy subset analysis of pertinent patient variables. variables include (1) the presence or absence of adjunctive treatment; (2) the presence or absence of fever; (3) status of pulmonary function; and (4) characteristics of sputum. duration oftreatment is generally 7-10 days. however, the optimal duration of therapy could be a main focus of evaluation. in comparative studies, patients should be assigned randomly to the test or "control" group, and insofar as possible, the study should be blinded. for patients who do not demonstrate clinical improvement (i.e., decreased dyspnea, cough, and volume and purulence of sputum production) or whose clinical conditions worsen after 3-5 days of treatment, clinical failure will be declared and such patients will be removed from the study. the addition of an antimicrobial agent that is not a study drug will also result in a designation of clinical failure. clinical response and results of pulmonary function tests and/or arterial blood gas analyses can be used to assess efficacy. the effect of treatment on sputum microbiology will be monitored. failure to eradicate a potential pathogen in a patient with a complete clinical response is common in this disease. the bronchial secretions of many patients remain "colonized" after the acute episode resolves. all patients entered into the study should be assessed on the basis of intent to treat. the clinical response in both the study and control group will be classified as (1) clinical cure, (2) clinical improvement (which requires measurement of an objective end point, e.g., volume and/or purulence of sputum), (3) clinical failure, and (4) indeterminate. patients will be evaluated at 3-5 days after initiation of treatment, and weekly thereafter. (1) definitions ofclinical response. clinical cureis the resolution of acute symptoms and signs to a baseline level of dyspnea, cough, sputum production, and, if elevated at enrollment, resolution of fever. clinical improvement is the subjective improvement in dyspnea, with reduction in cough, a quantified reduction in 24-hour volume or purulence of sputum, and a return of the temperature to normal if the patient is initially febrile. clinicalfailure is the lack of any resolution in the magnitude of the dyspnea, sputum purulence, or fever (if present) that prompted enrollment of the patient in the study. clinical response indeterminate should be substantitated by stated reasons. the clinical response definition may be supported by improvement or lack of improvement in sequential measurements of the patient's white blood cell count, oxygen saturation, and/or pulmonary function tests. (2) definitions ofmicrobiologic response. the categories of microbiologic response commonly encountered include eradication, persistence, relapse, reinfection, and superinfection consult general guidelines, section xiii.c, for detailed definitions. (1) initial history and physical examination should be performed just before enrollment. (2) chest radiography should be performed to rule out pneumonia. (3) hematologic, hepatic, renal, pulmonary function, and arterial blood gas studies (with room air) should be performed. (4) gram stain and ern 1992;15 (suppl 1) culture of sputum plus determintion of 24-hour sputum volume should be performed; nucleic acid probes and culture for mycoplasma and legionella may be included. (1) at a minimum, patients should undergo clinical evaluation 3-5 days after initiation oftherapy and weekly thereafter until completion of therapy. (2) for febrile patients the body temperature should be determined a minimum of four times daily. (3) quantitation of the volume of sputum produced daily and/or daily assessments of the degree of sputum purulence may assist in assessment of the patient's clinical response. (4) it is helpful to monitor patient's arterial blood gases and/or expiratory flow rates at periodic intervals. the precise frequency depends on the individual protocol (e.g., every 3-5 days for hospitalized patients and perhaps once during therapy for outpatients). (5) repeated chest radiographic, hematologic, hepatic, and renal studies are appropriate at 3-5 days after treatment has begun and within 48 hours after the end of treatment. (6) a sputum culture during therapy is indicated if there is evidence of clinical failure. in individual patients, such cultural data may be useful in identifying the emergence of bacterial resistance or in documenting failure to eradicate a potential bacterial pathogen (e.g., s. pneumoniae). patients should undergo clinical and microbiologic assessment within 48 hours, 7-14 days, and 21-28 days after completion of therapy. the clinical assessment should include assessment of cough, dyspnea, sputum volume, and sputum purulence. oximetry to determine oxygen saturation and spirometry should be performed. a chest radiograph is not required unless clinically indicated, since the presence of a pulmonary infiltrate precludes enrollment. sputum should be submitted for gram staining, culture, and sensitivity testing, or-in the case of mycoplasma or legionella infections -for nucleic acid probe tests. lower respiratory tract infections include bronchitis, bronchiolitis, and pneumonia and its complications. the relative frequency of isolation of various etiologic agents that cause community-acquired pneumonia differ according to age group, socioeconomic status, underlying disease, time of year, and possible concomitant viral illnesses. prospective studies of the causes of community-acquired pneumonia are often difficultto interpret because of imprecise methods of microbio-logic diagnosis, such as reliance on sputum culture and/or serologic testing. however, it is generally accepted that in north america viral agents (e.g., respiratory syncytial virus and parainfluenza virus type 3) are most important for children <5 years of age. the inability to obtain sputum from infants and children is a major deterrent to microbiologic diagnosis of pneumonia in this population. m. pneumoniae is considered to be a major cause of community-acquired pneumonias in north americans 5-25 years of age. in older individuals, mycoplasmas and viruses are less common causes, while bacterial agents are more prevalent. a majority (50%-90%) of cases of pyogenic pneumonia with acute onset in middle-aged or older adults are due to s. pneumoniae [59, 60] . pneumonias due to h. injtuenzae (either ampicillin-susceptible or ampicillin-resistant), s. aureus, mixed aerobicanaerobic bacteria, and aerobic facultative gram-negative bacilli such as klebsiella pneumoniae, in rank order, are less common. legionella species, (determined primarily on the basis of serologic studies) account for a variable proportion of cases of community-acquired pneumonia in adults (e.g., in 1% of patients not requiring hospitalization and in 5 %-20 % of those hospitalized). legionella species probably account for 10%-15 % of cases of so-called atypical pneumonia [61, 62] . other agents that cause nonpyogenic, or atypical, pneumonia include m. pneumoniae, c. bumetii, c. pneumoniae, and, rarely, chlamydia psittaci. in classic pneumonias, the isolation of certain pathogens can often be linked to certain specific conditions of the host (e.g., infection with group a ,6-hemolytic s. pyogenes, s. aureus, h. irfiuenzae, or s. pneumoniae following influenza). both typable and nontypable strains of h. influenzae are pathogenic primarily among smokers, patients with chronic obstructive pulmonary disease (copd), and some patients with lymphoma or other malignancies. aspiration pneumonia in the community is believed to involve mostly the normal oropharyngeal aerobic and anaerobic flora. in the nursing home or nosocomial setting, infections with aerobic gramnegative bacilli and s. aureus are additional considerations in aspiration pneumonia. data for nosocomial pneumonias prior to 1988 from the centers for disease control (cdc) may not be completely reliable because they appear to be based primarily on results of sputum cultures and cultures of endotracheal suction specimens. nonetheless, the rank order of pathogens in the last reported cdc survey of nosocomial infections is pseudomonas aeruginosa (16.9%), s. aureus (12.9%), klebsiella species (11.6%), and enterobacter species (9.4%), followed by escherichia coli, serratia marcescens, and proteus species [63, 64] . data based on the results of transtracheal aspiration performed on members of a high-risk population of elderly men in a veterans administration hospital and nursing home in the 1970s give a different perspective on nosocomial pneumonia. bartlettet al. [65] relied only on isolates from blood cultures, pleural fluid, andtranstracheal aspirates. they found gram-negative bacilliin about one-halfof 159 patients studied, anaerobes (peptostreptococcus species were the most common isolates) in about one-third, and s. pneumoniae in about one-fourth. klebsiella species were the most commonly isolated gram-negative aerobic bacilli. the isolates were polymicrobial in about one-half of the patients. gram-negative bacilli are morelikely to be involved in nosocomial pneumonia in high-risk populations, such as those in intensive care units, than in other patients. outbreaks of nosocomial pneumonia dueto someorganisms, including aerobic gram-negative bacilli and organisms not usually appreciated as nosocomial pathogens, may present particular problems. thelattergroupincludes s. pneumoniae, ampicillin-resistant h. infiuenzae, and m. catarrhalis [66] [67] [68] . the timely use of appropriate systemic antibacterial therapyshould eradicatethe pathogen in a largenumberof cases of pneumonia and lead to a reduction in morbidity as well as mortality. efficacy ofnewagents should at leastequalthose of established regimens when evaluated in prospective, randomized, controlled trials (active treatmentconcurrentcontrol) [69, 70] . if l3-lactamase-producing pathogens are suspected (e.g., h. influenzae), both the study and control drugs should have in vitro activity against such pathogens. theefficacy ratesfora newdrug for etiologic agents andclinicalsyndromes in which thereis noestablished therapy should at least equal those in recent historical controls. data from open studies may be useful in these instances. dataobtained frompartsoftheworldotherthanthe united states may be considered supporting evidence of efficacy. however, possible regional differences in resistance patterns mustbe notedandmay preclude directcomparison (e.g., appreciably higher resistance to penicillin g among s. pneumoniae strainsisolated in south africa and to erythromycin in spain than in north american isolates). the local antimicrobial susceptibility patterns will clearlybe the predominant influence on the choice of concurrentactive treatment control regimens. (1) clinical entities to be included are common communityacquired or nosocomial bacterial pneumonias. clinical entities not included are bronchitis, bronchiolitis, lowerrespiratory tract infections in patients withcystic fibrosis, lowerrespiratory tract infections caused by infrequent and/or difficultto-diagnose entities (e.g., infections withanaerobic bacteria; psittacosis, qfever, tularemia, andplague; andinfections with mycobacteria, viruses, or fungi). (2) microorganisms included in the guideline are s. pneumoniae (prototype), h. injluenzae, s. aureus, facultative aerobic gram-negative bacilli, pseudomonas species, m. pneumoniae, and legionella species. the diagnosis of infectious pneumonia combines clinical, laboratory, andmicrobiologic data. a compatible clinical picture (fever, cough, and/orauscultatoryfindings such as rales and/or evidence of pulmonary consolidation) together with confirmatory chestradiographic findings and isolationof the causative pathogen(s) from suitable respiratory specimens (e.g., expectorated sputum, transtracheal aspirate,bronchial washings or lavage, pleuralfluid) or bloodestablishes the diagnosis of bacterialpneumonia. pneumonia due to m. pneumoniae is identified by culture or nucleic acid probe and/or by documentation of a fourfold or greater rise in titer of complement-fixing antibody. detection of cold agglutinins does not establishthe diagnosis. the diagnosis of legionella pneumonia requires isolation of the organism from sputum, a bronchoalveolar lavage specimen, pleural fluid, or blood. alternatively, legionella antigen may be detected by immunofluorescence in respiratory secretions or byradioimmunoassay in urine. also, legionella maybe detected in respiratorysecretions withnucleic acidprobes. testing forantibody in acute-and convalescent-phase sera, except for antibody to l. pneumophila serogroup 1, is not specific enough for reliablediagnosis oflegionellosis, especially in areas of lowdisease prevalence. diagnostic methods for detection of c. pneumoniae are under development. the bacterialpathogens isolated shouldbe tested for susceptibility to antimicrobial agents by standardized methods. determinations of mbcs, postantibiotic effect, or effect of subinhibitory concentrations of antibiotics are notdone routinely andare notgenerally required forassessment of efficacy. when mycoplasma or legionella is isolated, antimicrobial susceptibility testing is not done routinely. selection of empiricantimicrobial therapy is based on the suspected pathogens and their anticipated susceptibility in vitro. penicillin g remainsthe drug of choice for almostall s. pneumoniae infections in the unitedstates [71] . ampicillin or a cogeneris the drug of choice for pneumonia due to non-sdactamase-producing h. injluenzae. aspiration pneumonia acquired in the community is treated with penicillin g, usually without the benefit of culture results. a lincosamide or a combination of a penicillin and a 13-lactamase inhibitor are alternatives. a macrolide (e.g., erythromycin) or tetracycline is preferred forpneumonia dueto m. pneumoniae or c. pneumoniae, and erythromycin is the choice for legionella infections [61, 72] . a semisynthetic penicillinaseresistantpenicillin is the treatment of choicefor pneumonia dueto methicillin-sensitive s. aureus. a combination ofa suitable cephalosporin or penicillin and an aminoglycoside is frequently employed for infections due to facultative gramnegative rods or to pseudomonas. in most other instances of community-acquired pneumonia, combination therapy is usually not required. oral preparations of the aforementioned parenteral compounds or oral drugs with comp~able in vi.tro activity can be used in milder cases. the optimal duration of therapy varies, but uncomplicated s. pneumoniae pneumonia is usually treated for 7-10 days [71] . for treatment of nosocomial pneumonias (e.g., associatew ith ventilator use), combination therapy with an extendedspectrum penicillin or cephalosporin and an aminoglycoside is commonly employed. initial therapy must be directed at the suspected pathogens in a given hospital and their known susceptibility profile. determination of the concentration of antimicrobial agent(s) in serum, other bodily fluids, or tissues is not done routinely. most often, cure is defined by clinical criteria alone. with resolution of the inflammatory process, the patient is unable to provide secretions from the lower airway for documentation of eradication of the causative pathogen. patients requiring tracheostomy or endotrachĩ ntubation may have persistent, presumably tracheal, colomzation with an etiologic organism after the criteria for clinical cure of pneumonia are met. relief of endobronchial obstruction and/or drainage of empyema fluid remains a mainstay of therapy for lower respiratory tract infections. the probability of cure for s. pneumoniae pneumonia is variable and ranges from 95 % in uncomplicated infection to (\)50%-80% with bacteremic disease [71] . relapse is not a significant problem with s. pneumoniae. newer methods for more precise microbiologic diagnosis of pneumonia, such as the use of semiquantitative cultures of protected endoscopic brushings or bronchoalveolar lavage specimens, are promising.· the practice of changing parenteral therapy to therapy with an oral agent such as a fluoroquinolone after 5-7 days is gaining increasing acceptance, as is the use of intravenous antimicrobial therapy in the home for follow-up management. it is likely that the number and precision of diagnostic techniques that rely on antigen detection or nucleic acid detection will increase. patients eligible for study are adults and children of both sexes with confirmed or presumptive diagnosis of communityacquired or nosocomial pneumonia. these guidelines may be adapted to treatment of patients in either a hospitalized or ambulatory setting or for patients that progress from hospital to an outpatient setting. (1) clinical criteria. patients must have signs and symptoms consistent with bacterial pneumonia (chest pain, cough, and/or ausculatory findings such as rales and/or evidence of pulmonary consolidation) with or without fever (oral temperature >38°c [100.4of]) or leukocytosis (blood leukocyte count >10,000/mm 3 or >15% band forms), and there must be radiographic or other laboratory evidence that supports the diagnosis (see below). (2) microbiologic and otheretiologic (noncultural) criteria. specimens obtained by expectoration or by endotracheal aspiration should be screened microscopically for suitability of culture (presence of >25 polymorphonuclear leukocytes and <10 squamous epithelial cells/low-magnification field [x 10]). suitable specimens should be cultured aerobically in appropriate media. blood specimens should be cultured for all patients, and pleural fluid, if present, should be aspirated, examined by microscopy, and cultured for both aerobes and anaerobes. the microbiologic diagnosis of infectious pneumonia is confirmed by the following criteria: (a) purulent expectorated sputum-identification of a predominant suspected pathogen by culture and/or microscopy (e.g., with s. pneumoniae by finding an average of >10 lancetshaped diplococci/oil-immersion field [x 1,000] for 10 fields examined) (material from endotracheal suctioning may also be used, and slides should be saved and made available as part of the case record) or (b) transtracheal aspirate, bronchial brushings, or biopsy material (obtained under direct visualizationwith a fiberopticbronchoscope, preferablydoublesheathed) -gram stain reveals neutrophils and a predominant pathogen is suspected by smear or culture; quantitative cultures of endobronchial brushes from potentially infected ventilator-dependent patients may be of value; (c) pleuralfluid or direct lungaspirateidentification of a predominant pathogen on gram stain or by culture; (d) positiveblood cultureyields a pathogen in a patient with a compatible clinical syndrome of bacterial pneumonia in the absence of another source of bacteremia. ifan organism is isolated, it should be susceptible to both the study and the control drug. clinical improvement or stabilization must be documented by 72 hours to permit retention in the study. (e) surrogate markersdetection of antigen or specific nucleic acid by non-culture methods may be used as a surrogate marker of infection. culture or other non-cultural methods for confirmation ofthe diagnosis of pneumonia must follow within 24-72 hours of starting therapy to retain the patient in the study. isolation by culture is not required for the diagnosis of pneumonia due to m. pneumoniae, legionella, or c. see general guidelines, section ii.d. use of accepted animal models for pneumoniacaused by specific pathogens is desirablefor evaluations of dosage, duration of therapy, achievable serum concentrations, and comparisons with other agents for efficacy and relative toxicity, as described in general guidelines, section ii.e. determinations of levels of antimicrobial agents in respiratory tract secretions and tissue are optionalsince there is a lack of accepted interpretation of results physicians should be available who are competent in the following procedures: bronchoscopy, endobronchial protectedbrush sampling, bronchoalveolar lavage, and thoracentesis. in addition to standard clinical microbiology, the laboratory should have access to nucleic acid probes for detection of legionella and mycoplasma, detectionof legionella species antigen, and determination of titers of specific antibody to mycoplasma and legionella. for most studies, adults (18-65 years of age) and elderly patients (~65 years of age) will be the prototype groups to be studied. additional potential study populations are neonates, infants, children, and immunosuppressed patients. male andfemale patients willbe included. pregnantor lactating women will be excluded. patientswith severeunderlying diseases (e.g., aids, metastatic tumor, shock) will be excluded. patients are excluded if they have received prior therapy witha potentially effective anti-infective agentfor~24 hours. see generalguidelines, sectionix, for additional details. it is notconsidered ethical to usea placebo control in studies evaluating the efficacy of a new anti-infective drug for treatment of pneumonia. active or historical controls are needed to assess the relative value ofthenewdrug. thehistoricalcure rate of uncomplicated (nonbacteremic) pneumonia due to s. pneumoniae in healthy hosts is 1'\j95 %. whenever feasible, the use of a control drug is desirable. the control anti-infective agent should be a drug, or one of several drugs, approved for pneumonia and still recognized by authoritative publications as "standard" treatment. other considerations are discussedin the general guidelines, section x. whenever possible, the study design shouldbe randomized, prospective, and double-blind. see general guidelines, sections x and xi, for details. the spectrumof organisms that causepneumoniais the result of the interplay of multiplehost factors and environmental factors. only somedeterminant factors in the host-parasite relationship are understood, e.g., the presenceor absence of oropharyngeal binding sites for microorganisms, patientage, immune status prior to infection, aspiration of oropharyngeal secretions, concomitant chronic diseases and/or organ failure, or damageto nonspecific or specific portions of the host defenses against microbial invasion. in a given patient, one or more factors may apply. (1) community-acquired vs. hospital-acquired pneumonia. thetraditional distinction between community-acquired and hospital-acquired pneumonia has blurred. traditional community-acquired pathogens, such as s. pneumoniae or l. pneumophila, are now recognized as causes of hospitalacquired pneumonia. patients with chronic diseases, e.g., lung, heart, renal, and/or hepatic failure, are cared for with increasing frequency outside of the hospital. these disease statesincrease the likelihood of colonization ofthe oropharyngeal secretions with facultative gram-negative bacilli and, hence, increase the risk of pneumonia due to this class of organisms traditionally associated withnosocomial pneumonia. (2) patient selection based on clinical category. because of this blurring between community-and hospital-acquired pneumonia, it is reasonable to select patients as trial candidateson thebasisofthe clinical picture. the greaterthehomogeneity of the randomized population of patients with pneumonia, the greaterthe likelihood thetrial results willhave clinicalimport. somepatientsmay fit in more than one category. suggested categories for patients with pneumoniaare presented in table 5. by necessity, the categories are arbitrary and will requireperiodic revision as new insights into pathogenesisemerge. in clinicaltrials ofpatientswhopresent with signsand symptoms of atypical pneumonia, mostpatients enrolled will be ambulatory. in trials of acute bacterial pneumonia, mostpatients willbehospitalized. atthetimeofpatient for statistical considerations, it is strongly recommended that patients be stratified into no more than three clinical categories of pneumonia. for example, in a comparative trial of two parenteral drugs with an appropriate spectrum of activity, patients could be categorized in one of three categories, i.e., acute bacterial pneumonia, aspiration pneumonia, or respirator-associated pneumonia, and then randomized. subsequent to the end of the study, patient response can be analyzed by type of infecting organism, presence of organ failure, severity of pneumonia, and other factors. alternatively, a trial may be designed to study the response of only those patients who meet the clinical criteria for atypical pneumonia. in this example, no stratification would occur prior to randomization. (3) compromised host. pneumonia, and other infections in the compromised host, is discussed in detail in the guide-lines on infections in the febrile, neutropenic patient. the compromised host mayor may not be neutropenic, have inadequate immunoglobulins, or exhibit abnormal lymphocyte function. a wide variety of opportunistic pathogens cause pulmonary infection in the compromised patient. development of a pulmonary infiltrate in a patient with a hematologic malignancy (e.g., leukemia or lymphoma) is a grave prognostic sign and requires an urgent, aggressive, and carefully planned approach to diagnosis and management. for example, local signs of infection in patients who are neutropenic often are fewer and less severe than those in the non-neutropenic person. frequently, neutropenic patients have distant sites of infection from which organisms may have disseminated to the lungs. no symptoms, signs, or roentgenographic features are specific for a given opportunistic infection in the compromised patient. noninfectious pulmonary pathologic conditions are common in this population and may mimic infection. these include radiation pneumonitis, drug toxicity, involvement by the underlying malignancy, pulmonary hemorrhage, pulmo-nary infarction, and congestive heart failure. concurrentand sequential infections of the lung are commonin this population, making the relationship of disease manifestations to a single pathogen difficult to ascertain. early diagnosis is often critical for these patients. guidelines used for diagnosis by examination of pulmonary infiltrates in healthy patients maynot be applicable for diagnosisin patients whoare compromised. for example, severely neutropenic patients may not have neutrophils in their sputum despite having significant bacterial or fungal pneumonia, and for some pathogens the sputum culture may be negative despitethe presenceof invasive lung infection (e.g., aspergillus pneumonia) ..the diagnosis of pulmonary infection in the compromised host may require the performance of an invasive procedure, e.g., percutaneous needle aspirationof the lung, transtracheal aspiration, bronchial lavage and brushingfor quantitative bacteriology, transbronchial biopsy, or open lung biopsy. pneumonia in the compromised patient may be rapidly fatal-hence, the need for empiric antimicrobial therapy. in addition, it is oftennecessaryto reduce the dosageof the immunosuppressive therapeuticagent. thus, the combinedexpertise of all involved physicians is desirable. the duration of treatmentvaries with the clinical category of pneumonia, with the results of blood cultures, and with the status of host defenses. for acute bacterial pneumoniain noncompromised hosts, it maybe desirable to treat until the patient's temperature has returnedto and remained in the normal range for a specific period, e.g., 3-5 days. the possible routesof administration and conversion fromone routeof administration to another are discussed in the general guideline, section xii. see general guidelines, section xii.f. clinical evaluation is based on resolutionor improvement of clinicaland laboratory signsof infectionsuch as fever and leukocytosis, purulent sputumproduction, and radiographic lung infiltrates. hospitalized patients will be assessed every day during treatment and within 5-7 days after completion of treatment. bodytemperaturewill be measuredat least every 8 hours during treatment, and the peak temperature for eachdaywillbe recorded. measurements ofvital signs(blood pressure, heart, and respiratory rates) will be obtained before enrollment and on each day at approximately the same time. the character of the sputum (color, consistency, volume, and number of neutrophils per low-magnification field [x 10d will be recorded when the patient enters the study and at regular intervals thereafter. arterial blood gas determinations will be performed as clinicallyindicated. a chest radiographwill be obtained 3 days after initiationof therapy, within 72 hours of completion of therapy, and at any other time the investigator deems necessary. the location and extent of pneumonic involvement (e.g., segmental, lobar) and the presenceof pleural effusion must be notedand recorded. whenever possible, the same radiologist (or a panel of radiologists) from the sameinstitutionshould interpret all radiographs. other special radiographic studies (e.g., ct scan) will be obtained as clinically indicated. repeated culturesof respiratory tract secretions,if obtainable, will be performed at48-72hoursafterinitiation oftherapy, within 72 hours of the completion of therapy, and whenever clinically indicated. standardized susceptibility testing (disk diffusion or broth dilution) will be performed on all isolates considered potentially significant. blood cultures will be repeatedif initially positiveor if the patient fails to respond to treatment. collectionof specimens that require the use of semi-invasive techniques (e.g., collection of pleural fluid, transtracheal aspiration, bronchoscopy) should be repeated onlyif the clinicalresponseis suboptimal. tests for surrogate markers will be repeatedif these were originally used for diagnosis. for all patients a posttherapy evaluation is necessary for collecting information that will assist in makinga precise assessmentof the patient's clinical and microbiologic response to therapy. patients who have received at least 5 days of therapy and at least 80% or more of prescribed medicationwill have an assessment of clinical response. (1) clinical cure is defined as complete resolution of all signs and symptoms of pneumonia and improvement or lack of progression of all abnormalities on the chest radiograph. (2) clinical failure is defined as anyof the following conditions: persistence or progression of all signs and symptoms after 3-5 days of therapy; development of new pulmonary or extrapulmonary clinical findings consistent with active infection; persistence or progression ofradiographic abnormalities; deathdue to pneumonia; or an inability to complete the study because of adverse effects. (3) indeterminate indicates that extenuating circumstances preclude classification as cure or failure. (2) definition of microbiologic response (1) microbiologic eradication is defined as elimination of the original causative organism(s) from the same site (e.g., expectoratedsputumor normally sterile body fluids such as pleural fluidor blood)during or upon completion of therapy. (2) presumed microbiologic eradication is defined as absence of appropriate material for culture (e.g., sputum or pleural fluid) for evaluation because the patient has improved clinically and does not produce sputum or because repeated aspiration of pleural fluid is not clinically justified. (3)microbiologic persistence is defined as failure to eradicate the original causative organism(s) from sites previously listed, whether or not signs or inflammation are present. (4) microbiologic relapse is defined as recurrence of pulmonary infection with the same organism(s) within 5 days after discontinuation of treatment or during treatment after two consecutive cultures have been negative. (5)superinfection is defined as development of a new lower respiratory tract infection (documented by fever, chest radiograph, and/or auscultatory findings) during treatment or within 3 days after treatment has been completed that is due to a new or resistant pathogen not recognized as the original causative organism(s). (6) colonization is defined as the development of a positive sputum culture that yields a bacterial strain other than the primary causative isolate that appears >48 hours after initiation of therapy, persists in at least two repeated cultures, and is not associated with fever, leukocytosis, persistence or progression of pneumonia, or evidence of infection at a distant site. (7) eradication and reinfection is defined as elimination of the initial infecting pathogen followed by its replacement with a new species or with a new serotype or biotype of the same organism in sputum, pleural fluid, or blood in the presence of signs or symptoms of infection after completion of therapy. for new or additional antimicrobial therapy because of continued infection at the original site in the absence of microbiologic data. (9) indeterminate is defined as circumstances in which it is not possible to categorize the microbiologic response because of death and the lack of opportunity to perform further cultures, the withdrawal of the subject from the study before follow-up cultures can be obtained, incomplete microbiologic data, or concurrent treatment of the patient with a potentially effective anti-infective agent that is not part of the study protocol. the name of the agent and the dose and duration of this therapy must be recorded. the duration of therapy will affect decisions about patient evaluability and outcome. (10) otherconsiderations-when more than one pathogen is present, a separate analysis must be made for each organism. (a) baseline assessment (1) blood for initial cultures, respiratory tract secretions (sputum), and/or pleural fluid, and/or surrogate markers of infection will be obtained. a complete history and physical examination will be performed. (2) tests of hematologic, re-nal, hepatic, and pulmonary function will be performed. (3) radiographic studies such as chest radiography or ct scanning will be performed. arterial blood gas determinations and other tests, such as a diagnostic bronchoscopy, will be done if clinically indicated. (1) culture of sputum will be repeated at 48-72 hours if available; blood cultures will be repeated at 48-72 hours if initially positive. semi-invasive tests will be repeated only if there is a suboptimal clinical response. (2) hematologic, renal, hepatic, and pulmonary function tests will be repeated on days 3-5 of therapy and at least every 5-7 days during therapy. (3) antimicrobial concentrations in blood will be determined if possible, but pharmacokinetic studies of respiratory secretions and other body fluids are optional. (1) if sputum is available, follow-up cultures should be done within 72 hours after completion of therapy. (2) hematologic, renal, hepatic, and pulmonary function tests will be repeated at 72 hours after completion of therapy. (3) chest radiography will be performed within 72 hours of completion of therapy, but other imaging (e.g., ct) and semi-invasive studies (e.g., bronchoscopy) will be performed only if the clinical response is suboptimal. response to therapy will be judged by a combination of clinical and microbiologic criteria and analyzed by intention to treat. clinical response is paramount. comprehensive review of morbidity and mortality trends for rheumatic fever, streptococcal disease, and scarlet fever: the decline of rheumatic fever streptococcal disease world-wide: present studies and prospects beta-hemolytic streptococcal diseases streptococcal pharyngitis pharyngitis: management in an era of declining rheumatic fever streptococcal pharyngitis in the 1980s effect of penicillin and aureomycin on the natural course of streptococcal tonsillitis and pharyngitis comparative effects ofpenicillin, aureomycinand terramycin on streptococcal tonsillitis and pharyngitis effect of treatment on streptococcal pharyngitis: is the issue really settled does penicillin make johnny's strep throat better? streptococcal pharyngitis: placebo controlled double-blind evaluation of clinical response to penicillin therapy the effect of penicillin therapy on the symptoms and signs of streptococcal pharyngitis prevention of rheumatic fever acute rheumatic fever: the come-back of a disappearing disease the fall and rise of rheumatic fever in the united states resurgence of rheumatic fever in the intermountain area of the united states adverse and beneficial effectsof immediate treatment of group a beta-hemolytic streptococcal pharyngitis with penicillin the group a streptococcal carrier state. a reexamination suitability of throat culture procedures for detection of group a streptococci as reference standards for evaluation of streptococcal antigen kits specificity study of kits for detection of group a streptococci directly from throat swabs identification of streptococcal pharyngitis in office laboratory: reassessment of new technology rapid strep tests: making sense of a crowded market. contemporary office visits to pediatricians. national ambulatory medical care service greater boston otitis media study group. epidemiology of otitis media during the first seven years of life in children in greater boston: a prospective, cohort study middle ear disease in samples from the general population. ii. history of otitis media and otorrhea in relation to tympanic membrane pathology, the study of men born in 1913 and 1923 frequency and severity of infection in day care disparate cultures of middle ear fluids the greater boston otitis media study group. otitis media in infancy and intellectual ability, school achievement, speech, and language at age 7 years otitis media in infants and children: management sulfisoxazole as chemoprophylaxis for recurrent otitis media -a double-blind crossover study in pediatric practice otitis media in infants and children: tympanocentesis bacteriology of the maxillary sinuses in patients with cystic fibrosis the diagnosis and management of sinusitis in children: proceedings of a closed conference acute maxillary sinusitis in children sinus infections etiology and antimicrobial therapy of acute maxillary sinusitis chronic maxillary sinusitis. definition, diagnosis and relation to dental infections and nasal polyposis etiology and antimicrobial treatment of acute sinusitis bacteriologic findings of acute maxillary sinusitis in young adults bacteriological study in chronic maxillary sinusitis anaerobic infection of the paranasal sinuses clinical characteristics of nosocomial sinusitis comparative effectiveness of amoxicillin and amoxicillin-elavulanatepotassium in acute paranasal sinus infections in children. a double-blind, placebo-controlled trial treatment of acute maxillary sinusitis in childhood-a comparative study of amoxicillin and cefaclor treatment of acute maxillary sinusitis -amoxicillin, azidocillin, phenylpropanolamine and pivampicillin beta-lactamase producing bacteria in head and neck infection short and long-term treatment results in chronic maxillary sinusitis endoscopic paranasal sinus surgery headaches and sinus disease. the endoscopic approach macroscopic purulence, leukocyte counts and bacterial morphotypes in relation to culture findings for sinus secretions in acute maxillary sinusitis sinusitis of the maxillary antrum management of acute and chronic respiratory tract infections role of infection in chronic bronchitis erythromycin in the treatment of acute bronchitis in a community practice a placebo-controlled, double-blind trial of erythromycin in adults with acute bronchitis role of infection in chronic bronchitis antibiotictherapy in exacerbations of chronic obstructive pulmonary disease community-acquired pneumonia and acutebronchitis prospective study of the aetiology and outcome of pneumonia in the community howcommonis legionnaire's disease? nationalnosocomial infection studyreport. annual summary hospital-acquired pneumonia bacteriology of hospital-acquired pneumonia nosocomialpneumococcal bacteremia a nosocomial outbreak of ampicillinresistant haemophilus influenzae type b in a geriatric unit a nosocomial outbreak of branhamella catarrhalis confirmed by restriction endonuclease analysis theclinicalevaluation of antibacterial drugs guidelines for evaluating new antimicrobial agents principles and practice of infectious diseases acute pneumonia key: cord-281418-mvgp6qfv authors: soccal, p. m.; aubert, j.-d.; bridevaux, p.-o.; garbino, j.; y., thomas; rochat, t.; rochat, t. s.; meylan, p.; tapparel, c.; kaiser, l. title: upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients date: 2010-07-15 journal: clin infect dis doi: 10.1086/653529 sha: doc_id: 281418 cord_uid: mvgp6qfv background. lung transplant recipients are frequently exposed to respiratory viruses and are particularly at risk for severe complications. the aim of this study was to assess the association among the presence of a respiratory virus detected by molecular assays in bronchoalveolar lavage (bal) fluid, respiratory symptoms, and acute rejection in adult lung transplant recipients. methods. upper (nasopharyngeal swab) and lower (bal) respiratory tract specimens from 77 lung transplant recipients enrolled in a cohort study and undergoing bronchoscopy with bal and transbronchial biopsies were screened using 17 different polymerase chain reaction—based assays. results. bal fluid and biopsy specimens from 343 bronchoscopic procedures performed in 77 patients were analyzed. we also compared paired nasopharyngeal and bal fluid specimens collected in a subgroup of 283 cases. the overall viral positivity rate was 29.3% in the upper respiratory tract specimens and 17.2% in the bal samples (p < .001). we observed a significant association between the presence of respiratory symptoms and positive viral detection in the lower respiratory tract (p = .012). conversely, acute rejection was not associated with the presence of viral infection (odds ratio, 0.41; 95% confidence interval, 0.20–0.88). the recovery of lung function was significantly slower when acute rejection and viral infection were both present. conclusions. a temporal relationship exists between acute respiratory symptoms and positive viral nucleic acid detection in bal fluid from lung transplant recipients. we provide evidence suggesting that respiratory viruses are not associated with acute graft rejection during the acute phase of infection. flex, and abnormal lymphatic drainage. nevertheless, our knowledge of the clinical effect of respiratory viruses is incomplete, particularly when detected by molecular assays applied to lower respiratory tract specimens. this is in part due to the retrospective design of most available studies, the use of different sampling sites (upper vs lower respiratory tract specimens), and the heterogeneity of diagnostic procedures (conventional vs molecular techniques) [1, 2, 4, [6] [7] [8] [9] [10] [11] [12] [13] . in addition to the direct consequences of any viral infection, subsequent cellular injury and altered host immunity could also initiate a cascade of immunologic events [14] [15] [16] , leading to acute and chronic allograft rejection. although several studies support the association between respiratory viruses and chronic lung rejection [1, 3, 10, 12, 14, [17] [18] [19] , the link between respiratory viruses and acute rejection has been studied mainly in small case series or by subgroup analysis [1, 2, 9-11, 20, 21] . on the basis of available evidence, the relationship between viral infection and acute rejection has not been established. the present investigation was specifically designed to assess the epidemiology of respiratory viruses in bronchoalveolar lavage (bal) fluid from lung transplant recipients and to analyze the relationship between these viruses and the presence of acute graft rejection. the most sensitive molecular assay methods were used to identify as many as 17 common respiratory viruses in not only the lower but also the upper respiratory tract. symptoms were carefully and prospectively described, and their association with either respiratory viruses or acute rejection was analyzed. during a 27-month study period from november 2003 through march 2006 (covering 3 winter seasons), lung transplant recipients were enrolled in a prospective cohort study [22] . patients were followed up at the 2 sites of a single transplantation network, including the university hospitals of geneva (geneva, switzerland) and the university hospital of lausanne (lausanne, switzerland). informed consent was required from each participant, and the study was approved by both institutional ethics committees. any lung transplant recipient who underwent bronchoscopy with bal and transbronchial biopsies was eligible, irrespective of the reasons leading to the procedure. on the basis of standardized guidelines, we perform routinely scheduled bronchoscopies at 1, 3, 6, and 12 months after transplantation and yearly thereafter. other indications for bronchoscopy with transbronchial biopsies include unexplained respiratory symptoms, functional deterioration with a у12% decrease in the forced expiratory volume in 1 s (fev 1 ), new chest radiologic infiltrate, and control procedure 1 month after treatment of any rejection of grade a3 or higher. thus, patients could undergo bronchoscopy for a variety of reasons with additional procedures during the study period. for technical reasons (eg, pathologist availability), transbronchial biopsies are not performed during weekends and holidays at our centers. for safety and ethical reasons, no additional bronchoscopy was performed only for the purpose of the study. the bal procedure and the real-time taqman reverse-transcription polymerase chain reaction (rt-pcr) assays for the detection of rna respiratory viruses (influenza viruses a, b, and c; respiratory syncytial viruses a and b; parainfluenza viruses 1, 2, 3, and 4; human rhinovirus; enterovirus; human metapneumovirus; and coronaviruses oc43, 229e, nl63, and hku1) were performed as described elsewhere [22] [23] [24] . the recently identified bocavirus was added. transbronchial biopsies were performed under fluoroscopic guidance using standard procedures [25] and analyzed according to published guidelines [26] by senior pathologists masked to the viral results. pooled nasopharyngeal and oropharyngeal swab specimens were obtained from patients who agreed to the procedure. for technical reasons and reasons of cost (110,500 rt-pcr assays were performed), the procedure (nasopharyngeal and oropharyngeal swabbing) was limited to 80% of the cases selected during the entire study period. swabs were immediately placed on appropriate transport medium and stored at ϫ80њc until further rt-pcr analysis. shortly before each bronchoscopic procedure, a specific case report form was completed and symptoms, reasons leading to the bal procedure, and the presumed diagnosis based on the available clinical data at the time were recorded. rhinopharyngitis was defined as the presence of acute respiratory symptoms with at least acute rhinorrhea with or without additional signs suggesting acute sinusitis and/or acute pharyngitis (sore throat confirmed by the presence of inflammatory signs on clinical examination). flulike illness was defined as the presence of a temperature 137.8њc plus 2 of the following 4 symptoms: cough, myalgia, sore throat, or headache. when available, lung functions measured 2-6 weeks before, at the time of, and 2-6 weeks after the bal procedure were recorded. fev 1 and maximal midexpiratory flow of 25%-75% were also recorded. impaired lung function was defined as a у12% decrease from the previous value. statistical analysis. each episode was classified into 4 categories according to the respective absence (a0 or a1 grade) or presence (a2, a3, or a4 grade) of acute rejection and/or respiratory virus. we considered only acute rejection grade a2 or higher in the analysis, because most centers would not recommend high-dose immunosuppressive treatment for a lower acute rejection grade. the fisher exact test was used to compare the frequency of respiratory symptoms in each category. generalized linear latent and mixed models (stata software, version 10; statacorp) were used for analysis of lung functions at each episode to take into account the repeated measures in each patient at the 2 study sites. odds ratios (ors) for acute rejection associated with respiratory viruses were calculated using the same statistical approach for repeated measures. to investigate a potential time-specific relationship between respiratory viruses and acute rejection, we repeated our analyses of episodes restricted to 4 key periods: 0-3 months, 4-6 months, 7-12 months, and 112 months after lung transplantation. seventy-seven lung transplant recipients underwent 343 bronchoscopies. patient characteristics are given in table 1 . all patients received induction immunosuppression with anti-interleukin 2 receptor antibodies (basiliximab) postoperatively, followed by a triple immunosuppression regimen of calcineurin or target of rapaof the 343 bronchoscopies, 229 (66.8%) were performed as routinely scheduled procedures, 85 (24.8%) were performed for a new clinical condition, and 29 (8.5%) were performed as a control biopsy after treatment for an acute rejection (grade a3 or higher) episode. some patients included for a routinely scheduled bronchoscopy presented with a new respiratory symptom with or without functional impairment and/or new radiologic infiltrate. taken together, 224 (65.3%) of 343 cases had at least 1 new respiratory symptom, and 54 (15.7%) had a new radiologic infiltrate. on the basis of the pretest evaluation, acute lung rejection and infection were suspected by the physician in charge, who was masked to microbiological results, in 176 cases (51.3%) and 99 cases (28.9%), respectively. for the remaining 68 cases (19.8%), the clinician did not suspect any particular diagnosis. rt-pcr assays. the rt-pcr viral assays performed on the 343 bal fluid specimens revealed an overall positivity rate of 17.2% ( ). eight bal specimens were discarded ben p 59 cause they had thawed during an electricity power outage. rhinovirus was the most frequently encountered virus, followed by coronaviruses and parainfluenza viruses ( table 2 ). the viral positivity rate for the 283 pooled nasopharyngeal and oropharyngeal specimens tested (82.5% of the total study population) was 29.3% ( ). the agreement between upper and n p 84 lower respiratory tract specimens according to each type of viral genus is shown in figure 1 . although influenza viruses, human metapneumovirus, and bocavirus were mostly (or even exclusively for bocavirus) found in the upper airway samples, other viruses (rhinoviruses and respiratory syncytial viruses) were found equally in the upper and lower respiratory tracts. no virus was exclusively found in the lower airways. association between clinical suspicion and final diagnosis. because bal fluid specimens were collected for a variety of clinical conditions, we were able to analyze the association among symptoms, the diagnosis suspected by the physician in charge, and the subsequent presence of a proven upper and/ or lower respiratory tract viral infection. we found a significant association between positive viral nucleic acid detection in bal fluid and the presence of at least 1 new respiratory symptom ( ), in particular cough ( ) and sputum pro-p p .012 p p .001 duction ( ). table 3 gives the clinical findings associated p p .04 with virus positivity for upper and lower tract specimens. physicians in charge suspected an infection in 20 (42.6%) of the 47 cases with virus-positive bal fluid, compared with 57 (24.2%) of the 236 cases that were virus negative. a history of rhinopharyngitis was documented in 20 (23.8%) of 84 cases with a virus-positive nasopharyngeal swab specimen, compared with 22 (11.1%) of 199 of those that were negative. of 343 biopsy specimens, 149 (43.4%) showed no evidence of acute rejection, 78 (22.7%) revealed minimal (a1) rejection, and 116 (33.8%) were graded a2 or higher. similar to the analysis given in table 3 , we first tested the accuracy of the pretest clinical evaluation performed by the physician in charge. when acute rejection was suspected by the clinician in charge, this was histologically confirmed in 64.1% of cases in the absence of respiratory virus and in 84.6% of cases when a respiratory virus was simultaneously identified in bal fluid (table 4 ). consistent with the results shown in table 3 , the presence of any respiratory symptoms, cough, or sputum was significantly associated ( for all) with the presence of a viral p ! .004 infection in bal fluid, irrespective of the presence or absence of acute rejection. impairment of lung function related to acute rejection was also significantly worsened by the presence of a viral infection (figure 2 ). in patients with simultaneous acute rejection and lower respiratory tract viral infection, the fev 1 recovery rate was significantly slower than in patients who had acute rejection without simultaneous viral infection ( ). p ! .05 we found that the or for acute rejection was !1 in the presence of a viral infection for each of the 4 periods studied (or for any period, 0.41; 95% confidence interval [ci], 0.20-0.88). the overall rate of viral infection in grade a0 and a1 cases was 25.4% (46 of 181), compared with 12.6% (13 of 103) in grade a2 or higher. thus, patients with a lower acute rejection grade were twice as likely to be positive for viral infection than those with a higher rejection grade (or, 2.0; 95% ci, 1.04-3.9). when repeating these analyses with the presence of virus in the upper respiratory tract as a predictor of acute rejection, we did not find an association between upper respiratory tract viral infection and acute rejection (or, 1.11; 95% ci, 0.6-2.05). in a supplementary analysis, we verified the occurrence of acute rejection during a 30-day and 90-day period after baseline bronchoscopy. at 30 and 90 days, the probability of acute rejection was associated with the presence of acute rejection at baseline using an extensive panel of molecular assays, we were able to show that 17.2% of prospectively collected bal fluid specimens from adult lung transplant recipients were positive for at least 1 respiratory virus. on the basis of a pretest clinical evaluation, patients testing positive were significantly more likely to present with respiratory symptoms (86.4%, compared with 60.9% with no identified virus). all analyzed individual symptoms were systematically more frequent in the presence of a respiratory virus, particularly cough and sputum, which were up to 3 times more common. consistent with these observations, the pretest clinical evaluation performed by the physicians in charge considered that an infection was more likely in those with a final positive viral detection. furthermore, lung function proved to recover significantly slower in the presence of a respiratory virus. all these findings corroborate that a positive association exists between positive viral nucleic acid detection in bal fluid and the presence of an acute respiratory illness in lung transplant patients. this is also concordant with other investigations that have linked the detection of respiratory viruses by rt-pcr to symptoms [1, 2, 4, 6, 10, 13, 27] . however, our investigation differs from these studies by its prospective design, the large panel of respiratory viruses screened, the systematic use of bal specimens, and the integration of pretest clinical evaluations performed by the physicians in charge. many of the previous studies limited the detection strategy to upper respiratory tract specimens and used an array of molecular tools that was often restricted to a subgroup of viruses (eg, rhinoviruses, coronaviruses, or bocaviruses were not systematically tested). finally, the link with pretest clinical conditions was not detailed in many of these reports. because of the availability of lung biopsy specimens in all cases, we were also able to assess the presence of acute rejection according to the presence of viral infection. a relationship between acute viral infection and subsequent acute rejection has been reported in small case series, but this possible association has not been appropriately confirmed. viral infection might trigger a chain of immunologically mediated events, leading to subsequent rejection or lung dysfunction. this is important because several studies have identified viral infection as a distinct risk factor for the development of bronchiolitis obliterans syndrome and chronic graft dysfunction [1, 3, 4, 10, 12, 14, 17, 18] . our investigation had the capacity to address this question because we were able to analyze a high number of lung biopsy specimens together with viral screening at the level of the lower respiratory tract. the biopsy analysis revealed that viral respiratory tract infections were not associated with simultaneous acute rejection. furthermore, we could show that the probability of acute rejection did not increase 30 and 90 days after a lower airways respiratory viral infection. when present, however, viral infections caused more severe lung dysfunction and significantly hampered the short-term functional recovery in the case of concomitant acute rejection. because the clinician in charge was masked to any viral result, a positive rt-pcr result did not modify the treatment of simultaneous acute rejection. thus, a less aggressive treatment of acute rejection in the presence of a virus cannot explain the slower recovery of lung function in these patients. this suggests that respiratory viruses per se do not promote acute rejection during the acute phase but could certainly worsen lung function and impair recovery from acute rejection episodes. paradoxically, we even observed a negative association between an episode of respiratory viral infection and the subsequent cumulative risk of developing acute rejection. this should not be considered a protective effect of viral infections; more likely, it is a lack of an association or a chance effect, and we refrain from drawing any other conclusions. however, this strongly suggests that when analyzing biopsy specimens according to reference guidelines [26] , pathologists should not be misguided by the presence of a viral infection. of note, our study design limited follow-up to a few weeks, and we cannot exclude the possibility that the viral infection triggered a chain of immunologically mediated events, leading to subsequent rejection or lung dysfunction. this is important because several studies have identified viral infection as a distinct risk factor for the development of bronchiolitis obliterans syndrome and chronic graft dysfunction [1, 3, 4, 10, 12, 14, 17, 18] . our investigation also provided the unique opportunity to compare viral detection in the upper (pooled nasopharyngeal and oropharyngeal swabs) and lower respiratory tract in a large number of paired specimens. the positivity rate in the upper respiratory tract was 29.6%, compared with 16.6% in the lower tract. this difference in recovery rate is similar to that observed in other smaller studies [12] , but, to the best of our knowledge, few or none of the previous investigations systematically analyzed paired specimens collected during the same procedure. an important observation is that when recovered only in the upper respiratory tract, respiratory viruses were less likely to be associated with lower respiratory symptoms. another interesting observation is that only 7.0% of negative nasopharyngeal specimens were associated with a discordant positive bal viral screening, thus suggesting a high negative predictive value. however, this value should be balanced with the relatively low prevalence of each individual respiratory virus in bal fluid specimens and the possible technical issues related to nasopharyngeal and pharyngeal swabbing that could negatively affect the recovery rate. our population was first selected on the basis of the need to perform a bal procedure; for this reason, we caution that a lower respiratory tract viral infection in lung transplant recipients cannot be definitely ruled out by a nasopharyngeal swab. yet this could be a reasonable initial screening strategy that needs to be confirmed in further studies and individually for each type of virus. in conclusion, our study demonstrates that there is a temporal relationship in lung transplant recipients between the emergence of acute respiratory symptoms and positive respiratory viral nucleic acid detection in bal fluid specimens. when detected only in the upper respiratory tract, viral infections are less likely to be associated with respiratory symptoms and graft dysfunction. we also provide solid evidence suggesting that respiratory viruses per se do not promote acute graft rejection, at least during the acute phase of infection, but that they do worsen graft function recovery when simultaneously present with acute rejection. clinical impact of communityacquired respiratory viruses on bronchiolitis obliterans after lung transplant impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients respiratory viruses and chronic rejection in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus respiratory viral infections in transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients absence of human bocavirus in 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syndrome community acquired respiratory viral infections after lung transplantation: clinical features and long-term consequences parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function the epidemiology of parainfluenza virus infection in lung transplant recipients treatment of respiratory syncytial virus pneumonia in a lung transplant recipient: case report and review of the literature influenza virus infection in adult solid organ transplant recipients respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection swiss paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study persistent hepatitis c viremia predicts late relapse after sustained response to interferonalpha in chronic hepatitis c lung rejection study group. revision of the 1990 working formulation for the classification of pulmonary allograft rejection influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients we thank patricia suter, sandra van belle, jean-marc fellrath, and lara turin for their excellent technical assistance and rosemary sudan for editorial assistance.financial support. swiss national science foundation (grant 3200b-101670 to l.k.).potential conflicts of interest. all authors: no conflicts. key: cord-295940-siy32dj7 authors: saito, makoto; adachi, eisuke; yamayoshi, seiya; koga, michiko; iwatsuki-horimoto, kiyoko; kawaoka, yoshihiro; yotsuyanagi, hiroshi title: gargle lavage as a safe and sensitive alternative to swab samples to diagnose covid-19: a case report in japan date: 2020-04-02 journal: clin infect dis doi: 10.1093/cid/ciaa377 sha: doc_id: 295940 cord_uid: siy32dj7 nan 2 dear editor. the diagnosis of covid-19 requires upper or lower respiratory samples. however, the problem of covid-19 is that around 70-80% of patients do not have productive cough. 1 to protect healthcare workers during sampling for diagnosis, the us cdc recommends not inducing cough to collect sputum samples, but rather the collection of nasopharyngeal and/or oropharyngeal swabs, or nasopharyngeal wash/aspirate or nasal aspirate. nasal swabs are reported to have higher viral titers than throat swabs; 2 accordingly, nasopharyngeal swabs are the preferred samples in japan. however, nasopharyngeal and oropharyngeal swabs cause discomfort to patients and can potentially increase the risk of direct exposure of healthcare workers by provoking coughing. moreover, the sensitivity for virus detection is low with these swabs; viral load is reportedly higher in sputum samples. 3 here we report a case in which gargle lavage samples yielded a positive pcr result. a 55-year-old man came to our hospital complaining of 5 days of fever (maximum 38.6 º c). he had a mild headache, but no respiratory symptoms. four days prior to his fever, he had had contact with a covid-19 infection cluster. on admission, his vital signs were within the normal range and his breathing sounds were normal. his blood tests on admission (day 6) revealed mild lymphocytopenia (720/μl) and slightly elevated c-reactive protein (0.88 mg/dl). although his chest x-ray was not remarkable, his ct-scan revealed patchy ground-glass opacities predominantly in left lower lobe (supplement figure 1, 2) . samples were taken to test for covid-19 by real-time reverse-transcriptase-polymerase-chain-reaction, using primers from the chinese center for disease control and prevention. 4 oropharyngeal swabs and gargle lavage (using 10 ml of normal saline) were collected because he did not produce sputum. additional gargle lavage samples and oropharyngeal swabs were collected and tested on days 8 and 9 and found to be positive, with a slightly higher amount of viral genome in the gargle lavage sample (supplement figure 3) . his pcr became negative on day 16 and 19, and discharged on day 19. for other respiratory pathogens, gargle lavage samples have been reported to be more sensitive than throat swabs. 5 gargle lavage can be done by patients themselves without putting healthcare professionals at increased risk, which is reportedly high in this outbreak. 1 gargle lavage thus offers a safer and possibly more sensitive alternative or additional option for diagnosing covid-19. y.k. is a co-founder of flugen, which is now developing a covid-19 vaccine. all other authors declare that they have no competing interests. clinical characteristics of coronavirus disease sars-cov-2 viral load in upper respiratory specimens of infected patients viral load of sars-cov-2 in clinical samples specific primers and probes for detection 2019 novel coronavirus comparison of gargle samples and throat swab samples for the detection of respiratory pathogens key: cord-309091-te15ahvw authors: larson, derek; brodniak, sterling l; voegtly, logan j; cer, regina z; glang, lindsay a; malagon, francisco j; long, kyle a; potocki, ronald; smith, darci r; lanteri, charlotte; burgess, timothy; bishop-lilly, kimberly a title: a case of early re-infection with sars-cov-2 date: 2020-09-19 journal: clin infect dis doi: 10.1093/cid/ciaa1436 sha: doc_id: 309091 cord_uid: te15ahvw nan a c c e p t e d m a n u s c r i p t 3 dear editor, it is with great interest that we read the first report of re-infection from sars-cov-2, which represented an important data point in the ongoing covid-19 pandemic [1] [2] [3] . questions have arisen regarding the timing and severity of re-infections, for which we offer a case report of symptomatic re-infection within 90 days. 42-year-old healthy male military healthcare provider presented with cough, subjective fever, and myalgias on 21 march following a workplace covid-19 exposure and tested positive by sars-cov-2 rt-pcr ( figure 1 ). physical examination was unrevealing and supportive outpatient management was pursued [4] . clinical resolution of illness occurred by day 10, and he returned to baseline excellent health for the following 51 days. on 24 may he presented with fevers, cough, shortness of breath and gastrointestinal symptoms, (snvs) were determined [5] . global lineage was determined using a subset of sars-cov-2 genomes available from the global initiative on sharing all influenza data repository (gisaid accessed, june 24, 2020). alignments were performed [6] and a maximum likelihood tree was generated [7] . the sars-cov-2 genome from the re-infection sample was deposited in ncbi genbank under accession the identification of specific products, scientific instrumentation, or organization is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the author, dod, or any component agency. this work was supported by [wun a1417] and [geis p0013_20_ah_01.01] to kbl for virus isolation, sequencing, and bioinformatic analyses; and idcrp-085 is funded by the defense health program. the authors of this study report no financial conflicts of interest. a c c e p t e d m a n u s c r i p t covid-19 re-infection by a phylogenetically distinct sarscoronavirus-2 strain confirmed by whole genome sequencing deep immune profiling of covid-19 patients reveals distinct immunotypes with therapeutic implications. science persistent positivity and fluctuations of sars-cov-2 rna in clinically-recovered covid-19 patients clinical outcomes of covid-19 with evidence-based supportive care an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform iq-tree 2: new models and efficient methods for phylogenetic inference in the genomic era tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus a c c e p t e d m a n u s c r i p t key: cord-298539-yncda1us authors: ruan, linhui; wen, min; zeng, qingrun; chen, chengshui; huang, shengwei; yang, su; yang, jianjing; wang, jingqiang; hu, yuhuan; ding, saidan; zhang, ying; zhang, hongxia; feng, yuanjing; jin, kunlin; zhuge, qichuan title: new measures for covid-19 response: a lesson from the wenzhou experience date: 2020-04-03 journal: clin infect dis doi: 10.1093/cid/ciaa386 sha: doc_id: 298539 cord_uid: yncda1us as the outbreak of covid-19 has spread globally, determining how to prevent the spread is of paramount importance. we reported the effectiveness of different responses of four affected cities in preventing the covid-19 spread. we expect wenzhou anti-covid-19 measures may provide experience for cities around the world that are experiencing this epidemic. to prevent the spread is of paramount importance. wenzhou, a coastal city in southern china with a resident population of 9.25 million, was the most heavily affected with respect to covid-19 infections outside of hubei province. this is the result of the more than 90,000 people who return to wenzhou from wuhan during on january 17 when the first case of covid-19 was confirmed, the wenzhou new coronavirus pneumonia prevention and control committee launched emergency epidemic prevention and control measures, which in general consisted of two phases. wenzhou's first-phase prevention and control measures (first-phase measures) were moderate preventive measures undertaken from january 17 to january 30, 2020. the main measures included (1) centralizing the confirmed and suspected patients in designated hospitals for treatment, (2) identifying and investigating all returnees from wuhan using a big data network and persuading them to undergo 14-day home quarantine, and (3) centralizing and medically observing individuals who had been exposed to a confirmed covid-19 patient. wenzhou's second-phase prevention and control measures (second-phase measures, also called "wenzhou anti-covid-19 measures") consisted of the most aggressive social distancing and quarantining measures and were carried out from january 31 to february 20, 2020. the measures were referred to as "25 emergency measures for wenzhou epidemic prevention and control" and mainly focused on pinpointing the infection source and completely cutting off any transmission routes. the measures are summarized as follows: 1) shutdown of city. all traffic in and around wenzhou, including airports, high-speed rail stations and bus stations, was cut off. only five access points to the expressway were open for transportation of prevention and control materials and necessities, and these were under strict monitoring. 2) lockdown of communities. all places of public gatherings were shut down, including malls, public entertainment facilities, group meetings, restaurants and hotels. the committee enforced crowd-grid management using communities and villages as individual units; se inspection stations were set up in major locations, and all entrants were interviewed and registered, with body temperature monitoring. in addition, all individuals were required to stay at home, and daily necessities were delivered in real time. 3) tracking and inspection. a combination of mobile big data, network searches, screening at city entrances, etc. was used to track and investigate individuals at risk of infection in a timely manner. all covid-19 contacts and suspected patients were isolated for quarantining. the confirmed cases were rapidly transferred to designated hospitals for treatment. the implementation of the first-phage measures significantly reduced the incidence of imported cases from other cities, suggesting that these measures were effective for import prevention, but did not block the covid-19 outbreak in the city. moderate preventive measures were also implemented in huanggang, xiaogan and jingzhou, cities in the region surrounding wuhan. the covid-19 onset time and populations in these three cities were similar to those in wenzhou, and thus the epidemic was occurring in parallel ( figure 1a ). however, after january 31, the incidence curves in the four cities were notably different. subsequent outbreaks were observed in huanggang, xiaogan and jingzhou but not in wenzhou. however, the numbers of new cases in these cities were meaningfully reduced after implementation of most aggressive social distancing and quarantining measures, which was similar to wenzhou second-phase measures on february 10 ( figure 1a) . these data suggest that the "wenzhou anti-covid19 measures" were effective in controlling the spread of covid-19. of the 504 patients in wenzhou, 434 patients were mild, and 62 were severe and 8 were critically severe, 502 patients had been discharged and 1 patient (0.2% mortality) had died, 1 patient was still in critical condition. 268 (53.2%) were male, and the median patient age was 47 (range, 2-93). the average time to diagnosis for the patients was 5.5 days (range from 1-21 days a comparative analysis was conducted in four cities. the incidence curves in these cities were very similar before january 31. wenzhou's first incidence peak appeared on january 29, and then it declined steadily. no second peak was found thereafter. however, huanggang, xiaogan and jingzhou all had their second and even third incidence peaks after january 31 ( figure 1b) . these three cities implemented rigorous measures that were similar to the "wenzhou anti-covid19 measures" on february 10, 14 and 15, respectively. as a result, the outbreak of covid-19 in these three cities was gradually placed under control. an r0 value of 3.11 (95% ci, 2.39-4.13), suggesting that covid-19 is very contagious. in general, an epidemic will increase as long as r0 > 1, and control measures aim to reduce the r0 <1. the r0 of sars has been estimated to be ~3 9 and sars outbreaks has been successfully controlled by isolation of patients and careful infection control 10 . therefore, wenzhou anti-covid-19 measures were the extremely rapidly response in covid-19 crisis. these data suggest that the most aggressive social distancing and quarantining measures (wenzhou anti-covid19 measures) in the second phase were effective in controlling the covid-19 spread. wenzhou anti-covid-19 measures may provide valuable experience for other cities around the world that are experiencing this epidemic. clinical features of patients infected with 2019 novel coronavirus in wuhan, china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia when will be the resumption of work in wuhan and its surrounding areas during covid-19 epidemic? a data-driven network modeling analysis (in chinese) update on the new coronavirus pneumonia outbreak as of 24:00 on 13 march national health commission of the people's republic of china update on the new coronavirus pneumonia outbreak as of 24:00 on 13 march wenzhou municipal health commission nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study novel coronavirus 2019-ncov: early estimation of epidemiological parameters and epidemic predictions dynamically modeling sars and other newly emerging respiratory ill-nesses: past, present, and future coronavirus infections -more than just the common cold hubei province after implementation of the 1 st (black arrow) and 2 nd phase measures the effect of different anti-covid-19 measures on the cumulative number of new cases of covid-19 in daily numbers of new cases by date of onset of symptoms (blue) after implementation of wenzhou anti-covid-19 measures and the number of estimated symptomatic cases (red line) without execution of wenzhou anti-covid-measures are plotted. three basic reproduction numbers (r0) were estimated with three stages (first stage this work was supported by grants from national natural science foundation of china (81771262，61976190) and international (regional) cooperation and exchange projects of the national natural science foundation of china (81820108011), and the key project of science technology department of zhejiang province (2017c03027) . key: cord-294853-8b0s5w4u authors: nolen, leisha d; seeman, sara; bruden, dana; klejka, joe; desnoyers, chris; tiesinga, james; singleton, rosalyn title: impact of social distancing and travel restrictions on non-covid-19 respiratory hospital admissions in young children in rural alaska date: 2020-09-05 journal: clin infect dis doi: 10.1093/cid/ciaa1328 sha: doc_id: 294853 cord_uid: 8b0s5w4u hospitalizations due to non-covid-19 respiratory illnesses decreased dramatically after social distancing was implemented in a high-risk population in rural alaska. our data from the past ten respiratory seasons show that this decline is unprecedented. this demonstrates the potential secondary benefits of implementing social distancing and travel restrictions on respiratory illnesses. a c c e p t e d m a n u s c r i p t 3 social distancing was implemented in many regions in the spring of 2020 to limit the spread of sars-cov-2, the virus responsible for coronavirus disease 2019 (covid-19). many reports have compared the respiratory illness rates during social distancing to one or two previous seasons; however, it is important to evaluate the difference over multiple seasons due to year to year variations in influenza, respiratory syncytial virus (rsv), and other respiratory illnesses [1] [2] [3] . people in the remote yukon-kuskokwim delta (ykd) region of alaska have a high burden of respiratory illnesses, with up to ten times the rate of infant hospitalization for pneumonia compared to the general us population [4] and the highest reported rates of infant rsv hospitalization in the united states, reaching 259 per 1,000 infants [5] . all ykd residents who require hospitalization go through the ykd regional hospital (ykdrh), allowing detection of all hospitalizations in this population. our team has conducted passive surveillance for rsv and acute respiratory infection (ari) hospitalizations in ykd children under 3 years of age since 1996 [6] . three social distancing mandates were issued by the governor of alaska in the spring of 2020. the first, on march 20 th , closed all schools, the second, on march 27 th , limited intrastate travel and travel to small communities, and the third, on march 28 th , closed nonessential businesses, prohibited public gatherings, and required people stay 6 feet apart from non-household members. these mandates limited both the transmission of pathogens within the ykd and the introduction of pathogens from outside regions. at that time, no cases of sars-cov-2 were documented within the ykd and there was no evidence it was circulating in this population. we compared the total, ari, and rsv hospitalization rate in ykd children <3 years in the first 5 months of 2020 to the previous ten respiratory illness seasons. one limitation of our study is the inability to distinguish what proportion of the decline in ari hospitalizations in ykd children is due to avoidance of care versus a decrease in circulation of respiratory pathogens secondary to the health mandates. numerous reports show people avoided medical care for a range of conditions during the peak months of the pandemic, resulting in in-home deaths and delayed diagnoses [7] [8] [9] . we hypothesize that if the precipitous decline was due only to avoidance of care, there would have been an increased number of children presenting late to care, leading to an increase in critically ill children and deaths due to ari. instead, only one child was hospitalized with respiratory failure and no respiratory related deaths were recorded in april or may. because of the lack of severe respiratory outcomes in this population, we conclude that the observed decline was primarily driven by the reduction in pathogen circulation due to travel restrictions and social distancing. our data show a large amount of variability in ari admissions year to year, therefore comparing the 2019-2020 season to one or two other seasons, as done in other publications, may be misleading. here we compare the 2019-2020 season to the ten preceding seasons and show a precipitous and sustained decline in ari admissions related to the start of social distancing mandates. these data compare ari hospital admissions during the recent 2019-2020 season with ten preceding seasons in a unique population in rural alaska with a history of very high rates of respiratory hospitalizations and limited medical accessibility. due to its extreme isolation, all children receive care from a single health center or are transferred by small admissions to veterans affairs hospitals for emergency conditions during the covid-19 pandemic covid-19 pandemic: impact caused by school closure and national lockdown on pediatric visits and admissions for viral and non-viral infections, a time series analysis impact of public health interventions on seasonal influenza activity during the sars-cov-2 outbreak in korea lower respiratory tract infection hospitalizations among american indian/alaska native children and the general united states child population severe respiratory syncytial virus disease in alaska native children. rsv alaska study group eighteen years of respiratory syncytial virus surveillance: changes in seasonality and hospitalization rates in southwestern alaska native children a population-based, observational study. the lancet public health the outbreak of novel coronavirus disease (covid-19) caused a worrying delay in the diagnosis of oral cancer in north-west italy: the turin metropolitan area experience effect of covid-19 epidemic on delay of diagnosis and treatment path for patients with nasopharyngeal carcinoma a c c e p t e d m a n u s c r i p t 8 airplane to higher-level care at a single tertiary medical center, thus allowing us to detect all hospitalizations. sars-cov-2 was not circulating in the region during the study period; key: cord-251945-v077hhgk authors: titanji, boghuma k; farley, monica m; schinazi, raymond f; marconi, vincent c title: response to correspondence: baricitinib as treatment of covid-19 friend or foe of the pancreas? cerda-contreras et.al date: 2020-08-14 journal: clin infect dis doi: 10.1093/cid/ciaa1212 sha: doc_id: 251945 cord_uid: v077hhgk nan a c c e p t e d m a n u s c r i p t dear editor, in response to our recent publication in your journal, cerda-contreras et.al present the case of a critically ill 72-year-old woman with covid-19 and multiple comorbidities. she was treated with baricitinib and steroids though subsequently succumbed to complications of acute pancreatitis. the authors hypothesized this complication could have been related to baricitinib. contrary to what is stated in their correspondence, acute pancreatitis has been described in several case reports as a manifestation of covid-19 1,2 . the ace-2 receptor used by sars-cov2 to bind to target cells is highly expressed in the pancreas making it an attractive target for infection by the virus. the cytopathic effects from local virus replication and significant inflammation associated with severe disease may contribute to pancreatic tissue damage in patients with covid-19. new onset type i diabetes mellitus and worsening type ii diabetes mellitus have been linked to damage of pancreatic islet cells in patients with covid-19 3 , further supporting the tropism of sars-cov2 for the pancreas and the potential damaging effects to that organ. we also note that in addition, the patient received treatment with steroids and propofol, both of which have been independently associated with acute pancreatitis 4-6 . furthermore, the patient was obese, which may have predisposed her to acute pancreatitis and contributed to adverse outcomes 7 . the abundance of confounding factors and alternative etiologies for acute pancreatitis in this case make it difficult to attribute the patient's complications exclusively to treatment with baricitinib. while one case of pancreatitis was reported in adverse events monitoring during clinical trials of baricitinib for the treatment of rheumatoid arthritis , long-term safety monitoring of up to 8.4 years has not identified additional cases 8 , which suggests that this is a rare occurrence. it was notable that the patient received substantial immunosuppression with both baricitinib and dexamethasone for treatment of covid-19. while plausible antiviral properties of baricitinib against coronaviruses have been hypothesized 9 and confirmed in primary lung cells, this is yet to be demonstrated in humans infected with sars-cov2. although speculative, it is possible that baricitinib in combination with dexamethasone without concurrent use of a proven antiviral agent, may have produced profound immunosuppression with enhanced viral replication resulting in multi-organ damage. the immunosuppressive effects of steroids are pleiotropic and mainly mediated m a n u s c r i p t by sequestration of cd4+ t lymphocytes in the reticulo-endothelial system and inhibition of gene expression for important cytokines and chemokines. in fact, the use of corticosteroids in viral infections has been fraught with controversy given the well described association with enhanced respiratory virus replication 10 . jak1/2 inhibitors such as baricitinib are more targeted but still highly potent immunosuppressive drugs that inhibit jak-stat signaling thus reducing downstream production of several important inflammatory cytokines. in targeting the hyper-inflammation that is associated with severe covid-19, timing of immunomodulatory therapy is key, and combining potent immuno-modulators requires caution to avoid tipping the balance in favor of enhanced viral replication. ongoing clinical trials will provide much needed clarification on the appropriate timing and choice of immune-modulatory therapies in the treatment of covid-19. a c c e p t e d m a n u s c r i p t covid-19 presenting as acute pancreatitis coronavirus disease-19 (covid-19) associated with severe acute pancreatitis: case report on three family members new-onset diabetes in covid-19 acute pancreatitis associated with intravenous administration of propofol: evaluation of causality in a systematic review of the literature acute pancreatitis associated with steroid therapy acute necrotising pancreatitis derived from lowdose corticosteroid use: an important reminder of clinical management obesity is a definitive risk factor of severity and mortality in acute pancreatitis: an updated meta-analysis fri0123 safety profile of baricitinib for the treatment of rheumatoid arthritis up to 8.4 years: an updated integrated safety analysis covid-19: combining antiviral and antiinflammatory treatments glucocorticosteroids enhance replication of respiratory viruses: effect of adjuvant interferon key: cord-288558-rthnj6wd authors: cheng, v. c. c.; hung, i. f. n.; tang, b. s. f.; chu, c. m.; wong, m. m. l.; chan, k. h.; wu, a. k. l.; tse, d. m. w.; chan, k. s.; zheng, b. j.; peiris, j. s. m.; sung, j. j. y.; yuen, k. y. title: viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome date: 2004-02-15 journal: clin infect dis doi: 10.1086/382681 sha: doc_id: 288558 cord_uid: rthnj6wd the role of severe acute respiratory syndrome (sars) coronavirus as an enteric pathogen was investigated in a cohort of 142 patients with sars who were treated with a standard treatment protocol. data from daily hematological, biochemical, radiological, and microbiological investigations were prospectively collected, and the correlation of these findings with diarrhea was retrospectively analyzed. sixty-nine patients (48.6%) developed diarrhea at a mean (± standard deviation [sd]) of 7.6 ± 2.6 days after the onset of symptoms. the diarrhea was most severe at a mean (±sd) of 8.8 ± 2.4 days after onset, with a maximum frequency of 24 episodes per day (median, 5 episodes; range, 3–24 episodes). a higher mean virus load in nasopharyngeal specimens obtained on day 10 after the onset of symptoms was significantly associated with the occurrence of diarrhea (3.1 log(10) vs. 1.8 log(10) copies/ml; p = .01) and mortality (6.2 vs. 1.7 log(10) copies/ml; p < .01). however, diarrhea was not associated with mortality. the lung and the gastrointestinal tract may react differently to sars coronavirus infection. additional investigation of the role of sars coronavirus in the pathogenesis of diarrhea in patients with sars should be conducted. the role of severe acute respiratory syndrome (sars) coronavirus as an enteric pathogen was investigated in a cohort of 142 patients with sars who were treated with a standard treatment protocol. data from daily hematological, biochemical, radiological, and microbiological investigations were prospectively collected, and the correlation of these findings with diarrhea was retrospectively analyzed. sixty-nine patients (48.6%) developed diarrhea at a mean ‫ע(‬ standard deviation [sd]) of days after the onset of symptoms. the 7.6 ‫ע‬ 2.6 diarrhea was most severe at a mean (‫ע‬sd) of days after onset, with a maximum frequency of 24 8.8 ‫ע‬ 2.4 episodes per day (median, 5 episodes; range, 3-24 episodes). a higher mean virus load in nasopharyngeal specimens obtained on day 10 after the onset of symptoms was significantly associated with the occurrence of diarrhea (3.1 log 10 vs. 1.8 log 10 copies/ml; ) and mortality (6.2 vs. 1.7 log 10 copies/ml; ). p p .01 p ! .01 however, diarrhea was not associated with mortality. the lung and the gastrointestinal tract may react differently to sars coronavirus infection. additional investigation of the role of sars coronavirus in the pathogenesis of diarrhea in patients with sars should be conducted. the severe acute respiratory syndrome (sars) pandemic has affected 18000 patients, with 774 fatalities [1] . sars is caused by a novel coronavirus, which was consistently isolated from patients with sars who had subsequent seroconversion [2] [3] [4] . similar histopathological findings of sars and seroconversion have been reproduced in cynomolgus macaques (macaca fascicularis) that were artificially inoculated with the same sars coronavirus [5] . subsequent detailed histopathological study of these infected macaques confirmed that the inflammatory changes were confined to the lungs [6] . thus, sars is largely regarded as a novel viral pneumonia. however, in a prospective clinical study of a cohort of patients with sars, diarrhea was noted in 1% of patients at hospital admission and in 73% during hospitalization [7] . viral genomes or the virus could be detected by rt-pcr or cell culture of stool samples obtained from these patients [8] . as illustrated by the example of enterovirus infection, shedding of virus in stool does not necessarily imply the presence of disease in the intestinal tract. nevertheless, presence of numerous coronavirus particles was demonstrated in the terminal ileum and colon in a patient with sars who had diarrhea [9] . it would be interesting to know whether the sars coronavirus indeed causes diarrhea, because histopathological examination of intestinal biopsy specimens does not reveal any inflammatory or cytolytic damages. we have previously shown that specimens obtained from the nasopharynx are useful for the diagnosis of rotavirus infection in children [10] . this is not unexpected, because an earlier study also demonstrated the presence of rotavirus in nasopharyngeal secretions from children with upper respiratory tract infection [11] . in this retrospective study, we attempt to correlate the virus load of sars coronavirus shedding from the nasopharynx, the upper end of the aerodigestive tract, with the presence of diarrhea in a cohort of patients with sars. demographic characteristics, clinical features, and hematological and radiological findings of patients with sars who had or did not have diarrhea were collected and analyzed. the clinical records for all 142 patients whose cases fulfilled the modified world health organization definition of sars [4, 7] and who were treated at the united christian hospital and caritas medical centre (hong kong) were analyzed. the daily clinical findings from history and physical examinations and hematological, biochemical, radiological, and microbiological investigations were prospectively collected and analyzed. in brief, the case definition includes fever (temperature, у38њc), cough or shortness of breath, and new pulmonary infiltrates noted on chest radiographs or high-resolution ct, in the absence of an alternative diagnosis to explain the clinical presentation. some of the clinical and virological results for the first 75 patients were previously reported [7] . all patients were treated with amoxicillin-clavulanate (1.2 g q8h iv) and azithromycin (500 mg q.d. po). in patients with a known allergy to penicillin, we administered 500 mg of oral levofloxacin every 24 h. as soon as the diagnosis of sars was established, ribavirin (a 4-g oral loading dose, followed by 1.2 g q8h or 8 mg/kg q8h iv if the patient could not tolerate oral treatment) was given for 14 days, in addition to a tailing regimen of hydrocortisone (starting dose, 200 mg q8h iv) for 10 days, followed by oral prednisolone (1 mg/kg for 5 days, 0.5 mg/kg for 3 days, and 0.25 mg/kg for 3 days) for 11 days. pulses of methylprednisolone (500-1000 mg q.d. iv for up to 3 g) were administered to patients with clinical deterioration. all hepatitis b surface antigen-positive patients were given prophylactic lamivudine (100 mg q.d. po) while taking corticosteroids. patients were prospectively monitored for development of diarrhea during hospitalization. diarrhea was defined as у3 bowel movements per day for у2 consecutive days. the occurrence of diarrhea in relation to the onset of symptoms of sars, the frequency of bowel movements, and the duration of diarrhea were recorded. investigation for other causes of diarrhea was performed, including culture for clostridium difficile, cell culture assay for detection c. difficile cytotoxin, and elisa (ideia rotavirus; dako) for detection of rotavirus. for the diagnosis of coronavirus infection, nasopharyngeal specimens and serum samples were obtained at hospital admission. the convalescent-phase serum sample was taken 14-28 days after the onset of symptoms. for all patients, qualitative and quantitative rt-pcrs for sars coronavirus were retrospectively performed using the nasopharyngeal specimens obtained at admission and 10 days after the onset of symptoms. stool and urine specimens were obtained for rt-pcr during the hospital stay. all virological diagnostic protocols, including performance of qualitative and quantitative rt-pcrs, rapid viral antigen detection tests, viral cultures, immunofluorescent antibody tests for detection for igg seroconversion against sars-associated coronavirus, and other microbiological diagnostic evaluations, were done in the manner described in our previous publications [4, 7] . statistical analysis. all data were calculated from the day of onset of clinical symptoms. we compared the demographic characteristics and laboratory values for patients with and without diarrhea by means of fisher's exact test for categorical variables and student's t test or the mann-whitney u test for continuous variables. a 2-tailed p value of !.05 was considered to be statistically significant. we used spss software, version 11.0 (spss), for all analyses. of the 142 patients recruited in this study, 138 (97.2%) were ethnic chinese, and the remaining patients were filipinos. the patients who presented with single-zone lesions, the right lower and left lower zones were more commonly involved than were the other zones. thoracic ct was performed for 34 patients with initial apparently normal chest radiograph findings. the lesions were mostly confined to the retrocardiac region in the left lower lobes. with regard to the microbiological evaluation for sars coronavirus, 137 patients (96.5%) had either a 4-fold increase in antibody to sars coronavirus or positive results of rt-pcr of nasopharyngeal, stool, or urine specimens (table 3) . fourfold increases in antibody titers to sars coronavirus were found in 26 of 28 patients who did not have any history of epidemiological exposure, and rt-pcr results were positive for the remaining 2 patients. during the course of infection, diarrhea occurred in 69 patients (48.6%). the distribution of episodes of diarrhea during the first 3 weeks of hospitalization is shown in figure 1 . diarrhea occurred days after the onset of symptoms and be-7.6 ‫ע‬ 2.6 came most severe on day , with a maximum frequency 8.8 ‫ע‬ 2.4 of 24 episodes per day (median, 5 episodes per day; range, 3-24 episodes per day). the diarrhea lasted for a duration of days in this cohort, resulting in prerenal azotemia in 3.8 ‫ע‬ 2.3 6 patients and electrolyte disturbances in 39 patients. the diarrhea was rather painless, with no blood or mucus present in the stool for any patients. none of the patients had positive results of culture for c. difficile or cell culture assay for c. difficile cytotoxin. all stool samples were negative for rotavirus on elisa tests. the clinical characteristics of patients with and those without diarrhea are summarized in table 4. there were no differences between the 2 groups with regard to age, sex, comorbidities, and chronic hepatitis b status. the manifestations on chest radiographs at hospital admission were also similar. there were no statistical differences in hematological and biochemical parameters between the 2 groups at presentation and on day 10 after the onset of symptoms. patients with diarrhea had lower potassium levels ( mmol/l) and higher mean sodium 3.3 ‫ע‬ 0.5 levels ( mmol/l). there was no difference in the mean 143 ‫ע‬ 3 virus loads in nasopharyngeal specimens (as measured by quantitative rt-pcr) at baseline between the groups with and without diarrhea. a higher mean virus load in nasopharyngeal specimens obtained on day 10 after the onset of symptoms was significantly associated with the occurrence of diarrhea (3.1 vs. 1.8 log 10 copies/ml; ) and mortality (6.2 vs. 1.7 log 10 p p .01 copies/ml; ). however, there were no differences be-p ! .01 tween patients with and those without diarrhea with regard to the incidence of intensive care admission, receipt of mechanical ventilatory support, length of hospital stay, and overall mortality. furthermore, the severity of diarrhea had no correlation with the virus load in nasopharyngeal specimens and overall mortality (figure 2). sars is predominantly a viral pneumonia with a rapid tempo of deterioration. however, extrapulmonary manifestations are not uncommon. skin rash [4] , petechial cutaneous bleeding with prolonged activated partial thromboplastin time and increased d-dimer [12] , impaired liver function, subclinical left ventricular diastolic dysfunction [13] , and rhabdomyolysis [14] were occasionally reported. one of the more common presentations other than respiratory symptoms is diarrhea [4, 7, 9, [15] [16] [17] [18] . our previous report that 170% of patients in the amoy garden cohort developed diarrhea showed that fecal excretion may be an important mode of shedding and transmission [7] . subsequent epidemiological investigation confirmed the importance of faulty sewage systems in propagating this massive outbreak of disease. retrospective virological examination of stool samples by rt-pcr confirmed the presence of the virus in 190% of the samples obtained between day 14 and 21 after onset of symptoms, which is almost twice the positivity rate of the corresponding nasopharyngeal samples [8] . it is therefore interesting to examine the role of sars coronavirus in the pathogenesis of diarrhea in patients with sars. diarrhea is not an uncommon symptom during hospitalization for patients with infectious diseases other than acute gastroenteritis. in fact, diarrhea has been well reported in pneumonic illnesses other than sars, such as legionnaires disease [19] , pneumocystis carinii pneumonia [20] , and influenza (especially in children) [21] . moreover, diarrhea is not uncommon in association with many systemic infections. for example, the rate of diarrhea was reported to be 34.5% among children with primary dengue fever [22] . the exact mechanisms leading to diarrhea in these systemic or pulmonary infections were not known, but they could be related to the cytokine profiles or metabolic changes associated with sepsis. in addition to primary viral illness, other potential causes of diarrhea include the use of drugs during hospitalization and c. difficile superinfection, because most patients with sars have been empirically treated with antibiotics that cover typical and atypical pneumonia. finally, diarrhea can be the direct result of viral cytolysis, because the virus can grow and produce a cytopathic effect in a large intestinal cell line, caco-2 [23] . a study of the correlation between the clinical parameters, microbiological findings, and outcomes for patients with sars with and without diarrhea may differentiate the relative importance of the suggested causative factors. in this cohort of 142 patients with sars, only 10.6% of the patients had diarrhea at hospital admission. during hospitalization, 69 (48.6%) of the patients had developed diarrhea at a mean time of days after the onset of symptoms. 7.6 ‫ע‬ 2.6 the most severe diarrhea occurred 1-2 days after the onset of diarrhea, with a maximum frequency of 24 episodes per day. in 6 patients, diarrhea was so severe that intravenous fluid hydration was warranted. the diarrhea was relatively painless and watery, as we have reported previously [7] . no correlation was found between diarrhea and hematological changes, radiological changes, use of antibacterials, or isolation of c. difficile and rotavirus from stool specimens. the only significant correlation was that there was a higher virus load in the nasopharyngeal specimens taken from patients with diarrhea on day 10 after the onset of symptoms. the finding that a higher virus load-but not the presence of diarrhea-predicted mortality in this group of patients is not unexpected. in 2 previous studies of animal models of sars [6, 24] , histological changes of acute respiratory distress syndrome (ards) and inflammation-characterized by diffuse alveolar damage (disruption of alveolar walls and infiltration with neutrophils and macrophages), alveolar hyaline membrane formation, as well as multinucleated syncytial cells-were found in a macaque monkey model artificially inoculated with sars coronavirus [6] . none of the monkeys developed diarrhea over a period of 16 days, despite the fact that rt-pcr results were positive for sars coronavirus in 1 of the stool samples. similarly, no histopathological changes could be detected in the intestines of the monkeys, despite there being concomitant florid pathological changes in the lungs [6] . these findings suggest that viral rep-lication triggers a different response and thus different functional impairment in the lungs and the gastrointestinal tract. a high virus load in the nasopharynx specimens either may predict severe pneumonia, ards, and mortality in some patients, or it may predict just diarrhea in other patients. this postulation is in keeping with the lack of significant difference in hematological markers of inflammation or increased cell turnover (lactate dehydrogenase level) between the groups with or without diarrhea. although suppression of inflammatory changes in the intestine resulting from steroid therapy is possible, the routine use of steroids and ribavirin as part of the treatment protocol did not allow us to control the effect of use of steroids or antivirals on diarrhea. moreover, the use of steroids has not affected the severe inflammatory reactions in the lungs of persons who have died of sars [25] . in our previous study, we showed that rotavirus can be detected by viral isolation in cell culture and by indirect immunofluorescent antigen detection [10] . this is not unexpected, because many viruses that affect the gastrointestinal tract also affect the respiratory tract, and vice versa. for instance, in several studies, rotavirus has been well reported to be associated with bronchiolitis and fatal pneumonitis in immunocompromised as well as immunocompetent hosts [11, 26, 27] . others reported that respiratory symptoms were found in 93% of patients with gastroenteritis caused by enteric adenovirus [28] . this virus has been found in samples from the upper respiratory tract and in stool samples obtained from these patients [29] . similar to rotavirus and adenovirus, the sars coronavirus can multiply and shed in the mucosa of the upper aerodigestive tract. in this regard, determination of the virus load in specimens from the nasopharynx may be useful for predicting the severity of illness in both the respiratory and the gastrointestinal tracts. from the laboratory perspective, rt-pcr is more likely to be affected by the abundant inhibitors present in stool than by nasopharyngeal secretions. moreover, the volume of diarrhea varies markedly from patient to patient. this may also affect the interpretation of data on the virus load in stool. therefore, we attempted to use nasopharyngeal specimens instead of stool specimens in this study. coronaviruses can cause respiratory or diarrheal diseases in both human and animals [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] . human enteric coronavirus has been seen in diarrheal stool samples, but culture methods and characterization are still problematic [30, 31] . human coronavirus oc43 and 229e are known to cause one-third of cases of the common cold [45, 46] . in the case of porcine coronavirus, a collection of transmissible gastroenteritis coronavirus (tgev) recombinants were generated to study the molecular basis of tgev tropism [47] . recombinants of group 1 had enteric and respiratory tropism, whereas group 2 recombinants infected the respiratory but not the enteric tract. a substitution in amino acid 219 of the s protein in group 1 recombinant was responsible for the loss of enteric tropism [47] . on the other hand, targeted recombination of the s gene of tgev resulted in a change from respiratory to enteric tropism and enhanced virulence [48] . however, no major deletion or mutation of functional significance had been identified by genomic sequencing of sars coronavirus isolated from the diarrheal patients [49] . we have clearly demonstrated the presence of a numerous virus in tissue biopsy specimens from the terminal ileum and colon of a sars patient with diarrhea [9] . the virus is capable of active replication inside the endoplasmic reticulum and adheres to the surface of enterocytes in both small and large intestines (as shown by electron microscopy), without causing significant villous atrophy or inflammatory cell infiltration in the mucosal or submucosal regions [9] . the absence of inflammation, cell necrosis, or microvilli atrophy does not exclude sars coronavirus as the cause of diarrhea in these patients. for astrovirus-associated diarrhea in turkeys, the animal model has demonstrated mild histological changes, with a surprising lack of inflammation due to increased activation of the potent immunosuppressive cytokine transforming growth factor b at the peak of diarrhea during astrovirus infection [50] . additional studies should be performed to ascertain the mechanism used by sars coronavirus in the pathogenesis of diarrhea in humans. 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n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome astrovirus induces diarrhea in the absence of inflammation and cell death key: cord-303330-zh8wzza5 authors: magleby, reed; westblade, lars f; trzebucki, alex; simon, matthew s; rajan, mangala; park, joel; goyal, parag; safford, monika m; satlin, michael j title: impact of sars-cov-2 viral load on risk of intubation and mortality among hospitalized patients with coronavirus disease 2019 date: 2020-06-30 journal: clin infect dis doi: 10.1093/cid/ciaa851 sha: doc_id: 303330 cord_uid: zh8wzza5 background: patients hospitalized with coronavirus disease 2019 (covid-19) frequently require mechanical ventilation and have high mortality rates, but the impact of viral burden on these outcomes is unknown. methods: we conducted a retrospective cohort study of patients hospitalized with covid-19 from march 30 to april 30, 2020 at two hospitals in new york city. sars-cov-2 viral load was assessed using cycle threshold (ct) values from a reverse transcription-polymerase chain reaction assay applied to nasopharyngeal swab samples. we compared patient characteristics and outcomes among patients with high, medium, and low admission viral loads and assessed whether viral load was independently associated with risk of intubation and in-hospital mortality. results: we evaluated 678 patients with covid-19. higher viral load was associated with increased age, comorbidities, smoking status, and recent chemotherapy. in-hospital mortality was 35.0% with a high viral load (ct<25; n=220), 17.6% with a medium viral load (ct 25-30; n=216), and 6.2% with a low viral load (ct>30; n=242; p<0.001). the risk of intubation was also higher in patients with a high viral load (29.1%), compared to those with a medium (20.8%) or low viral load (14.9%; p<0.001). high viral load was independently associated with mortality (adjusted odds ratio [aor] 6.05; 95% confidence interval [ci]: 2.92-12.52; p<0.001) and intubation (aor 2.73; 95% ci: 1.68-4.44; p<0.001) in multivariate models. conclusions: admission sars-cov-2 viral load among hospitalized patients with covid-19 independently correlates with the risk of intubation and in-hospital mortality. providing this information to clinicians could potentially be used to guide patient care. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a novel pathogen that has rapidly caused a devastating pandemic of coronavirus disease 2019 (covid19) . as of june 10, 2020, sars-cov-2 had infected more than 7 million people and killed more than 400,000 people throughout the world [1] . although the majority of patients who develop covid19 have mild presentations [2] , 18-33% of patients who are hospitalized require mechanical ventilation and up to 20% of hospitalized patients die [3] [4] [5] [6] [7] . investigations of risk factors for intubation and mortality with covid-19 in hospitalized patients have largely focused on patient characteristics, such as older age, obesity, and comorbidities, as well as presenting symptoms and laboratory parameters [5, [7] [8] [9] . in contrast, the impact of sars-cov-2 viral load on clinical outcomes in hospitalized patients has not been thoroughly investigated. in two studies of hospitalized patients in china, those with severe presentations of covid-19 had higher viral loads than those with mild presentations, but the impact of sars-cov-2 viral load on the risk of intubation or death was not evaluated [10, 11] . the current standard-of-care test to diagnose covid-19 is to collect a nasopharyngeal (np) swab and use a reverse transcription-polymerase chain reaction (rt-pcr) assay to detect sars-cov-2 rna [12] . these rt-pcr assays only report to clinicians whether sars-cov-2 is detected or not detected. however, these assays also contain quantitative information on cycle threshold (ct) values that are inversely correlated with viral load and are not reported clinically. we hypothesized that assessing sars-cov-2 viral load by analyzing ct values from an initial np swab sample could be a clinically valuable tool to identify patients at highest risk of intubation and death and provide insights into the pathogenesis of covid-19. we therefore conducted this retrospective analysis of sars-cov-2 viral loads on admission, clinical presentations, and outcomes at two affiliated new york city hospitals using a high-throughput rt-pcr assay. m a n u s c r i p t 6 this retrospective observational study consisted of all patients who were hospitalized at newyork-presbyterian hospital/weill cornell medical center and affiliated lower manhattan hospital and had a np swab sample collected and analyzed for sars-cov-2 by the cobas 6800 rt-pcr system (roche molecular systems, inc., branchburg, nj) between march 30, 2020 and april 30, 2020. the predominant np swab collection and transport kits used were the bd universal viral transport system (becton, dickinson and company, franklin lakes, nj) and the universal transport medium (hardy diagnostics, santa maria, ca). patients who did not have an np swab sample collected and analyzed within one day of hospital admission or whose sample was analyzed on a different diagnostic platform or at a different institution were excluded. the policy during the study period was to only perform sars-cov-2 tests in patients who were thought to require hospital admission; however, some patients who were tested were subsequently discharged from the emergency department (ed) without hospital admission. the cobas sars-cov-2 rt-pcr test received emergency use authorization approval by the united states food and drug administration and was performed according to the manufacturer's instructions [13] . this assay amplifies two different targets within the sars-cov-2 genome: orf1ab, a sars-cov-2-specific target and the e gene, a pan-sarbecovirus target that is present in sars-cov-2 and sars-cov, but not in seasonal coronaviruses or a c c e p t e d m a n u s c r i p t 7 middle east respiratory syndrome-cov. for routine clinical care, results are classified as detected if either the orf1ab or e gene is detected, or not detected if neither target is detected. however, the instrument also generates a ct value for each target that correlates inversely with quantitative viral load and is not released to clinicians. the ct value represents the number of replication cycles required for sufficient gene amplification to produce a fluorescent signal that crosses a predefined threshold. for this study, we reviewed ct values for both gene targets for all initial sars-cov-2 tests that were performed on np swab samples that were collected from study subjects for routine clinical care within one day of hospital admission. we separated the ct values for the sars-cov-2-specific target (orf1ab) into terciles based upon the quantitative values. we then designated high viral load samples as the lowest ct tercile, medium viral load samples as the middle tercile, and low viral load samples as the highest tercile. specimens that were designated positive for sars-cov-2, but for which only the e gene was detected, were designated low viral load samples. data were retrospectively abstracted manually from the electronic medical record using a quality-controlled protocol and entered into a redcap database [14] . all data collectors were trained and a random re-sampling of data previously showed high interrater reliability (mean cohen's kappa of 0.92) [5] . data included demographics, comorbidities, social characteristics, selected outpatient medications on admission, presenting symptoms on arrival to the hospital, oxygen supplementation required within three hours of presentation, laboratory parameters, chest radiograph findings, concurrent bloodstream infections, in-hospital complications, and inhospital mortality. clinical data after hospital discharge were not consistently available, and thus a c c e p t e d m a n u s c r i p t 8 only outcomes that occurred during the hospital admission were analyzed. the study was approved by the institutional review board (#20-03021681) at weill cornell medicine with a waiver of informed consent. we compared baseline characteristics and outcomes of hospitalized patients with covid-19 who had high, medium, and low initial viral loads using the non-parametric nptrend command in stata (statacorp, college station, tx) that tests for trend across ordered groups. continuous variables were represented with medians and interquartile ranges (iqr) and categorical variables were represented as proportions. a two-sided p value of ≤0.05 was used to designate statistical significance. the risk of in-hospital intubation and death was also compared across eight different numerical ct value ranges. we also constructed cox proportional hazard models to compare the cumulative risks of intubation and death during the inpatient admission among patients with high, medium, and low viral loads. we then identified baseline factors that were associated with in-hospital mortality and intubation using univariate logistic regression models. all variables that were statistically significantly associated with each outcome were then entered into separate multivariate logistic regression models. adjusted odds ratios of mortality and intubation were calculated for each of these variables with 95% confidence intervals (ci). analyses were conducted using stata, version 15.0. m a n u s c r i p t 9 a total of 678 np swab samples were available for analysis from unique hospitalized patients who met the study inclusion criteria ( figure 1 ). ct values for the orf1ab locus ranged the median age of patients with high, medium, and low viral loads was 72, 69, and 63 years, respectively (p<0.001; table 1 ). in addition to older age, patients with higher viral loads were more likely to have coronary artery disease, congestive heart failure, cerebrovascular disease, hypertension, chronic obstructive pulmonary disease (copd), chronic kidney disease, and active cancer. they were also more likely to be a former or current smoker or have received recent chemotherapy. patients with high viral loads had a median of 7 days from symptom onset until hospital admission, compared to 8 and 10 days for patients with medium and low viral loads, respectively (p<0.001). patients with higher viral loads were also more likely to a c c e p t e d m a n u s c r i p t 10 require oxygen by a non-rebreather, high-flow nasal cannula, or mechanical ventilation within three hours of presentation to the ed, but were less likely to present with fever, nausea, or vomiting. lymphopenia, anemia, and thrombocytopenia were more common among patients with higher viral loads; whereas, alanine aminotransferase elevations were less common. there were no differences in chest x-ray findings among patients with high, medium, or low viral loads. there were also no differences in viral loads among different racial or ethnic categories or between patients who did and did not use angiotensin-converting enzyme inhibitors (aceis), angiotensin ii receptor blockers (arbs), or hydroxychloroquine. the last day of study follow-up was june 8, 2020. by that day, 19.2% of patients had died during their admission, 75.8% had been discharged alive, 1.6% had been transferred to another hospital, and 3.4% were still hospitalized. the risk of intubation and death increased with higher viral loads. in-hospital mortality was 35.0% in patients with a high viral load, compared to 17.6% in patients with a medium viral load, and 6.2% in patients with a low viral load (p<0.001; table 1 ). the risk of intubation was 29.1% in patients with a high viral load, compared to 20.8% and 14.9% in patients with a medium or low viral load, respectively (p<0.001; table 1 ). these associations were also observed in time-based analyses (figure 2) , where compared to a low viral load, a high viral load was associated with a hazard ratio (hr) of in a multivariate model that adjusted for age, race, coronary artery disease, congestive heart failure, cerebrovascular disease, hypertension, copd, days of symptoms prior to admission, symptoms upon presentation, initial chest x-ray findings, and level of oxygen support within three hours of arrival to the ed (table 2) , having a high viral load was independently associated with increased risk of in-hospital mortality (adjusted odds ratio [aor] 6.05; 95% ci: 2.92-12.52; p<0.001) compared to having a low viral load. the risk of in-hospital mortality was also higher in patients with a medium viral load compared to a low viral load, but this association was not statistically significant (aor 2.06; 95% ci: 0.98-4.34; p=0.058). compared to those with a low viral load, having a high viral load was also independently associated with increased risk of intubation (aor 2.73; 95% ci: 1.68-4.44; p<0.001); whereas, the risk of intubation associated with a medium viral load did not reach statistical significance (aor 1.59; 95% ci: 0.96-2.63; p=0.07). patients with higher viral loads were also more likely to develop myocardial infarction, congestive heart failure, and acute kidney injury requiring hemodialysis (table 1) . this study demonstrated that patients who were admitted to the hospital with high sars-cov-2 viral loads, as assessed by ct values of np swab samples, were more likely to be intubated or die during their hospitalization. this association persisted even when adjusting for age, comorbidities, presenting symptoms, chest radiography findings, and degree of presenting hypoxia. while prior studies indicated that viral load correlates with severity of covid-19 presentation [10, 11] , our study of a larger cohort of hospitalized patients adds to this knowledge a c c e p t e d m a n u s c r i p t 12 base by identifying that admission viral load has important prognostic implications. reporting sars-cov-2 viral load based on ct values from admission np swab samples could therefore help identify patients who are at highest risk of adverse outcomes and who therefore may benefit from more intensive monitoring. identifying high viral load patients could also be helpful for allocating scare therapeutic interventions such as antiviral agents (e.g., remdesivir) [15] . our findings also suggest that stratification or adjustment for baseline viral load would benefit the design of clinical trials of antiviral agents for covid-19. it is also possible that viral load could be used along with other factors, such as age, comorbidities, and severity of symptoms and hypoxia to decide upon the need for hospital admission. however, additional studies that evaluate viral loads and clinical outcomes among all patients who present to the ed are warranted prior to pursuing this strategy clinically. older age and the presence of comorbidities such as hypertension, coronary artery disease, congestive heart failure, copd, and cancer are known to be associated with worse outcomes in covid-19 [2, 7, 16, 17] . such patients may have decreased cardiopulmonary reserve and thus are less likely to tolerate the physiologic insults caused by covid-19. our findings suggest these patients also have higher sars-cov-2 viral loads when they present to the hospital, which may contribute to the worse outcomes observed in these patients. reasons for higher viral loads specifically in these populations are not well understood and warrant further investigation. given that sars-cov-2 uses the angiotensin-converting enzyme 2 receptor (ace2) for entry into host cells [18] , there have been concerns that use of aceis and arbs may upregulate ace2 expression and lead to increased viral proliferation into host cells [19] . although patients with hypertension and congestive heart failure were more likely to have higher viral loads, use of aceis and arbs was not associated with higher viral load. our findings are consistent with those of observational studies that have not demonstrated worse outcomes in a c c e p t e d m a n u s c r i p t 13 patients who use aceis or arbs [20] [21] [22] and support the recommendations of professional societies of not discontinuing these medications in the setting of covid-19 [23] . another notable finding from this study is that there were no differences in admission sars-cov-2 viral loads or outcomes among different racial or ethnic groups. in the u.s., hispanic and black communities have been disproportionately affected by covid-19, with a greater proportion of deaths among these patients than what would be expected based on their population proportions [24] [25] [26] . our finding that admission viral loads were not different among race and ethnicity groups suggests that these disparities are not to related to viral load, but instead may be related to comorbid illnesses and non-biological factors such as social determinants of health. this further underscores the importance of studies that examine the impact of social determinants of health on outcomes during the covid-19 pandemic. we also found that patients with higher viral loads were more likely to develop myocardial infarction, congestive heart failure, and acute kidney injury. it is unclear whether these associations were from chance, were related to increased hypoxia in heart and kidney tissue, or were related to increased viral infection of these organs. a recent autopsy study demonstrated that sars-cov-2 frequently directly infects both the heart and kidney [27] and kidney injury and myocardial injury are commonly reported complications of severe covid-19 [28, 29] . additional studies are warranted to assess the relationship between viral loads in np swab samples, disease burden in the heart and kidney, and clinical outcomes. ct values as the cobas 6800 assay used in this study [30, 31] . thus, we suspect that our findings may also be applicable to other diagnostic platforms. we encourage others to evaluate the relationship between clinical outcomes and ct values using other diagnostic platforms and other patient populations. another potential role for reporting sars-cov-2 viral loads through ct values is to guide the use of isolation precautions, given that viral load correlates with infectivity [32] [33] [34] . our study did not assess this potential use of ct values, but we believe this is an important area for future investigation. another limitation is that our study was retrospective and relied on data that were documented in the electronic medical record, and thus could have misclassified patient characteristics or outcomes. however, our data abstraction process utilized a standardized protocol and our queries identified high interrater reliability for data collection. lastly, we focused on in-hospital mortality, and did not capture deaths that occurred after discharge from the hospital. in conclusion, we found that admission sars-cov-2 viral loads, as determined by ct values that are generated with standard-of-care diagnostic assays, are independently associated with intubation and death among hospitalized patients with covid-19. these a c c e p t e d m a n u s c r i p t 26 1 p values were calculated using the non-parametric nptrend command in stata, version 15.0, that tests for trend across ordered groups. 2 this variable was not assessed in all participants. the denominator is listed next to the variable. 3 ast elevation indicates a value >34 units/l. 4 alt elevation indicates a value >55 units/l. a c c e p t e d m a n u s c r i p t 27 covid-19): situation report -142 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the characteristics of clinical outcomes of adult patients hospitalized with covid-19 -georgia clinical characteristics of covid-19 in new york city epidemiology, clinical course, and outcomes of critically ill adults with covid-19 in new york city: a prospective cohort study characterization and clinical course of 1000 patients with coronavirus disease 2019 in new york: retrospective case series clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid-19 viral load dynamics and disease severity in patients with sars-cov-2 in zhejiang province viral dynamics in mild and severe cases of covid-19 interpreting diagnostics tests for sars-cov-2 cobas sars-cov-2 eua assay package insert v1 research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support emergency use authorization of remdesivir: the need for a transparent distribution process risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china cancer patients in sars-cov-2 infection: a nationwide analysis in china structure of the sars-cov-2 spike receptor-binding domain bound to the ace 2 receptor upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan, china renin-angiotensin-aldosterone system blockers and risk of covid-19 association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease 2019 (covid-19) addresses concerns re: using raas antagonists in covid-19 covid-19 fatalities tracker/nysdohcovid-19tracker-fatalities?%3aembed=yes&%3atoolbar=no&%3atabs=n. accessed on chicago's coronavirus disparity: black chicagoans are dying at nearly six times the rate of white residents, data show. chicago tribune hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states multiorgan and renal tropism of sars-cov-2 association of cardiac injury and mortality in hospitalized patients with covid-19 in wuhan, china renal involvement and early prognosis in patients with covid-19 pneumonia comparison of two high-throughput reverse transcription-polymerase chain reaction systems for detection of severe acute respiratory syndrome coronavirus 2 comparison of cepheid xpert xpress and abbott id now to roche cobas for the rapid detection of sars-cov-2 temporal dynamics in viral shedding and transmissibility of covid-19 predicting infectious sars-cov-2 from diagnostic samples viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious diseases wards key: cord-268809-plgip4h6 authors: bielecki, michel; züst, roland; siegrist, denise; meyerhofer, daniele; crameri, giovanni andrea gerardo; stanga, zeno giovanni; stettbacher, andreas; buehrer, thomas werner; deuel, jeremy werner title: social distancing alters the clinical course of covid-19 in young adults: a comparative cohort study date: 2020-06-29 journal: clin infect dis doi: 10.1093/cid/ciaa889 sha: doc_id: 268809 cord_uid: plgip4h6 background: social distancing and stringent hygiene seem effective in reducing the number of transmitted virus particles, and therefore the infectivity, of coronavirus disease 2019 (covid-19) and could alter the mode of transmission of the disease. however, it is not known if such practices can change the clinical course in infected individuals. methods: we prospectively studied an outbreak of covid-19 in switzerland among a population of 508 predominantly male soldiers with a median age of 21 years. we followed the number of infections in two spatially separated cohorts with almost identical baseline characteristics with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) before and after implementation of stringent social distancing. results: of the 354 soldiers infected prior to the implementation of social distancing, 30% fell ill from covid-19. while no soldier in a group of 154, in which infections appeared after implementation of social distancing, developed covid-19 despite the detection of viral rna in the nose and virus-specific antibodies within this group. conclusions: social distancing not only can slow the spread of sars-cov-2 in a cohort of young, healthy adults but can also prevent the outbreak of covid-19 while still inducing an immune response and colonizing nasal passages. viral inoculum during infection or mode of transmission may be key factors determining the clinical course of covid-19. covid-19 is a pandemic disease [1] transmitted from human to human [2] caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that emerged in late 2019 in wuhan, mainland china [3] [4] [5] . covid-19 can have a severe to fatal course, primarily in the elderly populations [6] , but it also affects children [7] and young adults [8] , in which the clinical course has been described to be mild or even asymptomatic [9] . physical interventions such as social distancing [10, 11] , wearing face masks [12] and implementing strict hygiene measures [13] reduce the rate of infection by reducing the transfer of respiratory viruses from infectious to susceptible persons through contact, droplets, or aerosols [14] . these interventions not only lower the chance of infection but also quantitatively reduce the viral inoculum received by the recipient [15, 16] and may change the route of transmission [17] from direct droplet-transmission in close proximity to the infecting person [18] to indirect transmission via contaminated surfaces [19] ) . higher nasal viral load is associated with worse clinical outcomes for severe acute respiratory syndrome [2, 20] , and higher initial viral exposure is associated with more severe disease [21] . to our knowledge, it is unknown if lowering the viral inoculum during infection with sars-cov-2 or altering the mode of infection by physical means can affect the clinical course of the disease. here, we present an outbreak at a swiss army base with two very similar groups infected prior and after the implementation of stringent social distancing and hygiene a c c e p t e d m a n u s c r i p t measures (sdhms). while both groups show evidence of infection, the rate of symptomatic covid-19 amongst the infected soldiers differed significantly amongst the two groups and was much lower in the cohort where infection happened after the implementation of these measures. we provide evidence that sdhms not only are effective in reducing transmission but also can alter the clinical course of covid-19 in infected individuals. we hypothesize that the difference in the clinical presentation of infected persons might be due to lower viral inoculum during infection or an altered mode of transmission of the virus, but further studies are needed to answer this question. a c c e p t e d m a n u s c r i p t we recruited soldiers stationed at a swiss army base in airolo between march 25 and april 14, 2020. patients not feeling fit for service were required to report to our clinic and were promptly isolated and tested for covid-19 by nasopharyngeal swabs. additionally, asymptomatic soldiers were sampled cross-sectionally as indicated in we observed a covid-19 outbreak at a swiss army base in airolo. three companies (508 soldiers) were stationed at the base (table 1) (table 1) were similar, with a median age of 20.4 years (range, 18-28 years). due to a stringent recruitment process, soldiers with severe health-related constraints are generally excluded from military service. company 1 consisted exclusively of male soldiers, while 12% of companies 2 and 3 were women. some of the soldiers, instructors, and other personnel at the base were stationed in separate units and therefore grouped as "other". they were excluded from further analysis due to group heterogeneity and segmentation into various subgroups with a very low sample size each. a c c e p t e d m a n u s c r i p t on march 11, 2020, we diagnosed the first patient suffering from covid-19 in company 3; we refer to this date as day 1 of the outbreak. in the following weeks, we observed an epidemic in companies 2 and 3 as depicted in figure 1a , while company 1 had no cases. on day 9, it became clear that the disease was widely spreading within companies 2 and 3; both units were put under quarantine, and hygiene measures were rigidly enforced across all three companies: soldiers had to keep a distance of at least 2 m from each other at all times, and in situations where this could not be avoided (e.g., military training), they had to wear a surgical face mask. a distance of 2 m was enforced between the beds and during meals. all sanitary facilities were cleaned and disinfected twice daily. symptomatic soldiers were immediately separated and required to report to our clinic where they were tested for covid-19 using nasopharyngeal swabs. strict separation of the unaffected company 1 from the other companies was enforced. until data censoring on day 54, 29% (102/354) of companies 2 and 3 suffered from pcr-confirmed symptomatic covid-19. none of the 154 soldiers from company 1 was diagnosed with covid-19 ( figure 1a ). all soldiers with symptoms compatible with a respiratory infection, or who did not feel fit for service, were required to present at our clinic. additionally, symptoms as well as vital parameters were assessed daily in the unaffected company 1. it is thus unlikely that we missed a symptomatic case of covid-19. we tested 15 symptomatic soldiers from company 1 for covid-19; all were negative. presented symptomatically in our clinic nor showed symptoms in our daily assessments during the following 19 days of follow-up despite daily assessment. viral concentrations were lower than in symptomatic patients ( figure 3 ) but still detectable. since 29% of the soldiers of companies 2 and 3 had previously presented symptomatically for covid-19, more than 30% of this population must have been infected asymptomatically but still developed a detectable immune response. infection of these soldiers is likely to have happened after the implementation of sdhms given that these measures were implemented 25 days prior to the testing date. the fraction of symptomatic patients with covid-19 amongst all soldiers with evidence of exposure to sars-cov-2 either by pcr or serology was significantly lower (p=0.02, fisher's exact test) in company 1 (0/13, 0%) than in companies 2 and 3 (45/113, 40%). a c c e p t e d m a n u s c r i p t companies also had a higher probability of developing covid-19 when infected. more than 50% of the soldiers of all companies could be sampled, however, 36% of company 1 and 42% of companies 2 and 3 either refused to participate or were not available ( figure 2 ). our sample is likely to be representative for all companies, since we we treated more than 100 young, previously healthy, adult patients with covid-19 at our clinic; all were treated symptomatically. no patient died, was admitted to the intensive care unit, or needed mechanical ventilation. one patient was referred to a hospital with interstitial pneumonia requiring oxygen supplementation for four days but recovered without obvious sequelae. despite the high reported prevalence of thromboembolic complications among severely ill patients with covid-19 [8] , we observed no thromboembolic complications in our population, although pharmacological thrombosis prophylaxis was only used in one case (the hospitalized patient). however, mechanical thrombosis prophylaxis was applied by encouraging physical training and involvement in cleaning and disinfection measures. we describe an outbreak of sars-cov-2 infections in young, healthy soldiers in two spatially separated groups with almost identical baseline characteristics but different clinical courses. while one cohort was heavily affected by covid-19, with 102 cases of suffered from covid-19. strict enforcement of sdhms prior to infection therefore reduced the rate of covid-19 amongst those infected. since we followed up the soldiers for 19 days after testing and soldiers were required to immediately report to our clinic if they became symptomatic during this period, we can exclude that any of the soldiers tested on that day later developed symptoms: 99% of cases become symptomatic before day 14 after infection [22] . while sdhms reduce the reproductive number [23, 24] , these non-pharmacological interventions have to our knowledge not been known to reduce the fraction of patients suffering of covid-19 amongst those who are infected prior to this study. a c c e p t e d m a n u s c r i p t although all three companies were very similar demographically, all members of unaffected company 1 were male soldiers, but approximately 10% of the affected companies 2 and 3 were female. a key role for gender in the spread of the disease is unlikely as other studies have reported no differences in viral shedding between males and females [25] . the literature on the ratio of asymptomatic courses is controversial ranging from 4% of a highly selected and exposed group in shanghai [26] , 18% on the diamond princess cruise ship [27] up to 75% [28, 29] of cross-sectional studies, some even reporting clusters of entirely asymptomatic cases [30] . this large range of the rate of symptomatic covid-19 amongst infected might reflect the differential implementation of measures to prevent exposure to the virus or the mode of infection; as observed between the two groups reported in this study. the companies 2 and 3 showed high infection rates approaching the proposed level of herd immunity of 70% [31] . in company 1, infected after the implementation of sdhm, the infection rates remained significantly lower. the epidemic might have ceased not only due to the implementation of sdhm but also due to herd immunity since both factors effectively lower the reproductive number of the virus. sdhms have been shown to quantitatively reduce the viral inoculum during infection [15, 16] . the route of transmission might also be changed by sdhms [17] from direct droplet-transmission in close proximity to the infecting person [18] to indirect a c c e p t e d m a n u s c r i p t transmission via contaminated surfaces [19] , although the hygiene measures implemented involved regular disinfection of potentially contaminated surface. our data show that sdhms not only slow infection with sars-cov-2 but also can attenuate the clinical course by reducing the rate of symptomatic patients amongst those infected. these findings suggest, that reducing the viral inoculum might not only lead to a reduced probability of infection but also could cause favor an asymptomatic infection while still being able to induce an immunological response at least in a proportion of the infected. however, our study has not directly studied the effect of viral inoculum on the clinical course of an infection with sars-cov-2 but shows the profound effect sdhms have thereon. since our study population consisted of young predominantly male adults, our findings might not be applicable to the general population (especially to the elderly and com a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t world health organization. who director-general's opening remarks at the media briefing on covid-19-11 early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china a new coronavirus associated with human respiratory disease in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention sars-cov-2 infection in children clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study rapid asymptomatic transmission of covid-19 during the incubation period demonstrating strong infectivity in a cluster of youngsters aged 16-23 years outside wuhan and characteristics of young patients with covid-19: a prospective contact-tracing study exploration of the effectiveness of social distancing on respiratory pathogen transmission implicates environmental contributions simulation suggests that rapid activation of social distancing can arrest epidemic development due to a novel strain of influenza risk factors for sars infection among hospital healthcare workers in beijing: a case control study handwashing and respiratory illness among young adults in military training1 1the full text of this article is available via ajpm online at www.elsevier.com/locate/ajpmonline physical interventions to interrupt or reduce the spread of respiratory viruses assessing the airborne survival of bacteria in populations of aerosol droplets with a novel technology a new transmission route for the propagation of the sars-cov-2 coronavirus a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient initial viral load and the outcomes of sars short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study temporal dynamics in viral shedding and transmissibility of covid-19 follow-up of asymptomatic patients with sars-cov-2 infection estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship the contribution of asymptomatic sars-cov-2 infections to transmission-a model-based analysis of the diamond princess outbreak asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility asymptomatic cases in a family cluster with sars-cov-2 infection herd immunity -estimating the level required to halt the covid-19 epidemics in affected countries repeated seroprevalence of anti-sars-cov-2 igg antibodies in a population a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-306495-o0ah1gj9 authors: haidar, ghady title: covid-19, organ transplantation, and the nuances of immunomodulation: lessons learned and what comes next date: 2020-08-11 journal: clin infect dis doi: 10.1093/cid/ciaa1193 sha: doc_id: 306495 cord_uid: o0ah1gj9 nan m a n u s c r i p t 2 the coronavirus disease 2019 (covid-19) pandemic has caused a seismic shift in transplant practices. many centers have suspended live donor transplants, and most have enforced significant restrictions to their deceased donor programs [1] . because of early observations suggesting a role of proinflammatory cytokines in the pathogenesis of severe covid-19 [2] , a prevailing thought has been that the anti-inflammatory effects of immunosuppressive medications may paradoxically diminish disease severity in solid organ transplant (sot) recipients with covid-19. there is precedent in the literature to support this line of speculation. for instance, kidney and liver transplant recipients with sepsis were shown to have lower mortality rates compared to non-transplant patients [3] , which was thought be partially due to a dampening of the destructive aspects of sepsis by immunosuppressive agents. additionally, calcineurin inhibitors modulate the expression of opportunistic infections such as cryptococcosis, protecting against mortality in sot recipients [4] . however, these optimistic notions were at odds with our knowledge that sot recipients with respiratory viral infections such as influenza develop more severe complications than the general population. additionally, transplant patients frequently suffer from the same comorbidities that have been associated with detrimental outcomes in covid-19. nonetheless, and despite early hints that mortality in sot recipients may be high, the outcomes of sot recipients with covid-19 have remained ill-defined. in this issue of the journal, kates and colleagues describe the outcomes of 482 sot recipients with covid-19 across over 50 transplant centers. while the majority of patients were kidney transplant recipients, this is nonetheless the largest study of sot and covid-19 to date and confirms the ominous findings from smaller cohorts: in short, sot recipients with covid-19 are at high risk for complications and death. the authors demonstrate that 78%, 34%, and 27% of sot recipients with covid-19 require hospitalization, intensive care, and mechanical ventilation, respectively. additionally, the inpatient mortality rate was ~20%, which is similar to the pooled weighed mortality rate of ~19% (range 8-33%) reported in studies from the general population with a similar median age and prevalence of comorbidities. indeed, over 90% of sot recipients with covid-19 had chronic medical conditions, and nearly a third were over 65 years of age. the authors should be commended for appropriately navigating the epidemiological quagmire of case fatality rates in covid-19, as comparisons with studies of younger and healthier patients-such as the chinese center for disease control study which reported a mortality rate of 2.3%[5]-would not have been suitable. the only predictors of mortality in the current study were age (> 65 years), heart failure, chronic lung disease, obesity, pneumonia, and lymphopenia. in contrast, the "net state of immunosuppression" had no impact on mortality, neither did time from transplant. thus, while morbidity and mortality related to covid-19 in sot recipients are substantial, a c c e p t e d m a n u s c r i p t 3 they appear to be driven by age and underlying medical conditions and unaffected by immunosuppression, corroborating the results of other studies in the general population. the study included only 30 lung transplant recipients was therefore unable to assess whether mortality in these patients is greatest (as is the case with sepsis [3] ), or whether covid-19 precipitates acute or chronic lung allograft rejection. furthermore, since all laboratory testing was done as standard of care, the study could not evaluate severe acute respiratory syndrome coronavirus 2 (sars-cov-2) viremia or the duration of sars-cov-2 pcr positivity, which may be longer than that of non-transplant patients. while prolonged viral shedding is common and does not imply infectivity [6] , critically ill sot recipients who are indeed, a recent study of deep immune profiling of patients with covid-19 revealed several "immunotypes" ranging from robust cd4 and/or cd8 responses to minimal lymphocyte responses to infection [8] . furthermore, the mortality benefit of dexamethasone among patients with covid-19 who require supplemental oxygen, particularly those who received the drug after their first week of illness [9] , lends some credence to the notion that several issues unique to transplantation will require re-evaluation as the pandemic evolves. first, programs need to be flexible in how they adapt to rising cases in their regions, relying on a tiered approach for adjusting transplant activity based on the burden of covid-19 in the region and hospital. moreover, centers that have remained unaffected by the pandemic may need to resort to temporary cessation of transplants if covid-19 cases surge. second, centers performing pre-operative sars-cov-2 pcr screening for asymptomatic transplant candidates must publish their experiences. these studies should focus on the impact that cancelling cases due to positive pcrs has on waitlist mortality, and on the optimal timing of reactivating a sars-cov-2-positive transplant candidate. third, universal sars-cov-2 testing of deceased donors, which is currently recommended by the ast[7], may one day be relaxed as herd immunity develops and once sars-cov-2 becomes seasonal circulating virus. finally, while it is permissible to use organs other than lungs and intestines from donors with influenza, whether organs from a donor with covid-19 can ever be transplanted warrants careful evaluation. issues to consider include a) the repercussions of sars-cov-2 viremia (which is uncommon and of unclear significance) [2] in the deceased donor and b) whether the use of any organ is safe, given the dissemination of sars-cov-2 to kidneys and other organs in some patients [10] . current guidelines recommend against the use of donors with a history of covid-19 unless a negative sars-cov-2 pcr result is documented[7]. since chronic nasopharyngeal shedding of ostensibly inert sars-cov-2 appears to be common [6] , these recommendations may be modified in the coming years. once an effective vaccine is developed and mass-administered, these paradigms and many others will need to be revisited. in the past decades, the transplant community has had to respond to sars-cov, pandemic h1n1, mers-cov, ebolavirus, zika virus, and others. while covid-19 is a pandemic of unparalleled proportions, transplant providers have learned to adjust to a new normal. looking beyond the current crisis, transplant research efforts should focus on pathogenesis and virology, immunosuppression strategies, donor and recipient screening issues, and vaccine and drug trials in sot. finally, we must prioritize protecting sot a c c e p t e d m a n u s c r i p t 5 recipients from sars-cov-2 infection by enforcing social distancing, implementing universal masking, and utilizing telemedicine services to provide care. funding: this work was supported by the national institutes of health through grant number kl2tr001856 awarded to g.h. early impact of covid-19 on transplant center practices and policies in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan, china inpatient mortality among solid organ transplant recipients hospitalized for sepsis and severe sepsis cryptococcus neoformans in organ transplant recipients: impact of calcineurin-inhibitor agents on mortality characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention virological assessment of hospitalized patients with covid-2019 deep immune profiling of covid-19 patients reveals distinct immunotypes with therapeutic implications dexamethasone in hospitalized patients with covid-19 -preliminary report renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china key: cord-301066-62qe4fb0 authors: chiu, susan s.; hung chan, kwok; wing chu, ka; kwan, see wai; guan, yi; man poon, leo lit; peiris, j. s. m. title: human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong, china date: 2005-06-15 journal: clin infect dis doi: 10.1086/430301 sha: doc_id: 301066 cord_uid: 62qe4fb0 background. human coronavirus nl63 (hcov-nl63) is a recently discovered human coronavirus found to cause respiratory illness in children and adults that is distinct from the severe acute respiratory syndrome (sars) coronavirus and human coronaviruses 229e (hcov-229e) and oc43 (hcov-oc43). methods. we investigated the role that hcov-nl63, hcov-oc43, and hcov-229e played in children hospitalized with fever and acute respiratory symptoms in hong kong during the period from august 2001 through august 2002. results. coronavirus infections were detected in 26 (4.4%) of 587 children studied; 15 (2.6%) were positive for hcov-nl63, 9 (1.5%) were positive for hcov-oc43, and 2 (0.3%) were positive for hcov-229e. in addition to causing upper respiratory disease, we found that hcov-nl63 can present as croup, asthma exacerbation, febrile seizures, and high fever. the mean age (± standard deviation [sd]) of the infected children was 30.7 ± 19.8 months (range, 6–57 months). the mean maximum temperature (±sd) for the 12 children who were febrile was 39.3°c ± 0.9°c, and the mean total duration of fever (±sd) for all children was 2.6 ± 1.2 days (range, 1–5 days). hcov-nl63 infections were noted in the spring and summer months of 2002, whereas hcov-oc43 infection mainly occurred in the fall and winter months of 2001. hcov-nl63 viruses appeared to cluster into 2 evolutionary lineages, and viruses from both lineages cocirculated in the same season. conclusions. hcov-nl63 is a significant pathogen that contributes to the hospitalization of children, and it was estimated to have caused 224 hospital admissions per 100,000 population aged ⩽6 years each year in hong kong. coronaviruses are enveloped rna viruses and are subdivided into 3 groups. human coronavirus 229e (hcov-229e) and human coronavirus oc43 (hcov-oc43) have been recognized since the mid 1960s as causes of upper respiratory disease, including the common cold, and are group 1 and group 2 coronaviruses, respectively. in 2003, a novel coronavirus (sars-cov) was identified as the causative agent of the severe acute respiratory syndrome (sars). its formal taxonomic status within the coronaviridae is not yet established [1] . recently, another group 1 human coronavirus, hcov-nl63, was reported in the netherlands [2, 3] . in one report, 8 adults and children infected with hcov-nl63 virus were documented; 3 patients had lower respiratory tract infection, 2 had an unknown clinical presentation, and the others had upper respiratory tract infection. a second report documented the same coronavirus in an 8-month-old boy with pneumonia [3] . one hundred thirty-nine other children and adults with respiratory tract infections whom tested negative for known viral pathogens underwent screening, which revealed the virus in 4 more children. however, a systematic study of the prevalence of hcov-nl63 infection or its clinical presentation is still lacking. furthermore, there is little information about the seasonality and clinical role of coronaviruses (other than sars-cov) in tropical or subtropical climates. in 2001 and 2002, we performed prospective clinical and virological studies of children (age, р18 years) with acute respiratory tract infection who were admitted to queen mary hospital (hong kong, china). data on human metapneumovirus (hmpv) infection, influenza, respiratory syncytial virus (rsv) infection, parainfluenza virus infection, and adenovirus infection were reported elsewhere [4] . in the present study, we use the clinical specimens to investigate the role of hcov-229e, hcov-oc43, and the newly discovered hcov-nl63 on childhood respiratory infections. hong kong island has a population of 288,371 persons aged р18 years, 84,018 of whom are р6 years old (based on 2001 census data [5] ). two public hospitals of the hospital authority of hong kong, queen mary hospital and pamela youde nethersole eastern hospital, provide 90% of all acute pediatric hospital care for hong kong island, with an admission ratio of 1:1.65. we studied a systematic sample of children (age, р18 years) with acute respiratory infection admitted to queen mary hospital during the period from august 2001 to march 2002. all children admitted to queen mary hospital with signs and symptoms of respiratory infection on one fixed day each week (tuesday) were included in the study. from april through august 2002, study enrollment times were increased to tuesday and thursday. all children with respiratory infection were included, regardless of other complaints. because we had previously documented an association between influenza virus a infection and febrile seizures, children with febrile seizures were also included [6] . nasopharyngeal aspirate (npa) specimens were collected for virological testing from all subjects. two aliquots of the npa specimen were frozen at ϫ70њc for subsequent molecular testing. at the end of the study, all medical records were reviewed by a pediatrician (s.s.c.). viral diagnosis. rapid diagnostic testing for rsv, influenza viruses a and b, adenovirus, and parainfluenza virus types 1-3 was performed with use of immunofluorescent antigen detection and culture, as previously described [4, 7, 8] . hmpv was detected by rt-pcr [4] . coronavirus rt-pcr. total nucleic acid was extracted from 220 ml of the npa clinical specimen using the qiaamp virus biorobot 9604 kit (qiagen), in accordance with the manufacturer's instructions. the total nucleic acid was eluted in 86 ml of the kit elution buffer and stored at ϫ70њc. cdna was generated using random hexamers, as described elsewhere [9] . pcr was performed using a set of consensus coronavirusreactive primers, as well as primers specifically designed to amplify hcov-229e, hcov-oc43, and hcov-nl63 [3, 10, 11] . the consensus primers coro in6-7 (forward, 5 -ggttgg-gactatcctaagtgtga-3 ; reverse, 5 -ccatcatcagat-agaatcatcata-3 ) amplified the conserved region of the coronavirus polymerase gene [9] . two ml of cdna was added to 50 ul of reaction mixture containing 1ϫ pcr buffer (roche diagnostic manufacturer, germany), 2.5 mmol/l each primer, 100 mmol/l dntp (roche), 3 mmol/l mgcl 2 , and 2.5 u of amplitaq gold polymerase (roche). samples were then heated to 94њc for 10 min, followed by 40 cycles of amplification as follows: 1 min each of denaturation, annealing, and extension at 94њc, 48њc, and 72њc, respectively, followed by final 10 min at 72њc. rt-pcr was performed in a multiplex format, as described elsewhere [11] with modification [12] . the pcr conditions were the same as those for the coro in6 and 7 rt-pcr described above, except that denaturation was for 2 min at 94њc, and annealing was for 1 min at 60њc. ten ml of pcr products were analyzed in a 2% (w/v) agarose gel by electrophoresis and stained with ethidium bromide, 0.5 mg/ml. the sizes of the pcr-amplified product for the coronavirus consensus (coro in6-7), 229e, and oc43 rt-pcr were 440 bp, 294 bp, and 469 bp, respectively. a real-time quantitative pcr specific to the n region of hcov-nl63 was used for specific detection and quantitation in npa specimens [3] . in brief, 2 ml of cdna was amplified in a 25-ml reaction containing 0.625 u of amplitaq gold polymerase (roche), taqman buffer a, 0.2 mmol/l dntp, 5.5 mmol/l mgcl 2 , 0.25 u of amperase ung, 0.8 mmol/l each primer, and 0.4 mmol/l probe. the primer sequences were forward, 5 -agga-ccttaaattcagacaacgttct-3 ; and reverse, 5 -gattac-gtttgcgattaccaagact-3 . the probe was 5 -6fam-tgt-tgtttgggttgctaag-mgbnfq-3 . reactions were first incubated at 50њ for 2 min, followed by 95њ c for 10 min, and they were then thermal-cycled for 40 table 2 . comparative results of rt-pcr using consensus and specific primers for human coronaviruses nl63 (hcov-nl63), oc43 (hcov-oc43), and 229e (hcov-229e) in nasopharyngeal aspirate specimens of 587 hospitalized children. rt a one patient with positive results using the coronavirus consensus rt-pcr had undetectable findings with the hcov-nl63-specific rt-pcr. this specimen (hk696) was confirmed to be hcov-nl63 using primers to the hcov-nl63 polymerase 1a region ( figure 3 ). all other specimens that were determined to be coronavirus positive with the consensus primers were also detected by the corresponding type-specific rt-pcr. cycles (95њ c for 15 s and 60њ c for 1 min). plasmid containing the target was used as a positive control. quantitative results are expressed as genome copies per reaction. one copy per reaction approximates to 326 copies per 1 ml of npa. standard precautions were taken to prevent pcr crosscontamination during specimen extraction and pcr analysis. positive and negative controls were included in each rt-pcr experiment. phylogenetic analysis of hcov-nl63. pcr-amplified products of the 1a gene [2] were genetically sequenced, and deduced viral sequences were analyzed and aligned with use of bioedit, version 5.0.9 (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). statistical analysis. hcov-nl63 loads per reaction were converted to log 10 values and correlated with the days from symptom onset using pearson correlation. unpaired t test was used for comparisons between children with high and those with low hcov-nl63 loads. the x 2 test was used to detect the association between hcov-nl63 load and the number of pathogens identified. a 2-sided p value of !.05 was considered to be statistically significant. data on clinical manifestations in patients with hcov-nl63, influenza a, and rsv infection were analyzed with 1-way analysis of variance, and significant results were further tested with a post-hoc tukey-kramer test. we detected coronavirus infections in 26 (4.4%) of 587 children studied during the 13-month period. fifteen children (2.6%) were positive for hcov-nl63 infection, 9 (1.5%) were positive for hcov-oc43 infection, and 2 (0.3%) were positive for hcov-229e infection (table 1) . for comparison, influenza a virus was detected in 7.7% of patients, rsv was detected in 8.2%, and adenovirus was detected in 7.2%, although some of these children had multiple viruses detected (table 1) . children aged р6 years accounted for 510 of the 587 specimens studied. from the identification rate of hcov-nl63 in our representative sample at queen mary hospital, we estimate that 71 children aged р6 years were admitted to this hospital over a 12-month period. with use of the known admission ratio between the 2 hospitals that provide care for 90% of patients on hong kong island, we estimated that 188 children aged р6 years were admitted because of hcov-nl63 infection, leading to an annual hospitalization rate of 224 hospitalizations per 100,000 population aged р6 years. table 2 shows the performance of the coronavirus consensus and type-specific rt-pcr. fourteen patients had cases detected using the consensus coronavirus primers. genetic sequencing of the pcr-amplified product revealed the infecting virus to be hcov-nl63 in 7 children, hcov-oc43 in 5, and 229e in 2. these results were confirmed by the virus type-specific primers in all patients, with the exception of 1 patient for whom the amplified sequence indicated infection with hcov-nl63 (table 2) . this additional patient's case was confirmed using third primer set targeted at the polymerase 1a region used for phylogenetic analysis. the human coronavirus type-specific primers detected an additional 8 and 4 patients infected with hcov-nl63 and hcov-oc43, respectively. therefore, in total, 15 patients had evidence of hcov-nl63 infection (table 2) . hcov-oc43 infection mainly occurred in the fall and winter months of 2001, whereas hcov-nl63 infections mainly occurred in the spring and summer months of 2002 (figure 1). the mean age (‫ע‬sd) of the 15 children infected with hcov-nl63 was months (range, 6-57 months). three 30.7 ‫ע‬ 19.8 children had no documented fever in the hospital. two of them had a history of fever at home. one totally afebrile child was hospitalized for stridor. the mean maximum temperature (‫ע‬sd) for the 12 children who were febrile in the hospital was , and the mean total duration of fever 39.3њc ‫ע‬ 0.9њc (‫ע‬sd) for all children who were ever febrile was 2.6 ‫ע‬ 1.2 days (range, 1-5 days). most children presented with respiratory symptoms, although 1 child presented with febrile seizure without obvious respiratory infection (table 3) . chest radiography was performed for 8 patients, but all findings were normal. five children (35%) had an alternative microbiologic diagnosis (influenza a h3n2 for 3 children, group a streptococcus species isolated from a throat culture for 1 child with scarlet fever, and campylobacter jejuni in the stool of a child with upper respiratory infection and diarrhea). one child with influenza a and hcov-nl63 coinfection also had escherichia coli urinary tract infection. the 11 children who had hcov-nl63 as the sole cause of infection and the patient with diarrhea who had respiratory symptoms not explained by c. jejuni infection were compared with age-matched children with influenza a or rsv infection. children admitted for influenza a had a longer mean duration of fever than did those infected with hcov-nl63 (table 4) . children with hcov-nl63 infection were less likely than those with rsv infection to have lower respiratory involvement (9% vs. 63.6%; ). p p .02 if we excluded patients for whom the presence of other respiratory pathogens was documented, the hcov-nl63 load in npa specimens appears to correlate inversely with the time after onset of symptoms ( ) ( figure 2 ). if patients were r p ϫ0.8 categorized into those with high viral load (у1000 copies/reaction) and low viral load (!1000 copies/reaction), the mean times (‫ע‬sd) between onset of symptoms (defined as onset of fever, if the child was febrile, or as onset of other respiratory symptoms, if the child was afebrile) and specimen collection were and days, respectively ( ). nei-1 ‫ע‬ 0. 6 2.25 ‫ע‬ 0.96 p p .02 ther peak temperature nor duration of fever was found to be significantly different between the 2 groups. six of 10 patients with hcov-nl63 as the sole identified pathogen had a high viral load, compared with 1 of 4 children infected with multiple viruses (p not significant). one child with hcov-nl63 upper respiratory tract infection had positive results of only the consensus primer pcr, so no viral load could be measured. phylogenetic analysis of hcov-nl63 1a gene recovered from 12 patients indicated that these viruses cluster into 2 evolutionary lineages. both lineages were represented in hong kong and appear to cocirculate in the same season ( figure 3) . the 9 patients with hcov-oc43 infection had a mean age (‫ע‬sd) of months (range, 14-57 months) (table 33.6 ‫ע‬ 14. 5 5) . two children remained afebrile while in the hospital: one was a 28-month-old boy with stridor who never had fever, even while at home, and the other was a 23-month-old girl with known early asthma who presented with exacerbation of asthma and a 6-day history of fever (temperature, у39.4њc) before hospital admission. the mean maximum temperature (‫ע‬sd) of febrile children in the hospital was , with a 39.3њc ‫ע‬ 0.4њc mean total duration of fever (‫ע‬sd) of days (range, 5 ‫ע‬ 2.4 1.5-8 days). the mean duration of hospitalization (‫ע‬sd) for this group of children was days. chest radiography 2.6 ‫ע‬ 1.5 was performed for 7 patients, and 3 had evidence of infiltrates. one of these children also had evidence of parainfluenza type 1 infection. the discharge diagnoses of the other 2 were viral pneumonia and atypical pneumonia (table 4) . two children had hcov-229e identified in their npa specimens. one was a 23-month-old girl who presented with rhinitis, cough, and fever and who was found to have acute otitis media and right lower lobe pneumonia during examination. blood culture subsequently yielded streptococcus pneumoniae. the other patient was a 13-year-old boy who had a brain tumor and who had undergone renal transplantation, with prolonged fever. tests of his npa were positive for hcov-229e on 3 occasions over the subsequent 3 months. he had minimal respiratory symptoms throughout and had no abnormalities detected by chest radiography. we documented that human coronavirus infection was a significant cause of hospitalization for children aged р18 years, accounting for 4.4% of all admissions for acute respiratory infections. the newly discovered hcov-nl63 was the most common coronavirus identified and was estimated to be as-sociated with a hospitalization rate of 210.6 hospitalizations per 100,000 population aged р6 years. a previous study showed that 8.2% of children aged !18 months who were admitted to a chicago, illinois, hospital with lower respiratory tract diseases had serological evidence of hcov-229e or hcov-oc43 infection [13] . of these children infected with coronavirus, 59% had pneumonia, 29% had bronchiolitis, and 5.8% had croup. in another study that used an elisa to detect hcov-229e and hcov-oc43 antigen in respiratory tract secretions obtained from 108 children with respiratory infection and 51 siblings, human coronavirus was found in 30% and 29% of the samples from the index children and their siblings, respectively [14] . this high level of detection could be biased by testing during the winter months and by performing tests mostly on specimens that tested negative for hcov-oc43 and hcov-229e are reported to have a winter seasonality in temperate regions, and preliminary data on the new hcov-nl63 from the netherlands also suggests a winter predominance [2, 10, 15] . in hong kong, we found that hcov-oc43 was predominant in the winter, whereas hcov-nl63 had a spring-summer peak of activity. there was little cocirculation of these 2 viruses. our data suggest that the seasonality of coronaviruses in tropical and subtropical regions is not restricted to the winter. we have described the clinical spectrum of this newly discovered hcov-nl63 in a cohort of hospitalized children. in addition to causing upper respiratory disease, we found that hcov-nl63 infection can present as croup, asthma exacerbation, and febrile seizures. the peak age for hospitalization with hcov-nl63 infection was 2-3 years, which is similar to that for hcov-oc43 infection. in comparison, the mean ages (‫ע‬sd) of children admitted to the hospital with influenza, hmpv infection, and rsv infection in the same cohort were , , and months, respectively 30.2 ‫ע‬ 22.6 31.7 ‫ע‬ 18. 7 19.5 ‫ע‬ 17.2 [4] . in contrast to an earlier report, the majority of children with hcov-nl63 infection in our study had no major underlying disorders [3] . a coinfecting pathogen was identified in 5 of the 15 children. the child with c. jejuni diarrhea also had symptoms of an upper respiratory tract infection that were not explained by the bacterial infection but were probably attributable to hcov-nl63. in the other 4 children, the coinfecting pathogen could fully explain the clinical presentation, and it is difficult to comment on the contribution of hcov-nl63. when age-matched children with influenza a or rsv infection and hcov-nl63 infection were compared, children with hcov-nl63 infection had a shorter duration of fever than did those with influenza a, and they were less likely to have lower respiratory tract involvement than were those with rsv infection. the duration of hospitalization seemed to be equivalent for all 3 infections. rapid virological diagnosis of rsv infection and influenza were available to clinicians, but diagnosis of hcov-nl63 infection was retrospective. availability of rapid diagnosis has been shown to result in shorter hospital stay [7] . the hcov-nl63 load in the nasopharynx is high during the first 1-2 days of symptoms and decreases in the next few days (figure 2). no correlation with disease severity was observed. these viral load data derived from cross-sectional data for a small cohort of patients and need to be further confirmed with sequential specimens. this pattern, with peak viral load around the onset of disease, is similar to that seen for influenza but different from that observed for sars [16, 17] . the quantitative rt-pcr missed 1 of the patients with hcov-nl63, suggesting that further optimization of the primer and probe sequence is required. hcov-oc43 was associated with exacerbation of asthma and pneumonia, as well as upper respiratory tract disease, in this study. in 1 study of 11 hospitalized us military recruits who only had evidence of coronavirus seroconversion, 36% had pneumonia [18] . asthma exacerbation was found in 14% of children with hcov-nl63 infection and in 11% of children with hcov-oc43 infections in this study [19] . hcov-229e was repeatedly detected in 1 immunocompromised child over 3 months. genetic analysis of a 305-bp region of the spike protein region of 229e revealed that these 3 specimens had identical genetic sequence (unpublished data). however, more contemporary hcov-229e sequences from hong kong would be needed to conclude whether the sequence identity in these 3 specimens provided evidence of virus persistence rather than reinfection. both reinfection with human coronavirus and prolonged shedding have been previously reported [14] . a number of children with human coronavirus infection had other pathogens detected in the same clinical specimen. multiple pathogens in the respiratory tract of patients with respiratory disease are being increasingly recognized [13] . it is difficult to establish whether these are true coinfections or whether one of the pathogens represents continued shedding from a previous infection. it was previously shown that human coronavirus was detectable by pcr in 0.4% of patients without symptoms of respiratory tract infection and that hcov rna could be detected by real-time rt-pcr up to 14 days after infection [12] . in patients with coinfection, the apparent association between influenza virus a with hcov-nl63 and parainfluenza virus type 1 with hcov-oc43 is likely due to the coincidence of the seasonality of these viruses. in conclusion, we demonstrate that human coronaviruses contribute to hospitalization of children with respiratory disease and that the newly recognized hcov-nl63 is at least as important as-if not more important than-hcov-229e and hcov-oc43 in this regard. we confirm earlier findings from the netherlands [2] that there are at least 2 lineages of hcov-nl63 and demonstrate that both variants may cocirculate in the tropics. a better understanding of human coronavirus epidemiology is of particular importance in view of potential interactions in pathogenesis and serodiagnosis of sars. severe acute respiratory syndrome coronavirus phylogeny: toward consensus identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans children with respiratory disease associated with metapneumovirus in hong kong population census influenza a infection is an important cause of febrile seizures cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients evaluation of the directigen flua+b test for rapid diagnosis of influenza virus type a and b infections rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) identification of a novel coronavirus in patients with severe acute respiratory syndrome detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction coronavirus infection in acute lower respiratory tract disease of infants epidemiology of coronavirus respiratory infections an outbreak of coronavirus oc43 respiratory infection in normandy, france performance of virus isolation and directigen flu a to detect influenza a virus in experimental human infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study coronavirus infections in military recruits: three-year study with coronavirus strains oc43 and 229e the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children we thank bonnie wong and chiu mei pang, for technical support, and wilfred h. s. wong, for statistical analysis. potential conflicts of interest. all authors: no conflict. key: cord-295270-6ptaxg74 authors: titanji, boghuma k; farley, monica m; schinazi, raymond f; marconi, vincent c title: response to correspondence: baricitinib – impact on covid-19 coagulopathy? jorgensen et. al. date: 2020-08-14 journal: clin infect dis doi: 10.1093/cid/ciaa1210 sha: doc_id: 295270 cord_uid: 6ptaxg74 nan a c c e p t e d m a n u s c r i p t this does not, however, preclude the possibility of an imbalance between arms that could emerge during the final trial analysis. baricitinib through its immunomodulatory effects as highlighted by jorgensen et.al may in fact be beneficial in terms of reducing coagulopathy in patients with covid-19, which is thought to be primarily mediated by hyper-inflammation and endothelial damage. all of the cohort studies of baricitinib for covid-19 treatment led to significant decline in inflammatory m a n u s c r i p t markers for patients who received the drug 2,3,8 . we agree that in the pursuit of effective therapeutics against covid-19, there is a need to balance the potential adverse effects of any intervention with its hypothesized benefits and to perform randomized, controlled trials. regarding baricitinib, actt2 should provide clarity on the vte issue in the near future and its role in the treatment of covid-19 in moderate to severe patients. a c c e p t e d m a n u s c r i p t thromboembolic safety reporting of tofacitinib and baricitinib: an analysis of the who vigibase. drug saf baricitinib restrains the immune dysregulation in covid-19 patients. medrxiv20135319 baricitinib therapy in covid-19: a pilot study on safety and clinical impact ruxolitinib in treatment of severe coronavirus disease 2019 (covid-19): a multicenter, single-blind, randomized controlled trial combination of ruxolitinib and eculizumab for treatment of severe sars-cov-2-related acute respiratory distress syndrome: a controlled study the janus kinase 1/2 inhibitor ruxolitinib in covid-19 with severe systemic hyperinflammation mechanism of baricitinib supports artificial intelligence-predicted testing in covid-19 patients use of baricitinib in patients with moderate and severe covid-19 fri0123 safety profile of baricitinib for the treatment of rheumatoid arthritis up to 8.4 years: an updated integrated safety analysis cardiovascular safety during treatment with baricitinib in rheumatoid arthritis vincent marconi and raymond f schinazi are partially funded by -emory university center for aids research (ai050409). raymond f schinazi is funded in part by nih grant 5-r01-mh116695. dr. vincent c. marconi has consulted or received research support from viiv, gilead, lilly and bayer. dr. raymond schinazi served as an unpaid consultant for eli lilly whose drugs are being evaluated in the research described in this paper. in addition, dr. schinazi owns shares in eli lilly and gilead, and is issued patents 20190134039, 10022378, and 9662332. the terms of this arrangement have been reviewed and approved by emory university in accordance with its conflict of interest policies. all other authors do not have any conflicts to declare. a c c e p t e d m a n u s c r i p t key: cord-300550-l28tadhn authors: luers, jan c; rokohl, alexander c; loreck, niklas; wawer matos, philomena a; augustin, max; dewald, felix; klein, florian; lehmann, clara; heindl, ludwig m title: olfactory and gustatory dysfunction in coronavirus disease 19 (covid-19) date: 2020-05-01 journal: clin infect dis doi: 10.1093/cid/ciaa525 sha: doc_id: 300550 cord_uid: l28tadhn coronavirus-disease-2019 (covid-19) caused by the severe-acute-respiratory-syndrome-coronavirus-2 (sars-cov-2) shows a rapid spread over-the-world. given scarce resources, non-laboratory diagnostics is crucial. in this cross-sectional study, two-thirds of european patients with polymerase chain reaction confirmed covid-19 reported olfactory and gustatory dysfunction, indicating the significance of this history in the early diagnostics. m a n u s c r i p t the rapid spread of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) urges clinicians and researchers to analyze the virus, characterize the disease, and find suitable treatment strategies. currently, fever, (dry) cough and fatigue are regarded as the main and most common symptoms in the early disease stage of especially with the transfer of the pandemic to europe, olfactory dysfunction has emerged as a new symptom of 3 interestingly, this symptom was not reported by most of the early studies from china 4,5 and still seems rather uncommon in chinese covid-19 patients, 1 accounting for only up to 5% of the patients. 6 herein, we show for the first time the high prevalence of reduced olfaction and decreased sense of taste in european covid-19 patients. this study was conducted by the university hospital of cologne, cologne, germany in adherence to the tenets of the declaration of helsinki and its later amendments. outpatients who had polymerase chain reaction (pcr) confirmed covid-19 in a deep throat-swab between 22 th and 28 th march 2020 were retrospectively identified by the records of the university hospital of cologne, cologne, germany. beside a covid-19 confirmed by polymerase chain reaction (pcr) in a deep throat-swab (which is the institutional screening standard for diagnosis of sars-cov-2), inclusion criteria were age ≥ 18 years and adequate command of the german language. exclusion criteria were hospitalization in the past or at the current time point caused by covid-19 or at the current time point due to other reasons. all included patients were asked using a standardized twosection questionnaire. section 1 requested demographic data and general information, including the date of positive pcr confirmed test and date of contact to confirmed covid-19 patients. section 2 assessed the detailed history of symptoms potentially associated with covid-19. first of all, patients were asked for the onset of fever, cough, sore throat, rhinitis, muscle aches, headache, diarrhea, reduced olfaction, and a reduced sense of taste during covid-19. if yes, date of the beginning of each symptom was recorded. furthermore, total nasal symptom score (tnss) was evaluated as the sum of scores for each of nasal congestion, sneezing, a c c e p t e d m a n u s c r i p t nasal itching, and rhinorrhea during covid-19 using a four-point scale (0-3). 7 a score of 0 indicated no symptoms, 1 documented mild symptoms that were easily tolerated, 2 described the awareness of symptoms which were bothersome but tolerable, and 3 matched severe symptoms that were hard to tolerate and interfered with daily activities or sleep. tnss was then calculated by adding the score for each of the symptoms to a total score between 0-12. all statistical analyses were performed using commercial software (spss version 26.0 for windows; spss, inc., chicago, il.). to investigate factors related to reduced olfaction as well as to a reduced sense of taste two general linear models were used with explanatory variables of age, gender, tnss, fever, cough, sore throat, rhinitis, and headache, respectively. furthermore, the associations between reduced olfaction and the sense of taste were investigated. the threshold for statistical significance was set at p < 0.05. 72 patients with polymerase chain reaction confirmed covid-19 agreed to participate and were enrolled in this study. none declined. out of these 72 participants, 41 were males and 31 females with a mean age of 38 ± 13 years (range, 21 -87 years; table 1 ). 44 of the 72 included patients (61%) had known contact with individuals with confirmed covid-19. the mean potential incubation time of these 44 patients was 3 ± 2 days. patients were asked 13 ± 3 days (range, 6 -22 days) after the first symptom occurred and 7 ± 1 days (range, 3 -9 days) after a positive covid-19 test. the most common general symptoms included headache (78%), cough (75%), and muscle aches (71%) while diarrhea was relatively rare (31%; table 1 ). reduced olfaction occurred in 53 covid-19 patients (74%) while reduced a sense of taste was present in 50 covid-19 patients (69%). 49 patients (68%) reported both symptoms, while one patient (1%) had only reduced sense of taste and four patients (6%) only reduced olfaction. both symptoms occurred on average at the fourth day after the first symptoms had been noted. however, nine patients (13%) noticed that reduced olfaction and loss of sense occurred together on the first day they realized any symptoms. one patient had reduced sense of taste alone at the first day he realized any symptoms. regarding nasal symptoms, mean tnss was 2.71 ± 2.50 (range, 0 -9; table 2 ). while a lot of patients had nasal congestion (54%), nasal sneezing (50%), and rhinorrhea (53%), nasal itching was rare (11%). none of the included participants reported new medical treatment potentially influencing the sense of smell and taste. a c c e p t e d m a n u s c r i p t the regression model for reduced olfaction was not significant (anova: = 0.610) while the model for reduced sense of taste was highly significant (anova: < 0.001). none of the investigated demographic factors including age and gender were associated with reduced olfaction or reduced sense of taste (p ≥ 0.05, respectively). in addition, fever, cough, sore throat, rhinitis, headache, and tnss were also not associated with reduced olfaction or reduced sense of taste (p ≥ 0.05, respectively). however, there was significance between the presence of reduced olfaction and a reduced sense of taste (p < 0.001). our study shows for the first time that both olfactory and gustatory dysfunction is very common in covid-19 patients, with olfactory dysfunction even leveling the symptom 'cough' at > 70%. we also demonstrate that rhinitis and many of its associated symptoms (nasal congestions, sneezing, and rhinorrhea) regularly occur in these patients, a finding that has not been reported before. others often see olfactory dysfunction as a standalone nasal symptom in covid-19 patients. 7 surprisingly, in participants of our study rhinitis was not associated with reduced olfaction. future studies must show whether there is really no pathophysiological link between the two but just a co-existence. in addition, patients were not tested for other respiratory tract pathogens and therefore other infections were not excluded. this fact and the missing matched control group are significant limitations of this study and have to be evaluated in further studies. according to the results of the tnss the measured nasal symptoms (not the olfactory dysfunction) are mostly mild in nature and not particularly bothersome to these patients. both, olfactory and gustatory dysfunction seem to develop in the early to mid-phase (average 4th day after the onset of symptoms) of the disease. there was only one participant of our study showing olfactory/gustatory dysfunction as the first symptom at all, but we would still regard it as a possible early-warning symptom, especially if it comes along without rhinitis. whether or not gustatory problems in covid-19 patients really target the sense of taste is unclear. the gustatory system (transmitted via the glossopharyngeal, facial and vagal nerve) only recognizes the basic tastes (sweet, sour, salty, bitter and umami (glutamate)), but most of the culinary experiences are recognized by the olfactory nerve. the fact that one of our patients experienced gustatory dysfunction alone with no smelling problems, indicates that more than one pathophysiological pathway might exist. many viruses affecting the upper aerodigestive tract (e.g. rhinovirus, parainfluenza epstein-barr virus, and some coronavirus) can lead to olfactory dysfunction, mostly through an inflammatory reaction of the nasal a c c e p t e d m a n u s c r i p t mucosa. the pathophysiology through which sars-cov-2 affects the olfactory system is unclear. a key receptor for the entry of sars-cov-2 into the host cells is angiotensin-converting-enzyme-2 (ace2). 8 this receptor is expressed in multiple organs (heart, lungs, kidney, intestines, buccal cavity, brain, etc.). a recent preprint study from india reported that inside the olfactory mucosa, the expression of ace2 is restricted to a subset of sustentacular and horizontal basal cells and not the olfactory sensory neurons. 8 thus, sustentacular and horizontal basal cells are thought to be highly susceptible to viral entry. nonetheless, although these cells play a role in the maintenance of the olfactory organ (i.e., metabolic and physical support to the olfactory mucosa as well as regeneration) they are not sensory in function. still little is known about which cells of the olfactory mucosa are affected by sars-cov-2 and whether the virus can directly attack sensory cells or the olfactory bulb directly. detailed clinical evaluation and functional tests regarding olfactory and gustatory dysfunctions including exact grading and duration of the complaints are now a high priority. a c c e p t e d m a n u s c r i p t notes: acknowledgment: all procedures performed in this were in accordance with the ethical standards of the institutional research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. formal approval to conduct this study was obtained from the ethics committee of the university of cologne (no. 20-1161) . informed consent was obtained from all study participants. none of the authors has a conflict of interest. all authors have no financial or proprietary interest in any material or method mentioned in the manuscript. all authors have control of all primary data and they agree to allow to review the data upon request. nasal sneezing, mean ± sd (range) none, n (%) mild, n (%) total nasal symptom score, mean ± sd (range) 2.71 ± 2.50 (range 13.38 ± 3.20 (range, 6 -22) a c c e p t e d m a n u s c r i p t key: cord-271269-0gimxteg authors: gourtsoyannis, john title: covid-19: possible reasons for the increased prevalence of olfactory and gustatory dysfunction observed in european studies date: 2020-05-31 journal: clin infect dis doi: 10.1093/cid/ciaa685 sha: doc_id: 271269 cord_uid: 0gimxteg nan a c c e p t e d m a n u s c r i p t dear editor, leurs et al describe the interesting observation that the reported prevalence of olfactory and gustatory symptoms seems to be substantially higher in european covid-19 cohorts compared to east asia cohorts. most of the studies from east asia report a prevalence of around 4-5%; 1-4 european studies report a prevalence of 34-79%. 4-8 however the authors did not sufficiently explore the possible reasons for this observed discrepancy. i think that it is worth doing so briefly here. some have postulated that one possible explanation for this discrepancy is a cultural difference in the epistemology of how patients and medical staff experience or report these symptoms. 7 whilst this is indeed possible, i feel that this is unlikely to account for such as striking difference in observed prevalence. there are two alternative and potentially more plausible and interesting explanations that i feel are deserving of more study. the first possibility is informed by the hypothesis that there are differences in ace2 receptor expression in the nasopharynx of east asians as compared to european populations. 9 increased expression of ace2 in european populations may contribute to a higher risk of olfactory and gustatory symptoms. the other intriguing possibility is that this emerging symptom amongst european populations is actually related to mutations in the virus itself; mutations which may be resulting in a clinical difference. new genomic analysis shows that the spike mutation (d614g; a g-to-a base change at position 23,403 in the wuhan reference strain) is found almost exclusively in europe 10 . this analysis also shows that it is likely to have originated in europe and has now spread across the continent. if this correlates with the increasing prevalence of olfactory/gustatory dysfunction then the possibility is therefore raised that the european mutation profile represent a clinically distinct strain, associated with a different symptom profile to the reference wuhan virus. this possibility would also raise further lines of inquiry with regards to tracking the evolution of sars-cov-2 virulence and its neuroinvasive potential. i have no conflicts of interest or funding sources to declare a c c e p t e d m a n u s c r i p t clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study neurological manifestations of hospitalised patients with covid-19 in wuhan ,china: a retrospective case series study alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection self-reported olfactory and taste disorders in patients with severe acute respiratory coronavirus 2 infection: a cross-sectional study olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study loss of smell and taste in combination with other symptoms is a strong predictor of covid-19 infection comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2. biorxiv key: cord-278045-hr3r17mz authors: yokota, isao; shane, peter y; okada, kazufumi; unoki, yoko; yang, yichi; inao, tasuku; sakamaki, kentaro; iwasaki, sumio; hayasaka, kasumi; sugita, junichi; nishida, mutsumi; fujisawa, shinichi; teshima, takanori title: mass screening of asymptomatic persons for sars-cov-2 using saliva date: 2020-09-25 journal: clin infect dis doi: 10.1093/cid/ciaa1388 sha: doc_id: 278045 cord_uid: hr3r17mz background: covid-19 has rapidly evolved to become a global pandemic due largely to the transmission of its causative virus through asymptomatic carriers. detection of sars-cov-2 in asymptomatic people is an urgent priority for the prevention and containment of disease outbreaks in communities. however, few data are available in asymptomatic persons regarding the accuracy of pcr testing. additionally, although self-collected saliva has significant logistical advantages in mass screening, its utility as an alternative specimen in asymptomatic persons is yet to be determined. methods: we conducted a mass-screening study to compare the utility of nucleic acid amplification, such as reverse transcriptase polymerase chain reaction (rt-pcr) testing, using nasopharyngeal swabs (nps) and saliva samples from each individual in two cohorts of asymptomatic persons: the contact tracing cohort and the airport quarantine cohort. results: in this mass-screening study including 1,924 individuals, the sensitivity of nucleic acid amplification testing with nasopharyngeal and saliva specimens were 86% (90%ci:77-93%) and 92% (90%ci:83-97%), respectively, with specificities greater than 99.9%. the true concordance probability between the nasopharyngeal and saliva tests was estimated at 0.998 (90%ci:0.996-0.999) on the estimated airport prevalence at 0.3%. in positive individuals, viral load was highly correlated between nps and saliva. conclusion: both nasopharyngeal and saliva specimens had high sensitivity and specificity. self-collected saliva is a valuable specimen to detect sars-cov-2 in mass screening of asymptomatic persons. since its discovery in wuhan, china in late 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has rapidly created a global pandemic of coronavirus disease 2019 . the fast evolution of this pandemic has been attributed to the majority of transmissions occurring through people who are presymptomatic or asymptomatic [1] [2] [3] . accordingly, detection of the virus in asymptomatic people is a problem that requires urgent attention for the prevention and containment of the outbreak of covid-19 in communities [4] . currently, the diagnosis of covid-19 is made by the detection of the nucleic acids of sars-cov-2 typically by real-time quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) testing of specimens collected by nasopharyngeal swabs (nps) [5, 6] . however, few data are available regarding the accuracy of qrt-pcr testing in asymptomatic persons upon which the implications of the current testing strategy depend. the sensitivity and specificity of pcr testing need to be elucidated in order to save unnecessary quarantine and contact-tracing, while minimizing new infections from presymptomatic persons. recently, specimen collection by nps has been under scrutiny, as this method requires specialized health care workers and the use of personal protective equipment (ppe) to mitigate the risk of viral exposure. consequently, self-collected saliva has been reported to have several advantages over nps. as the name implies, self-collection of saliva eliminates the close contact in sampling, obviating the need for ppe. additionally, providing saliva is painless and minimizes discomfort for the test subject. however, although we and others have shown the value of saliva as a diagnostic specimen in symptomatic patients [7] [8] [9] [10] [11] [12] , the utility of saliva in detecting the virus in asymptomatic persons remains to be elucidated. a c c e p t e d m a n u s c r i p t 6 we conducted a mass-screening study to determine and compare the sensitivity and specificity of nucleic acid amplification using paired samples (nps and self-collected saliva) for the detection of sars-cov-2 in two cohorts of asymptomatic individuals. the contact-tracing (ct) cohort included asymptomatic persons that have been in close contact with clinically confirmed covid-19 patients with a positive qrt-pcr by nps. close contact was defined as a person who was within approximately 2 meters of an infected person. contact-tracing was implemented by tracing the links of each infected person identified by two public health centers between june 12 and july 7, 2020. a separate cohort enrolled asymptomatic travellers arriving at tokyo and kansai international airports (airport quarantine [aq] cohort) between june 12 to june 23, 2020. in both cohorts, the subjects were requested to provide saliva in addition to mandatory nps sampling by the medical officers. saliva samples were self-collected in a sterilized 15ml polystyrene sputum collection tube (toyo kizai, warabi, japan) at partitioned booth. multiple partitioned booths enabled parallel sample collection with expedious flow of test subjects, with high feasibility of saliva testing especially in the context of mass-screening. all specimens were transported at 4°c and analyzed within 48 hours at the central laboratory (srl, tokyo, japan). all nps samples in the ct cohort were tested by qrt-pcr. the nps samples in the aq cohort was tested by either qrt-pcr or reverse transcriptase loop-mediated isothermal amplification (rt-lamp) [13, 14] at the discretion of the airport quarantine. all the method of collection for both saliva and nps was the same across all participants at all sites. all neat saliva specimens were self-collected in a sterilized 15ml polystyrene sputum collection tube (toyo kizai, warabi, japan) and transported at 4°c without transport media. while rt-lamp assay for saliva was performed at a central laboratory srl by using the same system and methods. regardless of the test site, all qrt-pcr tests for both nps and saliva were performed using the same methods, according to the manual by national institute of infectious diseases (niid, https://www.niid.go.jp/niid/images/epi/corona/2019-ncovmanual20200217-en.pdf). briefly, 5ul of the extracted rna was used as a template. one step qrt-pcr was performed using thunderbird® probe one-step qrt-pcr kit (toyobo, osaka, japan) and 7500 real-time pcr systems (thermo fisher scientific, waltham, usa). the cycle threshold (ct)-values were obtained using n2 primers (niid_2019-ncov_n_f2, niid_2019-ncov_n_r2) and a probe (niid_2019-ncov_n_p2). test value of qrt-pcr and rt-lamp methods were illustrated by scatter plots and kendall's coefficient of concordance w as nonparametric intraclass correlation coefficient taken non-linearity and censored value into consideration. the performance of diagnostic tests was evaluated by sensitivity se nps (nps)/ se saliva (saliva) and specificity sp nps (nps)/ sp saliva (saliva). sensitivity was positive probability in the infected population and specificity was negative probability in the non-infected population. to evaluate the concordance between nps and saliva test, true concordance probability was defined by ( ) , that p was the prevalence of sars-cov-2. although qrt-pcr using nps may be the best performing test available, it is not a "gold standard" without known clinical outcomes. therefore, the se nps , se saliva , sp nps , sp saliva and p were jointly estimated using a bayesian latent class model [15] [16] [17] of the 2,558 persons screened, consent was obtained from 1,940 persons (75.8%) and 1,924 persons were included for analysis ( figure 1 ). the most common reason for exclusion was the presence of symptoms (n=95; 33%) and declined consent (n=493; 22%) in the ct and aq cohorts, respectively. only 16 persons (0.82%) out of who agreed to participate were excluded due to insufficient saliva volume, confirming the feasibility of self-collection. (table 2a) . 114 persons were negative in both tests, which resulted in 152 of 161 matches. in the aq cohort, viral rna was detected in nps and saliva in five and four samples, respectively, out of 1763 individuals (table 2b ). the sensitivity of nps and saliva were 86% (90% ci: 77-93%) and 92% (90% ci: figure 3 , when the prevalence was varied from 0% to 30%, the point estimate for the true concordance probability ranged from 0.934 to 0.999 and the lower limit of the 90% ci was never below 0.9. true concordance probability with varying estimation constraints of sensitivity is shown to be very high (supplement 1), and therefore the qrt-pcr results from saliva and nps appeared to be sufficiently consistent. this study examined the accuracy of detecting sars-cov-2 by qrt-pcr using nps and saliva in a significant number (n=1,924) of asymptomatic individuals. our results showed that qrt-pcr in both specimens had specificity greater than 99.9% and sensitivity approximately 90%, validating the current practice of detecting infection by nucleic acid amplification. we report for the first time the accuracy of viral detection using natural clinical specimens of asymptomatic persons [18] , that the sensitivity is higher than the 52% to 71% reported in symptomatic patients [5, [19] [20] [21] [22] . covid-19 literature to date have been consistent in identifying the peak viral load at symptom onset with subsequent decline [7, 19, [23] [24] [25] [26] , suggesting the possibility of higher presymptomatic viral load. more recent studies have also shown that infectiousness peaks on or before symptom onset [27] , and that live virus can be isolated from asymptomatic individuals [28] . concomitantly, there have been reports of discrepancy between viral load as detected by qrt-pcr and contagiousness [28] [29] [30] , which may be of utmost importance in controlling outbreaks, as the potential to infect close contacts lends credibility to the current strategy of self-quarantine. although the a c c e p t e d m a n u s c r i p t 12 relationship of contagiousness and viral load is a subject in need of further investigation, abrogation of early infectiousness may also be an effective drug development target. the current study further extends that saliva may be a beneficial alternative to nasopharyngeal fluid in detecting sars-cov-2 in asymptomatic carriers. the comparison between paired samples have shown equivalent utility with similar sensitivity and specificity. however, self-collected saliva has significant advantages over nps sampling especially in the setting of mass screening. for example, saliva collection is non-invasive and does not require specialized personnel nor the use of ppe, which saves time and cost. additionally, providing saliva is painless and minimizes discomfort for the patient. these significant advantages became immediately apparent during our sample collection at the airport quarantine, where queue of international arrivals filtered smoothly through multiple collection booths. self-collection of saliva enables parallel sample collection, which is simply more conducive to simultaneous mass screening of large number of individuals, in settings such as social and sporting events. previous studies comparing the viral load between nps and saliva samples report investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series presumed asymptomatic carrier transmission of covid-19 the implications of silent transmission for the control of covid-19 outbreaks the promise and peril of antibody testing for covid-19 detection of sars-cov-2 in different types of clinical specimens comparison of nasopharyngeal and oropharyngeal swabs for sars-cov-2 detection in 353 patients received tests with both specimens simultaneously consistent detection of 2019 novel coronavirus in saliva saliva is a reliable tool to detect sars-cov-2 saliva as a noninvasive specimen for detection of sars-cov-2 swabs collected by patients or health care workers for sars-cov-2 testing comparison of sars-cov-2 detection in nasopharyngeal swab and saliva clinical evaluation of self-collected saliva by rt-qpcr, direct rt-qpcr, rt-lamp, and a rapid antigen test to diagnose covid-19 loop-mediated isothermal amplification of dna development of reverse transcription loop-mediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus 2 (sars-cov-2) bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard screening without a "gold standard": the hui-walter paradigm revisited estimation of sensitivity and specificity of multiple repeated binary tests without a gold standard false negative tests for sars-cov-2 infection -challenges and implications sars-cov-2 viral load in upper respiratory specimens of infected patients sensitivity of chest ct for covid-19: comparison to rt-pcr correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases profiling early humoral response to diagnose novel coronavirus disease (covid-19) temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) saliva is less sensitive than nasopharyngeal swabs for covid-19 detection in the community setting saliva or nasopharyngeal swab specimens for detection of sars-cov-2 temporal dynamics in viral shedding and transmissibility of covid-19 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards predicting infectious sars-cov-2 from diagnostic samples rapid isothermal amplification and portable detection system for sars-cov-2 rapid and visual detection of 2019 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal amplification assay validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of sars-cov-2 rna rt-lamp for rapid diagnosis of coronavirus sars-cov-2 a rapid, simple, inexpensive, and mobile colorimetric assay covid-19-lamp for mass on-site screening of covid-19 development and clinical application of a rapid and sensitive loop-mediated isothermal amplification test for sars-cov-2 infection we thank tokyo and kansai airport quarantine stations for their cooperation; we thank a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 24 figure 4 key: cord-313617-hh7lccet authors: sigel, keith; swartz, talia; golden, eddye; paranjpe, ishan; somani, sulaiman; richter, felix; de freitas, jessica k; miotto, riccardo; zhao, shan; polak, paz; mutetwa, tinaye; factor, stephanie; mehandru, saurabh; mullen, michael; cossarini, francesca; bottinger, erwin; fayad, zahi; merad, miriam; gnjatic, sacha; aberg, judith; charney, alexander; nadkarni, girish; glicksberg, benjamin s title: covid-19 and people with hiv infection: outcomes for hospitalized patients in new york city date: 2020-06-28 journal: clin infect dis doi: 10.1093/cid/ciaa880 sha: doc_id: 313617 cord_uid: hh7lccet background: there have been limited data regarding the clinical impact of covid-19 disease on people with hiv (pwh). in this study we compared outcomes for pwh with covid-19 disease to a matched comparison group. design: we identified 88 pwh hospitalized with laboratory confirmed covid-19 in our hospital system in new york between march 12 and april 23, 2020. we collected data on baseline clinical characteristics, laboratory values, hiv infection status, covid-19 treatment, and outcomes from this group and matched comparators (one pwh to up to five patients by age, sex, race/ethnicity and calendar week of infection). we compared baseline clinical characteristics and outcomes (death, mechanical ventilation, hospital discharge) for these two groups, as well as cumulative incidence of death by hiv status. results: patients did not differ significantly by hiv status by age, sex or race/ethnicity due to the matching algorithm. pwh hospitalized with covid-19 had high proportions of hiv virologic control on antiretroviral therapy. pwh had greater proportions of smoking (p<0.001) and comorbid illness than demographically similar uninfected comparators. there was no difference in covid-19 severity on admission by hiv status (p=0.15). poor outcomes for hospitalized pwh were frequent but similar to proportions in comparators; 18% required mechanical ventilation and ultimately 21% died during follow-up (compared with 23% and 20% respectively). there was similar cumulative incidence of death over time by hiv status (p=0.94). interpretation: we found no differences in adverse outcomes associated with hiv infection for hospitalized covid-19 patients compared to a demographically similar patient group. a c c e p t e d m a n u s c r i p t background sars-cov-2, the causative agent of coronavirus disease-19 , has infected millions of people worldwide since its identification in december 2019, resulting in >400,000 deaths to-date. [1] patients with immunocompromise have overall poorer outcomes from most serious infections, but this may vary based on the type of immune deficiency and the specific pathogens. hiv infection is one of the most common causes of immunocompromise globally, with over 1 million people with hiv (pwh) in the united states alone. [2] despite the substantial reach of the covid19 in this study we assessed the outcomes of covid-19 disease in pwh, comparing a hospitalized cohort of pwh from our nyc health system and a demographically matched group of comparator patients. we then evaluated factors associated with mortality for pwh hospitalized with covid-19. a c c e p t e d m a n u s c r i p t we identified all hospitalized patients with laboratory confirmed sars-cov-2 infection at five hospitals in the mount sinai health system admitted between march 12 and april 23, 2020. from this cohort we identified 88 patients with hiv-related diagnostic codes or those being treated with antiretroviral medications identified from inpatient or outpatient medication orders. we then selected an hiv uninfected comparator group (from the larger laboratory confirmed sars-cov-2 cohort) using a similar methodology to that used by the veterans aging cohort study for comparisons by hiv status. [5] we identified 405 hiv uninfected patients, matching one pwh to up to five patients by age (+/-2.5 years), sex, race/ethnicity, and calendar week of infection (to account for temporally associated changes in covid-19 management and differences in follow-up time). electronic health record (ehr) data were then collected including demographics, all diagnostic codes and procedures, as well as clinical laboratory measurements and outcomes (death, mechanical ventilation, discharge). smoking status was ascertained from a structured ehr element. oxygen supplementation requirements on admission were collected, verified via chart review and used to categorize covid-19 severity using published criteria. a c c e p t e d m a n u s c r i p t we then compared baseline characteristics, treatments and outcomes, testing to assess significant differences between pwh and comparators using the wilcoxon test for continuous variables and the chi-square test for categorical variables. we also stratified baseline laboratory measures by covid-19 severity for pwh, testing for differences in measures by severity category using ordinal logistic regression. we then compared differences in time to death to assess disease trajectory for hospitalized patients by hiv status by fitting unadjusted cumulative incidence function curves with hospital discharge as a competing risk. curves were compared using the test of pepe and mori. [7] to compare cumulative incidence of death by hiv status accounting for potential confounding factors we then fit a multivariable survival model using fine-grey competing risk methods, including demographics, covid-19 severity, comorbid conditions and laboratory values that differed by hiv status. [8] finally, we compared characteristics of pwh who died during hospitalization to those who were discharged or still alive at the end of follow-up. we explored the independent association of significant factors associated with death for pwh from covid-19 by fitting separate multivariable competing risk models using demographic factors and significant univariate predictors (one model for each predictor). based on our sample size, our primary analysis of proportion of hospital deaths by hiv status had 80% power to detect a 15% increase in the absolute risk of death for pwh compared to uninfected persons. all analyses were conducted using stata version 15. this study was approved by our institutional review board. a c c e p t e d m a n u s c r i p t of the 4,402 patients hospitalized during the study period for covid-19, 88 (2%) were pwh. the median age of pwh hospitalized with covid-19 disease was 61 (table 1; intraquartile range [iqr] 54-67) and most pwh were black (40%) or hispanic/latino (30%). patients did not differ significantly by hiv status when comparing age, sex, or race/ethnicity due to the matching algorithm. consistent with trends noted in hiv cohort studies, [9] pwh also had greater proportions of smoking (55% versus 23%; p<0.001) and comorbid illnesses than demographically-similar uninfected comparators, most notably chronic obstructive pulmonary disease (copd; 10% vs. 3%; p<0.001), cirrhosis (6% vs. 2%; p=0.02) and a history of cancer diagnosis (17% vs. 6%; p=0.001). pwh and uninfected persons had similar covid-19 severity on admission as measured by oxygen supplementation requirements (p=0.15). all pwh admitted for covid-19 were prescribed antiretroviral therapy and most (78%) were receiving integrase inhibitor-based regimens. the majority (58%) of patients with cd4 measurements on hospital admission had counts >200 cells/mm 3 (table s2; p=0.005). the majority of pwh were treated with hydroxychloroquine and azithromycin; experimental or expanded-access agents were used less frequently. most hospitalized pwh were discharged from the hospital during the follow-up period (figure 1 ). there was no significant difference in intensive care use by hiv status in our cohort. poor outcomes for hospitalized pwh were still frequent but similar to proportions in comparators; 18% required a c c e p t e d m a n u s c r i p t mechanical ventilation and 21% died during follow-up compared with 23% and 20%, respectively. competing risks analysis showed similar cumulative incidence of death over time by hiv status with no significant difference in the curves (p=0.94; figure 2 ). hiv was not significantly associated with risk of death in multivariable competing risks analysis after adjusting for demographics, copd, smoking, baseline ferritin level and baseline white blood cell count (table s3) . comparisons of pwh who died to those who were still alive or discharged at the end of follow-up revealed few differences in patients who died compared to those who did not. we did find, however, differences in the proportions who were organ transplant recipients who died versus those who had not died ( among patients from five hospitals in a large health system during the peak of the spring 2020 nyc sars-cov-2 epidemic, we found that pwh hospitalized with covid-19 had a substantial burden of comorbid illness and smoking but had good hiv disease control. although mortality and other adverse outcomes were high among pwh in our cohort, these were not worse than a demographically and temporally matched group with similar covid-19 severity at presentation and fewer comorbidities. our study represents the largest and most diverse cohort of pwh with covid-19 that has been described to date and adds to existing limited evidence that hiv might not be associated with more severe covid-19 disease course. concern that covid-19 disease might be more severe in persons with immunodeficiency or immune dysregulation has been raised since the emergence of the earliest cases. [10] uncontrolled series of immunosuppressed persons such as kidney transplant recipients and cancer patients have shown a c c e p t e d m a n u s c r i p t high mortality rates. [11] outcomes analyses for pwh during the covid-19 pandemic have been limited however, consisting of small case reports or series. [12] a single center in spain reported outcomes for five male pwh hospitalized with covid-19. four of the five men had been discharged and the fifth was in intensive care at the time of their report. [13] larger uncontrolled series from new jersey (13 hospitalized patients), new york city (31 hospitalized patients) and madrid, spain (28 hospitalized patients) also subsequently demonstrated covid-19 outcomes for pwh similar to those described for the general population. [14] [15] [16] this is broadly consistent with our finding that respiratory failure requiring mechanical ventilation and death were not more frequent in pwh when compared to demographically similar persons with covid-19. the lack of outcome differences is even more striking when noting that copd, cancer and smoking, risk factors linked to worse covid-19 outcomes in several previous studies, were far more prevalent in pwh in our cohort than in comparator patients. [17, 18] immunomodulatory effects of sars-cov-2 have been associated with severe sequelae including a cytokine release syndrome. [4] several factors have supported theoretical risks of worse covid-19 disease in pwh including incomplete immune reconstitution and evidence of persistent immune activation in many patients prior to the pandemic. [19, 20] furthermore, increased il-6 and d-dimer measures have been independently associated with chronic hiv infection [21, 22] and have also been closely linked with covid-19 severity in data from mostly hiv uninfected persons. [23, 24] neither biomarker differed on presentation for patients by hiv status nor was either associated with covid-19 severity at presentation for pwh. among pwh, cd4 decline was noted in the majority of patients with available data, consistent with existing immunologic data on covid-19 natural history, but the decrease in cd4 percentage was not large. [25] we did not find evidence of associations between immunologic measures (either decreases from pre-covid-19 values or low values at the time of presentation) and adverse covid-19 outcomes for pwh. organ transplantation was associated with death for pwh in our study, however, suggesting that non-hiv causes of immunodeficiency may be more prominent risks for severe outcomes. our study benefited from data collected from diverse patient groups from five hospitals within a nyc large health system that is one of the largest hiv care providers in the united states. our sample size of pwh was limited but we were nonetheless able to identify a large, well-matched comparison group for comparison of outcomes. to maximize our comparison group size, we limited our matching strategy to demographic and temporal factors using a similar method to the largest american hiv cohort study although this yielded differences in comorbidity profiles for the two groups. [5, 29] this difference in comorbidity profiles may have also been influenced by more frequent medical care for pwh, leading to an imbalance in the ascertainment of comorbid diagnoses. however, the marked difference in smoking history for pwh versus hiv uninfected persons supports likely differences in the true prevalence of these comorbid diseases by hiv status in our cohort. in addition, some measures such as laboratory data were missing for substantial portions of the sample, in proportions too large for imputation. nonetheless, our key exposures and outcomes were manually verified and provide important information for this vulnerable population. in conclusion, we found no differences in adverse outcomes associated with hiv infection for a c c e p t e d m a n u s c r i p t m a n u s c r i p t anti-retroviral therapy (art) 88 (100) -integrase 69 (78) -covid-19 projections the time is now to end the hiv epidemic do biomarkers of inflammation, monocyte activation, and altered coagulation explain excess mortality between hiv infected and uninfected people? the trinity of covid-19: immunity, inflammation and intervention veterans aging cohort study (vacs): overview and description ultra-high-throughput clinical proteomics reveals classifiers of covid-19 infection marginal or conditional probability curves in summarizing competing risks failure time data practical methods for competing risks data: a review excess clinical comorbidity among hiv-infected patients accessing primary care in us community health centers the virus that changed spain: impact of covid-19 on people with hiv covid-19 and kidney transplantation co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series covid-19 pneumonia in patients with hiv -a case series clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 description of covid-19 in hivinfected individuals: a single-centre, prospective cohort severity and mortality associated with copd and smoking in patients with covid-19: a rapid systematic review and meta-analysis cancer patients in sars-cov-2 infection: a nationwide analysis in china lack of mucosal immune reconstitution during prolonged treatment of acute and early hiv-1 infection hiv-associated chronic immune activation hiv status, burden of comorbid disease, and biomarkers of inflammation, altered coagulation, and monocyte activation markers of inflammation, coagulation, and renal function are elevated in adults with hiv infection risk factors for severity and mortality in adult covid-19 inpatients in wuhan clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study dysregulation of immune response in patients with covid-19 in wuhan, china a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 compounds with therapeutic potential against novel respiratory sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study aging and infectious diseases: do patterns of comorbidity vary by hiv status, age, and hiv severity? laboratory values -median (iqr) a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t >50 copies/ul 1 (9) 11 (21) a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-297239-or6h6p9p authors: ridgway, jessica p.; bartlett, allison h.; garcia-houchins, sylvia; cariño, sean; enriquez, aurea; marrs, rachel; perez, cynthia; shah, mona; guenette, caroline; mosakowski, steve; beavis, kathleen g.; landon, emily title: influenza among afebrile and vaccinated healthcare workers date: 2015-06-01 journal: clin infect dis doi: 10.1093/cid/civ163 sha: doc_id: 297239 cord_uid: or6h6p9p background. to prevent transmission of influenza from healthcare workers (hcws) to patients, many hospitals exclude febrile hcws from working, but allow afebrile hcws with respiratory symptoms to have contact with patients. during the 2013–2014 influenza season at our hospital, an influenza-positive hcw with respiratory symptoms but no fever was linked to a case of possible healthcare-associated influenza in a patient. therefore, we implemented a temporary policy of mandatory influenza testing for hcws with respiratory symptoms. methods. from 3 january through 28 february 2014, we tested hcws with respiratory symptoms for influenza and other respiratory pathogens by polymerase chain reaction of flocked nasopharyngeal swabs. hcws also reported symptoms and influenza vaccination status, and underwent temperature measurement. we calculated the proportion of influenza-positive hcws with fever and prior influenza vaccination. results. of 449 hcws, 243 (54%) had a positive test for any respiratory pathogen; 34 (7.6%) hcws tested positive for influenza. an additional 7 hcws were diagnosed with influenza by outside physicians. twenty-one (51.2%) employees with influenza had fever. among influenza-infected hcws, 20 had previously received influenza vaccination, 18 had declined the vaccine, and 3 had unknown vaccination status. there was no significant difference in febrile disease among influenza-infected employees who had received the influenza vaccine and those who had not received the vaccine (45% vs 61%; p = .32). conclusions. nearly half of hcws with influenza were afebrile prior to their diagnosis. hcws with respiratory symptoms but no fever may pose a risk of influenza transmission to patients and coworkers. nosocomial transmission of influenza is an important cause of morbidity and mortality among patients during the influenza season each year [1] . indeed, 17% of influenza cases are acquired in a healthcare setting [2] . sick healthcare workers (hcws) serve as a reservoir for influenza and may transmit the virus to vulnerable patients [3, 4] . to prevent transmission of influenza and other respiratory viruses, the centers for disease control and prevention (cdc) recommends that hcws with fever and respiratory symptoms be excluded from work until at least 24 hours after they are afebrile without the use of antipyretics [5] . in contrast, the cdc suggests that hcws with respiratory symptoms but no fever be allowed to work, provided that they wear a face mask during patient care activities and adhere to proper respiratory etiquette and standard precautions. such afebrile hcws are generally considered to be at low risk of transmitting influenza to patients. however, a recent event that occurred at our 600-bed hospital in chicago raised concern for influenza transmission by an afebrile hcw. in december 2013, our hospital's infection control program was alerted to a case of possible healthcareassociated influenza in an inpatient. a patient was diagnosed with influenza after being hospitalized for 11 days. further investigation of all potential contacts found that a hcw with respiratory symptoms but no fever had cared for the patient in the days before the potential case. this hcw tested positive for influenza and was the only identified source of infection for the affected patient. in accordance with the cdc's guidelines, our hospital's routine sick policy at the time prohibited febrile hcws from working, but not those with respiratory symptoms in the absence of fever. given the risk of influenza transmission to patients from afebrile employees with influenza, we implemented a temporary mandatory influenza-testing policy for all hcws with respiratory symptoms. from 3 january through 28 february 2014, the following policy for mandatory influenza testing was in place. hcws without fever but with respiratory symptoms (including cough, sore throat, runny nose, or congestion) were required to undergo influenza testing to continue working. hcws with fever and respiratory symptoms were not allowed to work, in accordance with the usual sick policy, but were also given the option of being tested for influenza. to test for influenza, flocked nasal swabs were collected from both nares. swabs were then analyzed by the filmarray respiratory panel (biofire, salt lake city, utah), a multiplex polymerase chain reaction (pcr) assay that tests for respiratory viral and bacterial pathogens, including influenza, adenovirus, coronavirus, parainfluenza, and respiratory syncytial virus (rsv), among others. at the time of testing, hcws also completed a screening questionnaire describing their symptoms and influenza vaccination history and had their temperature measured to assess for fever. hcws whose tests were positive for influenza or who did not undergo testing were required to refrain from work for 7 days or until symptoms resolved, whichever was longer. work restrictions were also implemented for employees who tested positive for other viruses depending on work area-for example, hcws with rsv were not allowed to work in the neonatal intensive care unit. hcws with any respiratory symptoms were not allowed to work in the stem cell transplantation unit until symptoms completely resolved, regardless of test result. afebrile employees with negative tests were allowed to continue to work in all other units if they felt well enough to do so, but were required to use a mask at work at all times until their respiratory symptoms had resolved. febrile employees with negative tests for influenza were allowed to return to work when they had been without fever for >24 hours without the use of antipyretics. the χ 2 test was used to compare results. stata 13 statistical software was used for analyses (statacorp, college station, texas). over the 2-month screening period, 449 hcws underwent 458 respiratory virus panel tests; 243 (54%) hcws had a positive test for any respiratory pathogen. the most common viruses isolated were coronavirus (142 positive results), influenza (35 positive results), and rsv (33 positive results). fourteen hcws were coinfected with 2 respiratory viruses. eighty (18%) hcws reported fever or had fever measured during their evaluation. see table 1 for the frequency of symptoms present among individuals infected with the most frequently identified respiratory pathogens. among the hcws with influenza, 33 tested positive for influenza a(h1n1), and 1 tested positive for the h3 subtype of influenza a. one of the hcws with h1n1 simultaneously tested positive for influenza b. figure 1 illustrates the proportion of positive influenza tests over time. an additional 7 hcws were diagnosed with influenza a(h1n1) via pcr testing performed by their primary physicians outside the employee screening program, bringing the total number of influenza-infected employees to 41. these additional hcws were also asked about symptoms of fever and influenza vaccination status. only 21 (51.2%) employees with influenza reported history of fever or were found to be febrile during evaluation. among influenza-infected hcws, 20 had received the influenza vaccine for the 2013-2014 season prior to their influenza diagnosis, 18 had declined the vaccine, and 3 had unknown vaccination status. there was a trend toward fever being more common among influenza-infected employees who had not received influenza vaccination compared with employees who had received influenza vaccination, but this result was not statistically significant (61% [11/18] vs 45% [9/20]; p = .32). of note, our institutional policy expects hcws to receive yearly influenza vaccination, but does not mandate it. overall influenza vaccination compliance among staff at our institution was 68% for the 2013-2014 influenza season. we have characterized the symptoms associated with a variety of respiratory viruses in the context of a mandatory influenza screening program for symptomatic hcws. although a higher percentage of individuals with influenza experienced fever compared with individuals with other respiratory viruses, fever was present in only half of influenza-infected employees. previous studies have similarly reported that a sizeable proportion of individuals infected with influenza a are afebrile, ranging from 32% to 56% [6] [7] [8] . the absence of fever among many influenza-infected individuals raises serious concern about the current practice of using fever as the criteria for excluding hcws from work. fever is often used as a proxy for possible influenza in hcws with respiratory symptoms. in accordance with the cdc's recommendations, many hospitals allow afebrile employees with respiratory symptoms to continue to have contact with patients. because fever is only present half of the time among employees with influenza, using fever as the main exclusion criteria for work is not sufficient to prevent employees with influenza from caring for patients. it is possible that afebrile hcws with influenza may be less contagious than those who have fever. the magnitude of influenza viral shedding is lower in infected individuals with fewer symptoms compared with more highly symptomatic individuals [7] . one study found that the higher a person's temperature, the higher the rate of influenza viral shedding [7] . however, it is not known if the level of viral shedding perfectly correlates with the risk of influenza transmission. afebrile employees with influenza may still shed virus and pose a risk of influenza transmission to patients and coworkers [9] . theoretically, hcws with respiratory symptoms should wear masks and practice hand hygiene, and so the risk of transmission of respiratory viruses to patients should be limited. however, hcw compliance with face masks and other personal protective equipment is self-reported to be around 60% but often observed to be less than this [10] . it is unlikely that hcws with respiratory viral illnesses would have a much higher compliance with this policy. until better hand hygiene and personal protective equipment compliance is demonstrated across multiple healthcare settings, it would be inadvisable to rely solely on these measures to preclude the spread of influenza in hospitals. to prevent healthcare-associated influenza, hospitals should consider more stringent infection control measures for hcws with respiratory symptoms, even if no fever is present. a mandatory influenza testing program for all hcws with respiratory symptoms is one such measure, but is admittedly expensive and labor-intensive. the filmarray respiratory panel alone can cost the laboratory up to $200 per panel, including labor and equipment. a more limited screening program may be sufficient, only testing employees with direct patient care or those with certain symptoms, such as cough. we found that 100% of employees with influenza disclosed having a cough. during the screening program at our hospital, 276 employees reported coughing; if we had only screened hcws with cough, we could have reduced the number tested for influenza by 40%. many hcws with influenza had been vaccinated for influenza in the months prior to their diagnosis. although not statistically significant, a higher percentage of hcws who had not received the influenza vaccine were febrile than hcws who had received the influenza vaccine (61% vs 45%). the influenza vaccine has been shown to reduce the incidence of influenza among healthy adults, and may reduce the severity of illness among vaccinated individuals who do develop influenza [11] [12] [13] [14] . if vaccination predisposes to subclinical or less severe influenza, it may actually contribute to hcws working with influenza because they have mild illness. although several studies have shown that vaccination of hcws may decrease the risk of nosocomial influenza [15] [16] [17] [18] , our findings highlight the importance of not relying solely on influenza vaccination of hcws for prevention of nosocomial influenza transmission. other infection control precautions are necessary, such as careful evaluation of sick employees and use of masks and hand hygiene. our study does have limitations. the temporary policy required influenza testing for afebrile hcws with respiratory symptoms, but not for febrile hcws as they were expected to stay home from work regardless of their test results. hcws with fever and more severe symptoms may not have chosen to be tested, and so we may have underestimated the proportion of influenza-positive hcws with fever and severe symptoms. conversely, there were likely asymptomatic hcws or those with mild symptoms who were not tested, in which case we would have overestimated the proportion of hcws with influenza with fever. other hcws may have been diagnosed with influenza or other respiratory viruses by outside clinicians and not have reported their results to their employer. an additional limitation is that vaccination status was collected based on self-report. it is possible that sick hcws' self-report of vaccination status was not entirely accurate, but there is no reason to believe that febrile hcws with influenza would systematically report vaccination status differently than afebrile hcws with influenza. another potential limitation is that the determination of "fever" was also partially based on self-report. some hcws who reported fever may not have had an objectively measured temperature >37.8°c (100.0°f). however, hospitals' sick policies rely on hcws' self-assessment of fever to determine whether or not they are eligible to work, and so self-report of fever more accurately reflects the true condition of fever identification among hcws in the workplace. pcr may not be 100% sensitive for detection of influenza, and we may have missed some cases of influenza if individuals had a low viral load or if specimens were not properly collected. however, pcr is more sensitive than other influenza diagnostic tests including viral culture [19, 20] . finally, these data were collected only during the 2013-2014 influenza season when the h1n1 strain was the predominant circulating strain; it is unknown if our findings are generalizable to other strains of influenza. we have described the symptoms associated with respiratory viruses among hcws in a large urban hospital. most strikingly, we found that afebrile employees with respiratory symptoms, figure 1 . results of screening tests for influenza over time. including those previously vaccinated, are potential sources of nosocomial influenza transmission. these findings can inform infection control practices and sick leave policies during the influenza season. influenza in the acute hospital setting healthcare-associated influenza in canadian hospitals from molecular characteristics of outbreaks of nosocomial infection with influenza a/h3n2 virus variants nosocomial influenza in children prevention strategies for seasonal influenza in healthcare settings clinical signs and symptoms predicting influenza infection viral shedding and clinical illness in naturally acquired influenza virus infections clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china influenza b in households: virus shedding without symptoms or antibody response the use of personal protective equipment for control of influenza among critical care clinicians: a survey study efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis vaccines for preventing influenza in healthy adults the relative efficacy of trivalent live attenuated and inactivated influenza vaccines in children and adults evaluation of trivalent, live, coldadapted (caiv-t) and inactivated (tiv) influenza vaccines in prevention of virus infection and illness following challenge of adults with wild-type influenza a (h1n1), a (h3n2), and b viruses the influence of staff and resident immunization rates on influenza-like illness outbreaks in nursing homes influenza vaccination of health care workers in long-term-care hospitals reduces the mortality of elderly patients effect of influenza vaccination of nursing home staff on mortality of residents: a clusterrandomized trial influenza vaccination of healthcare workers in acute-care hospitals: a case-control study of its effect on hospital-acquired influenza among patients rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 comparison of the directigen flu a+b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection acknowledgments. we thank the university of chicago microbiology laboratory, respiratory therapy, and occupational medicine for their assistance in implementing the employee influenza surveillance program.financial support. this work was supported by the university of chicago medicine.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-290133-4ou7ubb4 authors: weiss, martin m.; weiss, peter d.; mathisen, glenn; guze, phyllis; henderson, donald a.; inglesby, thomas v.; o'toole, tara title: rethinking smallpox date: 2004-12-01 journal: clin infect dis doi: 10.1086/425745 sha: doc_id: 290133 cord_uid: 4ou7ubb4 the potential consequences of a competently executed smallpox attack have not been adequately considered by policy makers. the possibility of release of an aerosolized and/or bioengineered virus must be anticipated and planned for. the transmission and infectivity of variola virus are examined. arguments for and against pre-event vaccination are offered. the likely morbidity and mortality that would ensue from implementation of a mass pre-event vaccination program, within reasonable boundaries, are known. the extent of contagion that could result from an aerosolized release of virus is unknown and may have been underestimated. pre-event vaccination of first responders is urged, and voluntary vaccination programs should be offered to the public. two defenses against a vaccine-resistant, engineered variola virus are proposed for consideration. methisazone, an overlooked drug, is reported to be effective for prophylaxis only. the extent of reduction in the incidence of smallpox with use of this agent is uncertain. it is useless for treatment of clinical smallpox. n-100 respirators (face masks) worn by uninfected members of the public may prevent transmission of the virus. conventional wisdom holds that smallpox presents an unlikely threat to the public health [1] [2] [3] [4] . that wisdom asserts that the virus is sequestered in 2 secure sites [5] and that, even if it somehow released, vaccine will abort any potential epidemic [3] . moreover, conventional wisdom holds that promising drugs are in development to treat smallpox [6] [7] [8] [9] and that the virus is not highly contagious [3] . such considerations could prove to be overly optimistic and do not take into account the many uncertainties regarding transmission and infectivity of the smallpox virus. in addition to the possible existence of more-virulent "weaponized" strains, further advances in genetic engineering may permit construction of strains able to evade the current vaccine. australian workers markedly increased mousepox virulence by splicing a mouse il-4 gene into a laboratory strain [10] ; similar constructions might be assembled using human smallpox virus (variola major) or another pox virus (e.g., monkeypox virus) and human genes [11] . this article critically examines some of the current tenets of public health policy and highlights the uncertainty of much of the data. we also examine potential defenses against a release of smallpox virus and make recom-mendations regarding immunization and the development of prophylactic medications. smallpox may be transmitted via respiratory droplets or via fine-particle aerosol. the distinction between the two has critical public health implications. respiratory droplets (i.e., sputum and saliva) have a range likely no more than 2 m (∼6 ft) and are therefore a threat only to persons in the immediate vicinity of the affected patient. epidemiological studies support the finding that respiratory droplet spread is the prime route of transmission; the geographic locus of transmission is described as being almost always at the bedside, rather than public areas [12, 13] . freefloating aerosolized virions, on the other hand, would have a considerably more extensive range. in 1962, dixon [14] reviewed the evidence for the alternative mode-aerosolized spread-and concluded that true airborne infection was extremely rare. nonetheless, epidemiological evidence suggests that transmission by means of aerosolized particles may be a real occurrence. in 1970, persons on 3 floors of a german hospital developed smallpox, despite isolation of the coughing, smallpox-infected patient in a single room [15] . seventeen cases of smallpox developed; none of the patients had direct contact with the initial patient. subsequent "smoke" testing demonstrated air flows consistent with an aerosol spread [15] . the last recorded death due to smallpox, according to world health organization investigators, was likely associated with virus that had been transmitted by aerosol [16] . in 1978, janet parker, a medical photographer at the university of birmingham medical school in england, became ill with smallpox and subsequently died. her darkroom was 1 story above and several rooms down the hall from the laboratory of dr. henry bedson, a prominent smallpox researcher. smallpox virus can also be transmitted by fomites, such as clothing and bedding [14] . laundry workers have developed smallpox. one study found a much higher recovery of smallpox virus from pillows and bedclothes than from air samples of the patient's coughs [17] . the length of time that these objects remain infectious is unclear, but on the basis of the historical pattern of epidemics, it is likely no more than a few days. current wisdom holds that smallpox, contrary to its popular reputation, is not a highly infectious disease [3, 12] . examinations of outbreaks in india and pakistan in the 1960s showed that each case of smallpox gave rise to only 3 new cases during the infectious (dry) season and to 1 new case during the humid season [3] . such observations-along with the long incubation period of smallpox (mean, 12-14 days; range, 7-21 days)suggest that there would be adequate time to vaccinate the public and prevent a more widespread outbreak. not revealed in these reports is the extent to which the affected public had already been vaccinated. if the percentage of the population that had been vaccinated was high, the aforementioned findings may merely reflect the population's immune status, rather than a low attack rate. another report placed the vaccination level in india at that time at 80% [18] . if accurate, this would support the reason for the low attack rate as being a consequence of a public protected by immunization, rather than due to a virus with low inherent infectivity. indeed, there are data that smallpox is highly contagious. during the period of endemic smallpox, in field studies in africa, 30% of susceptible contacts became infected [19] . other sources report attack rates of anywhere from 37% to 88% among unvaccinated contacts [20] . potentially fatal reactions to smallpox vaccination include encephalitis, progressive vaccinia, eczema vaccinatum, and myopericarditis. postvaccinial encephalitis or encephalomyelitis has been reported to occur at an incidence of 1 case per 300,000 vaccinations [21] . in recent data from an ongoing department of defense (dod) study, there was 1 case of encephalitis reported among 623,244 vaccinations [22] ; the patient recovered. there was no evidence by either viral culture or pcr for vaccinia being the etiology. progressive vaccinia (a postvaccination viral dissemination with subsequent shock and localized gangrene) occurs in persons with immunodeficiencies, and eczema vaccinatum (a generalized spread of vaccinia to skin beyond the vaccination site) occurs in persons with atopic dermatitis; neither was reported in the dod study [22] . fifty cases of contact transfer of vaccinia occurred, primarily in spouses and adult intimate contacts [23] . the lower-than-expected incidence of adverse events may reflect more-careful screening of vaccination candidates for immunosuppression and eczema (for whom vaccination is contraindicated), the generally healthy status of the population being vaccinated, the previous vaccination in up to two-thirds of vaccine recipients, and covering of the vaccination site, which reduces inadvertent inoculation of contacts. (in previous vaccination campaigns, the vaccination site was left exposed.) an unexpected finding in the dod study above was the occurrence of 83 cases of myopericarditis [23] [24] [25] . there was 1 death among these cases [25] . other than for that fatality, in all 64 cases for which there was follow-up cardiac testing, there was normalization of electrocardiograms, echocardiograms, exercise testing, and functional status [25] . there was no increased incidence of coronary events in the dod program [22, 25] , but in the much smaller civilian vaccination program (involving 36,217 vaccinees), the number of myocardial infarctions observed (5 cases) was higher than would have been expected (2 cases) [26] . plaque-purified tissue culture vaccines are in clinical trials and may have a lower incidence of adverse reactions than does the standard calf lymph vaccine [27] . in addition, attenuated and dna subunit smallpox vaccines are under development [28] and may prove to be safer for immunocompromised persons. there have been attempts to answer the question of how many deaths would arise from preemptive mass vaccination of the public. depending on the percentage of the population vaccinated, the number of deaths is estimated to be in the range of 125-500 [3, 29, 30] . the likely deaths and morbidity that would ensue from a vaccination program must be weighed against the likelihoodand consequences-of a smallpox attack. the conventional wisdom, as noted above, is that smallpox "does not spread rapidly under natural conditions" and, in fact, spreads at a "leisurely" pace [3, p. 492 ]. transmission usually requires "close prolonged contact" for spread [3, p. 492 ]. each case of smallpox "gives rise to (only) about three new cases" [3, p. 492 ]. the long incubation period of 1-3 weeks "provides the time to intervene and limit secondary spread" [12, p. 460 ]. we can "readily stop outbreaks within two infective generations (about 4 weeks) after recognition of the initial cases" [3, p. 492 ]. there is a problem in basing public policy on these principles. even if the above is an accurate representation of the contagiousness of smallpox, this paradigm reflects the spread of natural smallpox. unfortunately, any future smallpox epidemic would likely be an unnatural, man-made event. the natural history of an unnatural event may not be natural. a second misconception regards vaccination. contrary to the widely held belief that vaccination is equally successful after implantation of the variola virus, "postexposure vaccination is at best of limited effectiveness" [31, p. 1923 ]. the most optimistic report on postexposure vaccination, plotting efficacy against time, utilized a presumed average incubation period. it concluded that postexposure vaccination reduced the clinical case rate by 50% when administered up to 5 days postexposure [32] . concerns over the effectiveness of postexposure vaccination have been raised by others [33, 34] . bozzette et al. [35] calculate that there would be 150,000 deaths in a "high-impact airport attack," despite the presence of an aggressive postevent immunization program. it could be argued that his calculation may be an underestimation. in their model, bozzette et al. [35] used a pattern of spread based on outbreaks that occurred after world war ii in a largely smallpox-immune population. vis-à-vis smallpox, the immune status of the older portion of our population is uncertain. it was generally accepted that the immunity provided by vaccination deteriorates with time. two-thirds of persons with smallpox in the 1960s had preexisting vaccination scars [19] . however, hammarlund et al. [36] found substantial humoral and or cellular immunity against vaccinia persisting in persons who had been vaccinated 25-75 years earlier and cite epidemiological studies that argue for long-term protection. regardless, the immune status of our younger population (i.e., those aged !37 years), with regard to smallpox, probably resembles the status of the aztec, inca, and 17th century american indian populations, rather than that of a vaccinated people. it is possible, therefore, that each index case would give rise to considerably more than just the 3 secondary cases in the outbreaks that occurred after world war ii. as is reported in a consensus statement by smallpox authorities, "a clandestine release of smallpox, even if it infected only 50-100 persons to produce the first generation of cases, would spread rapidly in a now highly susceptible population, expanding by a factor or 10-20 times or more with each generation of cases" [22, p. 2132 ]. this pattern of spread likely occurred in the population of central mexico, which, according to aztec tribute rolls taken before their exposure to smallpox in the early 1500s, was 25 million. the spanish, in 1620, estimated that the population was 1.6 million, but other factors, including measles, also probably played a role in the decline [37] . bozzette et al. [35] ascribe a mortality rate of 22.5% to the unimmunized population. however, there are data showing a mortality rate of 52% in an unvaccinated population [38] . the same long incubation period that some authorities hold to be an advantage in control of the disease [12] could actually prove to be our achilles' heel. even within the limits of the shortest possible incubation period (7 days), high-impact attacks could be repeated-at the same site or at different sites, with no one aware that attacks were taking place. to make a cogent assessment of the consequences of a smallpox attack, several questions must be answered. otherwise, we are engaged in no more than guesswork. the questions are these: (1) can smallpox virus be aerosolized? (2) if it can be aerosolized, for how long does it remain viable, and how far can it be carried? (3) even if it can remain aerosolized and viable for a prolonged period of time, just how infectious is it by this route? smallpox virus can be aerosolized [21] . however, the current opinion on how long the virus can remain viable in this state is that the viability rapidly decreases after 60 min ("no more than 20%-30% survived" [31, p. 1923] ), implying that there is nil viability left soon thereafter and, thus, that aerosolization does not represent much of a threat [31] . unfortunately, closer scrutiny of the science underlying that assertion shows less reason to be sanguine. the great majority of the loss in variola virus viability was already present when first measured 5 min into the study. thereafter, there was but modest further decline over the remaining 60-min length of the study [39] . the virus may therefore persist at a relatively stable level of viability for hours. how long the virus can actually remain aerosolized is unknown, as is its infectivity in this mode. if one extrapolates from the results of studies of vaccinia, aerosolized variola virus that is protected from uv light survives for 24 h [21] . a critical caveat that was not addressed above is that the discussion has been limited to natural smallpox in a natural setting. the soviet union is known to have engaged in an active program to aerosolize bioweapons, including smallpox, for use in bioweapons [40] . if modified or attached to the appropriate carrier, variola virus could possibly remain suspended and infectious for a considerable period. on the other hand, dissemination of variola virus into the air (e.g., via crop dusters or bomblets) subjects the virus to variables such as uv light, thermal factors, humidity, and wind. the virus might not survive, or it might be dispersed in the atmosphere into such low concentrations that it is no longer infective. because the minimal infective dose has not been determined, the efficacy of such dissemination is unknown. the current bush administration sought widespread preevent vaccination of the public over concern as to whether an effective vaccination program could be implemented after an attack on an unvaccinated public [41] . the public health com-munity, however, citing safety issues, has opposed immunizing the public [41] . animal studies demonstrate that cidofovir (vistide; gilead) has activity against poxvirus infections [42] [43] [44] [45] , but only when it was administered either concurrently or, in one study, within 3 days after the initial challenge with the virus. if its effectiveness extends to humans, this drug would have a prophylactic effect only. it would not be of benefit for treatment of established clinical smallpox. cidofovir has been modified to render the drug bioavailable by the oral route. this modification (adding a lipid tail to produce hexadeclyoxypropyl-cidofvir [hdp-cidofovir]) resulted in a new drug, which, in vitro, is 100 times more effective against variola than is unmodified cidofovir [46] . methisazone, a thiosemicarbazone, has been reported to be effective for smallpox prophylaxis. a clinical trial in india in the 1960s involving 15000 contacts claimed a 96% reduction in the incidence of the disease ( ) [47, 48] . however, p ! .001 this study has been criticized elsewhere [49] . treatment and control groups were incompletely randomized, with a possible bias in favor of methisazone. a subsequent fully randomizedbut considerably smaller-trial reported favorable but lessimpressive results (the incidence of smallpox in the control group was almost double that in the methisazone group) [50] . this finding did not reach statistical significance. methisazone was stated to be effective prophylaxis in the eighth edition (from 1977) of harrison's principles of internal medicine [51] , but it is doubtful that many made note of it. by then, smallpox had essentially been eradicated, and there was little reason to pay much attention to the entry. later editions of harrison's virtually eliminated the smallpox chapter, along with discussion of the drug. the agent has since fallen off of our radar screens [52] . a panel of smallpox authorities assessed methisazone and determined that it had only modest benefit, probably reducing the incidence of smallpox by only 30%-40% [53] . this reduction should not be dismissed as inconsequential. in the event of a smallpox attack with an engineered virus, even such modest efficacy could prove critical. not addressed, however, is the question of just how effective methisazone would be without coadministration of vaccine. (vaccine could be useless in an attack with a modified virus.) in all of the aforementioned studies, contacts simultaneously received postexposure vaccination and methisazone. one study, on a related poxvirus, suggests an answer to this question. methisazone was investigated as prophylaxis for variola minor (alastrim), where contacts were not vaccinated, and was found to be effective for the prevention of alastrim at a significance level of .01 [54] . methisazone is not without side effects. nausea and vomiting have been reported in one-tenth to two-thirds of persons who receive the drug [52, 54] . for prophylaxis, the drug must be given within 8 days of the initiation of infection with variola major [55] . methisazone has a significant weakness: without patent protection, it is essentially an orphan. a pharmaceutical company is unlikely to expend research effort or promotion on such a drug. that weakness, however, is also a strength: in the public domain, it would likely be inexpensive to produce. the smallpox virus is 200-300 nm in size. n-100 respirators, with ulpa (ultra-low penetration air) filters, are 99.999% efficient in filtering particles of у120 nm in size [56] . the retail cost of these masks is $7. n-95 respirators, which are less effective respirators, have been reported to be protective in preventing transmission of severe acute respiratory syndrome coronavirus (size, 100 nm) in health care workers [57, 58] , but use of these respirators failed to prevent a cluster of cases in one hospital [59] . concerns have been raised over leakage around the mask, especially in the absence of fit testing [60] . nonetheless, these masks, if distributed to the public, could prove to be critical for the control of a smallpox epidemic that was overwhelming our health care system, and they might also prove to be effective in limiting contagion of smaller viruses, such as influenza virus (either natural virus, as in 1918, or engineered virus [61] ). additionally, an aerosolized smallpox attack would likely paralyze our cities. availability of masks might allow some measure of confidence for essential services to continue. a focus on the hazards of smallpox vaccination without consideration of the potential consequences of a competently executed smallpox attack may lead to skewed analyses and flawed decisions. in particular, the use of a more virulent, "weaponized" strain of smallpox virus could mean that the epidemic would outrun the currently planned postevent vaccination/isolation measures. although conventional wisdom suggests that smallpox, in its natural state, is largely limited to spread via respiratory droplets, concern about the potential for aerosol transmission is real and might be a greater problem in a developed society with large urban populations. despite the potential hazards, we believe that greater efforts should be made to promote pre-event immunization-especially in emergency providers and health care workers. furthermore, consideration should be given to allowing the public voluntary access to the vaccine. with proper informed consent and careful screening to minimize the risk of adverse side effects, such a program could reduce the risk of a runaway epidemic. because of the possibility of an attack involving bioengineered smallpox virus that is resistant to the current vaccine, methisazone should be reexamined, and research should be continued on other antiviral agents. also, an adequate supply of masks should be assured. although unlikely at the present time, the possibility of a future bio-engineered attack using smallpox should not be arbitrarily rejected. because of scientific advances (the polio virus has recently been synthesized de novo) [62] and ready access to those advances (complete genomes for viruses, including variola, are available on the internet), we face a potential vulnerability. although that threat may not be immediate (variola would be a complex genome to synthesize, and its dna requires the activity of associated proteins to be infectious) [63] , it may not be long in coming. board on health promotion and disease prevention, institute of medicine. review of the centers for disease control and prevention's smallpox vaccination program implementation: letter report #4 evaluation of 21st-century risks of smallpox vaccination and policy options smallpox vaccine policy is bad science harrison's principles of internal medicine bio-terrorism, nih-cdc grand rounds us hunting antiviral drug to use in case of smallpox ammo for the war on germs in vitro activity of potential anti-poxvirus agents expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox smallpox: anything to declare? 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ecmaj effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) sars among critical care nurses cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada interim domestic guidance on the use of respirators to prevent transmission of sars influenza as a bioweapon active poliovirus baked from scratch a not-so-cheap stunt key: cord-273840-jjm7y07m authors: vabret, astrid; dina, julia; gouarin, stéphanie; petitjean, joëlle; corbet, sandrine; freymuth, françois title: detection of the new human coronavirus hku1: a report of 6 cases date: 2006-03-01 journal: clin infect dis doi: 10.1086/500136 sha: doc_id: 273840 cord_uid: jjm7y07m background. human coronavirus hku1 (hcov-hku1), a new group 2 coronavirus, was first characterized in 2005 from 2 adults with pneumonia in hong kong, china. to the best of our knowledge, there is no other report to date about the detection of this new virus. we report a molecular method allowing for the detection of hcov-hku1 and also report the clinical presentation of 6 infected patients. methods. we screened 141 specimens (135 nasal samples and 6 stool samples) received in february and march 2005 in our laboratory and obtained from 135 hospitalized patients (61.5% of whom were <5 years old and 34.1% of whom were >20 years old) for hcov-hku1. results. hcov-hku1 was detected in 6 (4.4%) of the 135 nasal specimens and in 2 (33.3%) of the 6 stool samples; the positive samples were obtained from 6 patients (5 children and 1 adult). the clinical presentation of these 6 patients was as follows: 3 were admitted to the hospital for acute enteric disease resulting in severe dehydration associated with upper respiratory symptoms; 1 had fever, otitis, and febrile seizure; 1 had a sample obtained to investigate failure to thrive; and 1 had a sample obtained for exploration of x-linked agammaglobulinemia and hyperleucocytosis. conclusion. hcov-hku1 can be detected in respiratory and stool samples from children and adults in a part of the world other than hong kong. our results suggest that hcov-hku1 could be associated with respiratory and enteric diseases, and its detection can be related to a persistent asymptomatic infection in patients with poor underlying conditions. was detected by rt-pcr of the pol gene of coronaviruses with use of conserved primers in the nasopharyngeal aspirates of patients. the complete genome was sequenced, and its general organization concurs with those of other coronaviruses. it contains the he gene, which characterizes the group 2 coronaviruses, and has a very intriguing feature consisting of a variable number of tandem copies of a 30-bp repeat region that codes for a highly acidic domain (atr) in orf1a (nsp1 region). despite the high number of cell cultures inoculated with respiratory samples, hcov-hku1 could not be recovered from cell culture [4] . no cytopathic effect was observed, and rt-pcr performed on the culture supernatant and cell lysates showed a lack of viral replication. isolating hcov on cell culture is very difficult. hcov prototype strains 229e, oc43, and nl63 can replicate in mrc5 cells, hrt18 cells, and llc-mk2 cells, respectively, but there are still few primary isolates. woo et al. [4] reported that sars-cov can be recovered only from !20% of patients infected with sars-cov. there is no other report to date concerning the detection of hcov-hku1. in our laboratory, we conducted a preliminary study of 135 re-spiratory samples to know if this new coronavirus can be detected in a part of the world other than hong kong. the purpose of this article is to present the method used for the detection of hcov-hku1 and report the clinical presentation associated with this infection. we screened arbitrarily for hcov-hku1 135 huh7 cell culture showing an extensive lysis at day 4 after onset of infection. huh7 cells were inoculated with respiratory samples collected from 135 patients hospitalized in february and march 2005 at the university hospital of caen (caen, france). the age distribution of the tested patients was as follows: 83 patients (61.5%) were !5 years of age, 6 patients (4.4%) were 6-20 years of age, and 46 patients (34.1%) were 121 years of age. the tested respiratory specimens were obtained from patients with respiratory symptoms and then were sent to the laboratory for viral diagnosis. the patients or, in the case of minors, the parents or legal guardians of the patients consented to having their samples tested for respiratory viruses, including coronaviruses. the samples were processed routinely for immunofluorescence direct antigen assay using monoclonal antibodies against influenza a and b viruses; respiratory syncytial virus; parainfluenzavirus types 1, 2, and 3; and adenovirus (imagen). all samples had negative results. it is part of our strategy to inoculate samples with negative results onto a cell culture system (huh7 cell line) [5] . in this protocol, huh7 cells were grown in rpmi-1640 medium with glutamine with 5% fetal calf serum (gibco; invitrogen), penicillin (100 u/ml), and gentamycin (50 mg/ml), and were distributed in 48-well tissue culture microplates. when huh7 cells were 80% confluent, the growth medium was discarded and each well was inoculated with 100 ml of the clinical samples. microplates were centrifugated at 1500 rpm for 30 min at 30њc. samples were then removed, and each well was filled with 1 ml of the same culture medium supplemented with 2% fetal calf serum (gibco; invitrogen). by day 4 after onset of infection, huh7 cells were examined for cytopathic effect and the supernatant were used for amplification techniques. cytopathic effect characteristics are as follows: small cells, no refringent, cells scattered on the culture, and a progressive lysis of the cell sheet. the rna was extracted using the high pure rna isolation kit (roche) and tested with rt-pcr techniques previously described and used for the detection of rhinovirus, enterovirus, influenza virus c, hcov-229e, hcov-oc43, and hcov-nl63 [6, 7] . a 1-step hcov-hku1 rt-pcr amplification of a 443-bp fragment was performed using the onestep rt-pcr kit (qiagen) and the following primers set defined in n gene from the genbank sequence database (accession number ay597011): hku1 sens (5 -accaatctgagcgaaattaccaaac-3 ) and hku1 antisense (5 -cggaaacctagtagggatagctt-3 ). the reac-tion was undertaken using onestep rt-pcr kit (qiagen, courtaboeuf) according to the manufacturer's protocol. each rt-pcr test was performed using the usual precautions to avoid contamination. no cross-reaction of this rt-pcr assay was observed with the other classical hcov. the assay was performed without a positive control. rt-pcr products were processed on an agarose gel stained with ethidium bromide and visualized under uv light. the positive results were confirmed by sequencing from the amplified product and by rt-pcr performed directly with use of the respiratory specimens. for 2 patients with results positive for detection of hcov-hku1 in respiratory specimens, stool samples obtained at the same time were available and have been tested. two other rt-pcr assays were performed using the specimen that was positive for hcov-hku1 detection, one of which involved amplification of a 713-bp fragment with s1 gene-specific primers hku1-s1 sens (5 -accacagttcctcgcataagt-3 ) and hku1-s1 antisense (5 -agatattggcgtttagac-3 ), the other amplifying a variable size fragment with primers defining on both sides of the highly acidic domain in orf1a (atr-hku1 sens: 5 -aatggcctctcgtatgtat-3 ; atr-hku1 antisense: 5 -ttacaagtaacacagaacgca-3 ). the rt-pcr products were sequenced, and the obtained nucleotide sequences of the partial s1 gene were compared with the prototype strain sequences available in genbank. the alignments were prepared using clustal x, version 1.83. a phylogenetic tree was constructed by the neighbor-joining method, and bootstrap values were determined by 1000 replicates. we used hcov-oc43 as an outgroup. the medical records were examined retrospectively for the 6 patients with test results positive for hcov-hku1. among the 135 cell cultures tested with rt-pcr, the positive samples consisted of 10 coronaviruses (6 positive for hcov-hku1, 2 positive for hcov-nl63, and 2 positive for hcov-oc43), 23 rhinoviruses, 1 enterovirus, and 1 influenza virus c. there were 2 codetections, one of which consisted of hcov-hku1 and influenza virus c, and the other of which consisted of rhinovirus and hcov-oc43. to eliminate cell culture contamination, the n gene hcov-hku1 rt-pcr was performed directly on the respiratory specimens obtained from the 6 patients. all of these specimens, as well as the stool samples available for 2 of these patients, were found to have positive results. the 443-bp amplicon resulting from the amplification of the partial n gene was sequenced for 6 of the 8 specimens with positive results (genbank accession numbers dq131635-dq131641) (figure 1). our isolates shared 98%-100% nucleotide identity with the same region of the hcov-hku1 reference strain. we then sequenced the 713-bp amplicon corresponding to partial spike gene of 4 positive specimens. genetic sequences from other positive specimens could not be obtained, because the primers used for the amplification of the partial spike gene did not amplify a pcr product of sufficient quantity to allow for reliable genetic sequencing analysis. the phylogenetic analysis of 603-bp of these sequences (genbank accession numbers dq131642-dq131645) shows that our isolates form a group containing sequences with different markers and that there is no cross-contamination between the different isolates. three of these isolates clustered with the prototype strain described in hong kong. one isolate (dq131645) had characteristics of an outlier (figure 2). to determine if our isolates contained a variable number of tandem copies of 30bp repeat region-the significant feature of hcov-hku1-we sequenced amplicon resulting from a rt-pcr of the region of orf1a. to confirm the number of atrs, this test was conducted several times for 2 isolates. the results show that there were 7 and 8 atrs in the 2 isolates tested, compared with 11 and 14 atrs recorded in the 2 hong kong isolates [4] . hcov-hku1 was detected in nasal and stool samples obtained from 6 patients, 4 of whom were male, and 2 of whom were female (table 1). the age of the infected patients ranged from 8 months to 5 years, with the exception of one 19-yearold man (patient 5). the age distribution of the patients infected with hcov-hku1 and the age distribution of tested patients were closed. the medical records of all 6 patients were examined. three of them (patients 1, 2, and 6) were admitted to the hospital for severe dehydration due to fever, vomiting, and diarrhea. upper respiratory symptoms (rhinitis, pharyngitis, or otitis) were also reported. unfortunately, no stool samples for these patients were available in the hospital. patient 4 had a febrile seizure and otitis. both respiratory and stool samples tested positive for hcov-hku1. patients 3 and 5 were admitted to the hospital for a complete investigation and did not suffer from acute symptoms; patient 3 was an 8-month-old girl with failure to thrive, and patient 5 was a 19-year-old man with bruton agammaglobulinemia and hyperleucocytosis with a final diagnosis of chronic myeloid leukaemia. a stool sample obtained from patient 3 was available and tested positive for hcov-hku1. hcov-hku1 was first characterized in hong kong in january 2005 in the respiratory specimens of 2 adults with pneumonia. hcov-hku1 is a previously unrecognized group 2 coronavirus. the clinical significance of this viral infection was made evident in the index patient by the combined evolution of clinical symptoms, viral loads in the respiratory samples, and development of a specific antibody response. this index patient was a 71-year-old chinese man with poor underlying conditions (pulmonary tuberculosis complicated by bronchiectasis, a history of being a chronic smoker, and obstructive airway disease). he was admitted to the hospital for pneumonia and was discharged after 5 days of hospitalization. however, respiratory, stool, and urine samples were collected from him weekly for a 5-week period. the viral load in respiratory specimens decreased during the second week of the illness and was undetectable in the third week. the decrease in viral load was accompanied by recovery from the illness and development of a specific antibody response. hcov-hku1 was detected only in respiratory specimens, not in stool and urine samples [4] . there is no other report to date about the geographic and temporal distribution and the clinical symptoms associated with this virus. in our study, the arbitrary decision was made to screen 135 huh7 cell cultures showing a cytopathic effect at day 4 after onset of infection and inoculated by nasal specimens collected during 2 winter months, february and march 2005. the choice of huh7 cells was made because some studies showed that these cells appear to have a broad spectrum, allowing for the detection of a wide range of viruses, including coronaviruses 229e, oc43, and mhv [8] [9] [10] . unfortunately, we failed to amplify hcov-hku1 on huh7 cells by serial passage. at first passage, we could see a cytopathic effect on day 4. however, because of the lack of specific antibodies against hcov-hku1, we could not test huh7 cells to know whether there were infected cells showing viral replication. so, the hcov-hku1 rna detected by rt-pcr may be a residual virus from a clinical sample or a virus amplified in cell culture in the first passage on huh7 cells. the duration of incubation is limited to 4 days, which may be insufficient for the growth of coronaviruses. more experiments are being conducted to adapt hku1 strains to cell line. six patients were infected with hcov-hku1, including 5 children and 1 adult. because samples were obtained mainly from pediatric departments, 61.5% of the tested patients were children. so, the high frequency with which hcov-hku1 figure 2 . phylogenetic analysis of a 603-bp region of partial spike gene of 4 human coronavirus (hcov)-hku1 isolates from france. the phylogenetic tree was constructed by the neighbor-joining method, and bootstrap values were determined by 1000 replicates in clustal x, version 1.83. to construct the tree, the reference strain hcov-hku1 (nc006577) was included. hcov-oc43 is used as the outgroup. [2, 8, 9, 10] . our study only suggests that hcov-hku1 could also have a winter seasonality in these regions. a better understanding of the epidemiology of different hcov in different parts of the world could contribute to better management of the molecular and serological diagnosis of future emerging or reemerging viruses, such as sars-cov. because of the lack of a systematized selection of the tested samples, the patients with positive results are presented as case reports. it should be noted that one-half of the patients were admitted to the hospital not for respiratory illnesses, but for gastroenteritis resulting in severe dehydration associated with upper respiratory symptoms. in effect, previous studies have suggested that coronaviruses other than sars-cov may be involved in enteric diseases, despite the fact that there is no clear evidence that they cause enteric illness [9, [11] [12] [13] . this would not be surprising in view of the clear involvement of sars-cov and some animal coronavirus strains in severe diarrheal diseases. we have shown that hcov-hku1 can be detected in stool samples. nevertheless, further studies must be conducted to confirm the potential dual tropism (respiratory and enteric) of this group 2 coronavirus. hcov-hku1 was also detected in 2 patients without acute symptoms and with poor underlying conditions (patients 3 and 5). although hcov (except sars-cov) were generally associated with acute and self-limiting respiratory infections, this detection of viral rna can be related to a persistent or asymptomatic chronic infection. chiu et al. [14] reported a repeated detection of hcov-229e in an immunocompromised child 13 months old. the genetic analysis of a 305-bp region of the spike protein region of 229e revealed that these 3 specimens had identical genetic sequences, providing evidence of virus persistence rather than reinfection. patient 6, who tested positive for hcov-hku1, had an influenza c virus detected in the same clinical specimen. it is not possible to determine whether this is true coinfection or whether 1 of the viruses represents continued shedding from a previous infection. it is interesting to note that the genome of group 2 coronaviruses and influenza c virus both contain the he gene. phylogenetic analysis suggested that the he genes of coronaviruses and influenza c have a common ancestral origin. because hcov and influenza c virus infect similar tissues, the significant sequence homology between the he genes of the 2 viruses suggests that coinfection followed by recombination could have occurred in the past [4, 15] . the expression of the he gene has been shown to be heterogeneous in different species of group 2 coronaviruses. the biological significance of he protein is not well understood. further experiments have to be performed to determine the essentiality and function of he in hcov-hku1, especially in cell tropism. in conclusion, we report a molecular method allowing for the detection of the new coronavirus hku1. we are able to detect hku1 in respiratory samples and stool specimens, indicating that this virus can be excreted in this way. our results indicate that hcov-hku1 is present in hospitalized patients with mild respiratory and enteric tract illness. determining whether or not hcov-hku1 can cause enteric diseases will require further investigations. phylogenetic analysis of our isolates suggest that both variants of hcov-hku1 may cocirculate. ours is the first observation and report of hcov-hku1 infection in france. future studies will yield more-accurate data about the circulation of this virus. severe acute respiratory syndrome coronavirus phylogeny: toward consensus identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterization and complete genome complete sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in huh7 hepatocarcinoma cell line development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses human coronavirus nl63 mouse hepatitis virus strain jhm infects a human hepatocellular carcinoma cell line coronavirus 229e-related pneumonia in immunocompromised patients an outbreak of coronavirus oc43 respiratory infection in normandy, france human coronavirus nl-63 infections in children: a 1-year study biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child association of coronavirus infection with neonatal necrotizing enterocolitis human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong, china the hemagglutinin/esterase gene of human coronavirus strain oc43: phylogenetic relationships to bovine and murine coronaviruses and influenza c virus potential conflicts of interest. all authors: no conflicts. financial support. the european commission episars contract (sp22-ct-2004-511063) and the programme de recherche en réseaux franco-chinois (épidémie du sras : de l'émergence au contrôle). key: cord-294335-qnu19ru5 authors: yousaf, anna r; duca, lindsey m; chu, victoria; reses, hannah e; fajans, mark; rabold, elizabeth m; laws, rebecca l; gharpure, radhika; matanock, almea; wadhwa, ash; pomeroy, mary; njuguna, henry; fox, garrett; binder, alison m; christiansen, ann; freeman, brandi; gregory, christopher; tran, cuc h; owusu, daniel; ye, dongni; dietrich, elizabeth; pevzner, eric; conners, erin e; pray, ian; rispens, jared; vuong, jeni; christensen, kim; banks, michelle; o'hegarty, michelle; mills, lisa; lester, sandra; thornburg, natalie j; lewis, nathaniel; dawson, patrick; marcenac, perrine; salvatore, phillip; chancey, rebecca j; fields, victoria; buono, sean; yin, sherry; gerber, susan; kiphibane, tair; dasu, trivikram; bhattacharyya, sanjib; westergaard, ryan; dunn, angela; hall, aron j; fry, alicia m; tate, jacqueline e; kirking, hannah l; nabity, scott title: a prospective cohort study in non-hospitalized household contacts with sars-cov-2 infection: symptom profiles and symptom change over time date: 2020-07-28 journal: clin infect dis doi: 10.1093/cid/ciaa1072 sha: doc_id: 294335 cord_uid: qnu19ru5 background: improved understanding of sars-cov-2 spectrum of disease is essential for clinical and public health interventions. there are limited data on mild or asymptomatic infections, but recognition of these individuals is key as they contribute to viral transmission. we describe the symptom profiles from individuals with mild or asymptomatic sars-cov-2 infection. methods: from march 22 to april 22, 2020 in wisconsin and utah, we enrolled and prospectively observed 198 household contacts exposed to sars-cov-2. we collected and tested nasopharyngeal (np) specimens by rt-pcr two or more times during a 14-day period. contacts completed daily symptom diaries. we characterized symptom profiles on the date of first positive rt-pcr test and described progression of symptoms over time. results: we identified 47 contacts, median age 24 (3-75) years, with detectable sars-cov-2 by rt-pcr. the most commonly reported symptoms on the day of first positive rt-pcr test were upper respiratory (n=32, 68%) and neurologic (n=30, 64%); fever was not commonly reported (n=9, 19%). eight (17%) individuals were asymptomatic at the date of first positive rt-pcr collection; two (4%) had preceding symptoms that resolved and six (13%) subsequently developed symptoms. children less frequently reported lower respiratory symptoms (age <18: 21%, age 18-49: 60%, age 50+ years: 69%; p=0.03). conclusions: household contacts with lab-confirmed sars-cov-2 infection reported mild symptoms. when assessed at a single time-point, several contacts appeared to have asymptomatic infection; however, over time all developed symptoms. these findings are important to inform infection control, contact tracing, and community mitigation strategies. severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the virus responsible for coronavirus disease 2019 , continues to cause significant morbidity and mortality worldwide [1, 2] . rapid recognition of covid-19 symptoms is vital for timely clinical diagnosis, management, and for public health interventions such as contact tracing activities and infection prevention and control measures. understanding the frequency of asymptomatic infections in the community setting is also important to inform mitigation efforts focused on reducing viral transmission. as the covid-19 pandemic progresses, our understanding of the clinical spectrum of covid-19 is quickly evolving. however, the majority of our current information on the clinical presentation of covid-19 comes from patients requiring hospitalization [3] [4] [5] and from special populations such as those in outbreak investigations (e.g., cruise ships) that only capture symptom information at a single point in time [6] [7] [8] [9] and in long-term care facilities [10] . while the clinical characteristics and symptoms of individuals with more severe covid-19 have been described, there remains relatively little detailed information on the natural progression of clinical and symptom profiles for individuals with mild illness, or people with no symptoms but laboratory evidence of infection. here we describe a cohort of household members (hereafter referred to as household contacts) who tested positive for sars-cov-2 following exposure to someone else in their home with laboratoryconfirmed infection. we describe their demographic and clinical characteristics, time from exposure to symptom onset, symptom profiles, and the evolution of symptoms over time. m a n u s c r i p t 5 individuals with covid-19 identified through routine public health surveillance and their household contacts were enrolled in a household transmission investigation. we enrolled households from march 22 to april 22, 2020, in the milwaukee, wisconsin and salt lake city, utah metropolitan areas, as previously described in detail (n. lewis, v. chu, d. ye, et al., manuscript in preparation). only the household contacts of source individuals were included as the study population for this analysis; no household contacts were hospitalized prior to or during the 14-day study period. we interviewed household contacts using a standardized questionnaire to obtain demographic and clinical characteristics, along with detailed symptoms that contacts may have experienced prior to enrollment as well as symptoms experienced on the day of enrollment. on the first day of the study period (day 0, i.e. day of enrollment), we collected nasopharyngeal (np) swab specimens from all 198 enrolled household contacts. we observed household contacts for 14 days following enrollment and requested that they record daily measured temperatures and symptoms in a symptom diary. on day 14 (the final close-out visit), we returned to the household and collected np swab specimens from all household members and retrieved the daily symptom diaries. during the 14-day follow-up, an investigation team returned to the household for interim np swab collections from all household contacts if any previously asymptomatic household contact developed new symptoms. we tested np specimens using the cdc 2019-ncov real-time polymerase chain reaction (rt-pcr) assay [11] . contacts with sars-cov-2 infection confirmed by rt-pcr on at least one np were included in this analysis. a c c e p t e d m a n u s c r i p t 6 we assessed symptoms reported by household contacts on the collection date of their first rt-pcrpositive np specimen (figure 1 , subset a), and categorized symptoms as constitutional (fever, chills, myalgia, or fatigue), upper respiratory (runny nose, nasal congestion, or sore throat), lower respiratory (cough, difficulty breathing, shortness of breath, wheezing, or chest pain), neurologic (headache, loss of taste, or loss of smell), and gastrointestinal (nausea/vomiting, diarrhea, or abdominal pain). we calculated proportions for each category of symptoms and stratified these proportions by sex, age, race, ethnicity and presence of self-reported underlying medical conditions. underlying medical conditions included diabetes mellitus, immunocompromising conditions, and any chronic lung, cardiovascular, kidney, liver, neurologic, or other chronic disease. we also evaluated the co-occurrence of various symptom combinations. differences between groups were assessed using a fisher's exact test. we identified and prospectively followed household contacts who were asymptomatic at the time they initially tested positive for sars-cov-2 by pcr ( figure 1 , subset b) to see if they developed symptoms during the study period. we also reviewed symptom data to identify any prior symptoms. to examine evolution of symptoms over time, we described in detail the symptom diaries of a subset of household contacts who were negative on enrollment (day 0) but tested positive for sars-cov-2 during the two-week longitudinal follow-up period ( figure 1 , subset c). limiting this part of the analysis to this subset of positive contacts ensured their reported symptoms were likely due to acute covid-19 and allowed for a granular description of day-by-day symptom evolution. we used a survival function to estimate the median days from exposure, defined as symptom onset in household source cases, to symptom onset in the corresponding household contacts. we performed analyses using sas software, version 9.4 (sas institute inc., cary, nc, usa) and r-studio of the 47 household contacts with laboratory-confirmed sars-cov-2 infection by rt-pcr, the majority were female (n=29, 62%), white (n=35, 74%), non-hispanic (n=42, 89%), and with a variable age distribution: <18 years (n=14, 30%); 18-49 years (n=20, 43%); 50-64 years (n=10, 21%); 65 years or older (n=3, 6%). half (n=24, 51%) of the household contacts with sars-cov-2 had an underlying medical condition, with the most prevalent conditions being any chronic lung disease (n=9, 19%) and any cardiovascular disease (n=6, 13%) ( table 1 ). the proportion of contacts with one or more underlying medical condition increased with age (<18 years: n=4/14, 29%; 18-49 years: n=11/20, 55%; 50-64 years: n=6/10, 60%; 65+ years: n=3/3, 100%). in figure 2 we present symptoms reported on the date of first positive rt-pcr and symptoms reported throughout the illness for the 47 rt-pcr positive household contacts (see supplemental table for more detail). the most commonly reported symptom categories on the date of first positive rt-pcr were upper respiratory (n=32, 68%) followed by neurologic (n=30, 64%). for symptoms experienced throughout the illness, the percent of household contacts reporting neurologic symptoms increased to 94% (n=44), predominated by headache (n=41, 87%), followed by upper respiratory symptoms (n=42, 89%). nasal congestion and runny nose were the most commonly reported upper respiratory symptoms at both date of first positive rt-pcr test (n=17, 47% and n=39, 83% respectively) and throughout the illness (n=20, 43% and n=32, 68% respectively). symptoms on the date of first positive rt-pcr stratified by sex, age, comorbidity status, and race are shown in figure 3 (and in more detail in supplemental table) . a majority of both sexes reported upper respiratory or neurologic symptoms with no statistically significant differences found between the sexes. similarly, contacts with and without underlying medical conditions most commonly reported upper respiratory or neurologic symptoms with no significant differences between the two groups. among the different age groups, the most common symptoms were as follows: upper respiratory symptoms in children <18 years (n=10, 71%), neurologic symptoms in adults 18-49 years (n=14, 70%), and upper respiratory symptoms in adults 50 years or older (n=11, 85%). there was a significant difference in the percentage of household contacts reporting lower respiratory symptoms with increasing age (age <18 years: 21%, age 18-49 years: 60%, age 50+ years: 69%; fischer's exact p=0.03). co-occurrence of the symptoms reported on the date of first positive rt-pcr is displayed in figure 4 . the median duration of illness with any symptom was at least 16 days (iqr: 11-21); n=14, 30% of individuals were still symptomatic at study close-out. among rt-pcr positive household contacts, 25% developed symptoms three days (95% ci: 2-4) after exposure (symptom onset in the presumed household source case) and the estimate increased to 50% at four days (95% ci: [3] [4] [5] , and 75% at six days (95% ci: 5-9) post exposure. in addition, 50% of the household contacts tested positive by rt-pcr six days (95% ci: 5-7) after the onset of symptoms in the household contact, increasing to 75% at eight days (95% ci: 7-11). the symptom profiles and demographic characteristics of our cohort of sars-cov-2 rt-pcr positive household contacts differ from those described in inpatient populations [3] [4] [5] 12] . our findings indicate that mild upper respiratory and neurologic manifestations may be more common and findings such as fever and cough may be less common among the non-hospitalized population than previously appreciated. additionally, we observed no continually asymptomatic individuals in our study; six (13%) individuals who had no symptoms at the collection date of first positive rt-pcr all went on to develop symptoms during follow up. this has important implications for diagnosis and community mitigation strategies such as clinical case definitions, symptom screening, temperature screening, testing, and return to school policies. our findings also emphasize the importance of widespread preventative measures since individuals with mild symptoms are difficult to identify without testing but may still be a source for spread of infection. we compared the demographic characteristics and symptom profiles of our cohort of household contacts to those of inpatients described by the covid-19-associated hospitalization surveillance network (covid-net) [12] . we found that our population were younger (28% vs 75% age 55 years or older), less likely to be male (38% vs 54%), and had fewer individuals with one or more underlying health conditions (51% vs 89%) [12] . we compared our cohort's symptoms on date of first positive rt-pcr to the symptoms on day of admission described in covid-net; we found that our cohort was less likely to report cough (43% vs 86%), fever or chills (19% and 6% vs 85%), or difficulty breathing/shortness of breath (11% vs 80%). covid-net and additional studies have also described gastrointestinal symptoms in a significant proportion of hospitalized patients with covid-19 [12] [13] [14] [15] with covid-net reporting 27% and 24% of inpatients having diarrhea and nausea/vomiting, respectively [12] . in contrast only 13% of our cohort reported diarrhea, and 9% nausea/vomiting at collection date of first positive rt-pcr; 36% and 19% reported having ever had diarrhea or nausea/vomiting, respectively. a c c e p t e d m a n u s c r i p t 11 the symptoms that were most commonly reported by our cohort at the date of first positive rt-pcr were upper respiratory (primarily nasal congestion and runny nose), and neurologic (primarily headache). only 19% of our cohort reported fever (subjective or objective) on the collection date of first positive rt-pcr and 53% reported ever having had fever during the 14-day observation period. when comparing symptom profiles by age group, we found that children under 18 years were more likely to be asymptomatic compared to persons 18 years or older, and symptomatic children were most likely to report upper respiratory symptoms. several studies have noted that the inpatient covid-19 population tends to be predominantly male, and that males have a higher morbidity and mortality when hospitalized for covid-19 [12, 16] . however, we did not observe any statistically significant differences in reported symptoms stratified by sex. we also identified a significant proportion of individuals (13%) who were asymptomatic on the collection date of first positive specimen. this proportion of asymptomatic individuals is similar to that found in other younger, more healthy populations such as navy service members where 20% of covid-19 cases were asymptomatic [17] . however, it is important to note that all the asymptomatic individuals in our population went on to develop symptoms over the 14-day follow-up period. this is consistent with another longitudinal study that found only 2% of individuals who were asymptomatic at diagnosis remained asymptomatic throughout a 14-day observational period [18] . in contrast, a review of 16 covid-19 observational studies found that 40-50% of individuals with covid-19 were asymptomatic (although only 5 of the 16 cohorts provided longitudinal data); the five studies with longitudinal data found that very few asymptomatic individuals (~10-15%) went on to develop symptoms [19] . notably, our prospective design included asking each contact about 18 different symptoms daily for 14 days. other observational or retrospective studies likely identified symptoms differently, possibly less granular and/or sensitive. it is possible that individuals classified as asymptomatic in other studies may be classified as symptomatic using the methodology in our study. understanding the spectrum of the natural history of covid-19 is important, but even so, a c c e p t e d m a n u s c r i p t 12 there may continue to be challenges identifying covid-19 cases early due to non-specific or mild symptoms. the study findings presented here must be interpreted in light of several potential limitations. first, symptom data were self-report and may be subject to recall bias, when symptom onset preceded the day 0 visit. also, symptoms are subjective by definition and hence individuals may experience and report symptoms differently. second, by the time we reached the households, 89% of rt-pcr positive household contacts were already positive (i.e. positive by rt-pcr on day 0). prior symptom data were captured but not recorded daily. to allow for a granular description of day-by-day symptom evolution we limited our sample to household contacts who were negative on enrollment sarscov-2 rt-pcr test compared to symptoms reported throughout the illness *nasal congestion variable was present for 36/47 symptom diaries, denominator n=36 for these estimates. bloss of smell: partial n=7, 50% and complete n=7, 50%. closs of taste: partial n=9, 64% and complete n=5, 36%. dcough: dry n=12, 60% and productive n=8, 40% esubjective and objective rt-pcr test, stratified by sex, age, underlying medical condition, and race (n=47)a *aconstitutional = fever, chills, muscle aches, fatigue, upper respiratory = runny nose, nasal congestion, sore throat, lower respiratory = cough, discomfort in chest, shortness of breath, wheezing, chest pain, neurologic = headache, loss of taste, loss of smell, gastrointestinal = nausea, diarrhea, abdominal pain; bother race = american indian/alaska native, asian, other; p-values calculated using fisher's exact test figure 4 : combinations of covid-19 symptoms reported by household contacts on the date of first positive sars-cov-2 rt-pcr test (n=47)a *aconstitutional = fever, chills, muscle aches, fatigue, upper respiratory = runny nose, nasal congestion, sore throat, lower respiratory = cough, discomfort in chest, shortness of breath, wheezing, chest pain, neurologic = headache, loss of taste, loss of smell, gastrointestinal = nausea, diarrhea, abdominal pain figure 5 . timeline of symptom onset in household contacts who changed from negative for sars-cov-2 (by rt-pcr) on day 0 to positive during follow-up (n=5) and contacts who were asymptomatic at collection date of 1st positive specimen (n=6) but developed symptoms later *afirst household exposure (defined as symptom onset in the household source case) to enrollment (day 0) covid-19) cases in the us world health organization, coronavirus disease (covid-2019) situation reports clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in clinical characteristics of 60 discharged cases of 2019 novel coronavirusinfected pneumonia in taizhou public health responses to covid-19 outbreaks on cruise ships -worldwide estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship initial investigation of transmission of covid-19 among crew members during quarantine of a cruise ship -yokohama covid-19 in 2 persons with mild upper respiratory tract symptoms on a cruise ship covid-19 in a long-term care facility centers for disease control and prevention 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms clinical characteristics of covid-19 patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study enteric involvement in hospitalised patients with covid-19 outside wuhan gender differences in patients with covid-19: focus on severity and mortality sars-cov-2 infections and serologic responses from a sample of u.s. navy service members -uss theodore roosevelt coronavirus disease outbreak in call center prevalence of asymptomatic sars-cov-2 infection: a narrative review covid-19) social distancing covid-19) childcare, schools, and youth programs covid-19) how to protect yourself & others clean hands save lives. 2020 covid-19) cleaning and disinfecting your facility a c c e p t e d m a n u s c r i p t 15 a c c e p t e d m a n u s c r i p t 18 key: cord-007075-sl45z4i0 authors: marty, francisco m; chemaly, roy f; mullane, kathleen m; lee, dong-gun; hirsch, hans h; small, catherine b; bergeron, anne; shoham, shmuel; ljungman, per; waghmare, alpana; blanchard, elodie; kim, yae-jean; mckevitt, matt; porter, danielle p; jordan, robert; guo, ying; german, polina; boeckh, michael; watkins, timothy r; chien, jason w; dadwal, sanjeet s title: a phase 2b, randomized, double-blind, placebo-controlled multicenter study evaluating antiviral effects, pharmacokinetics, safety, and tolerability of presatovir in hematopoietic cell transplant recipients with respiratory syncytial virus (rsv) infection of the lower respiratory tract date: 2019-12-03 journal: clin infect dis doi: 10.1093/cid/ciz1167 sha: doc_id: 7075 cord_uid: sl45z4i0 background: presatovir significantly reduced nasal viral load, signs, and symptoms of respiratory syncytial virus (rsv) infection in a human challenge study. we evaluated presatovir in hematopoietic-cell transplant (hct) recipients with rsv lower respiratory tract infection (lrti). methods: patients with confirmed rsv in upper and lower respiratory tract and new chest x-ray abnormalities were randomized (1:1), stratified by supplemental oxygen and ribavirin use, to receive oral presatovir 200 mg or placebo every 4 days for 5 doses. the primary endpoint was time-weighted average change in nasal rsv viral load through day 9. secondary endpoints included supplemental oxygen-free days, incident respiratory failure requiring mechanical ventilation, and all-cause mortality. results: from january 31, 2015, to march 20, 2017, 60 patients from 17 centers were randomized (31 presatovir, 29 placebo); 59 received study treatment (50 allogeneic, 9 autologous hct). in the efficacy population (29 presatovir, 28 placebo), presatovir treatment did not significantly reduce time-weighted average change in viral load (−1.12 vs −1.09 log(10) copies/ml; treatment difference −0.02 log(10) copies/ml, 95% confidence interval: −.62, .57; p = .94), median supplemental oxygen-free days (26 vs 28 days, p = .84), incident respiratory failure (10.3 vs 10.7%, p = .98), or all-cause mortality (0 vs 7.1%, p = .19) versus placebo. adverse events were similar between arms (presatovir 80%, placebo 79%). resistance-associated substitutions in rsv fusion protein emerged in 6/29 presatovir-treated patients. conclusions: presatovir treatment was well tolerated in hct patients with rsv lrti but did not improve virologic or clinical outcomes versus placebo. clinical trials registration: www.clinicaltrials.gov, nct02254421; eudract, #2014-002475-29 from 2003 to 2015, no patients with radiographic abnormalities consistent with lrti but with rsv detected in upper respiratory tract samples only ("possible" rsv lrti) died, but 28-day mortality was 26% in hct recipients with probable or proven rsv lrti [3] . despite rsv disease burden in hct recipients and other high-risk adults, options for rsv prophylaxis or treatment in adults are limited. aerosolized ribavirin is not indicated for rsv treatment in adults and is associated with concerns regarding difficulty of administration, adverse effects, and high cost [8, 9] . although some centers report using aerosolized or oral ribavirin to treat rsv lrti in adult hct recipients, efficacy has not been demonstrated in a randomized controlled clinical trial [10, 11] . palivizumab is used for prevention of severe rsv disease in high-risk children ≤24 months of age but is not effective as treatment for established infection in children or adult hct recipients [12] [13] [14] . thus, there is an unmet need for specific treatment for adults at risk for severe rsv infection. presatovir is a novel, orally available rsv fusion inhibitor under investigation for treatment of rsv [15] . presatovir has a favorable safety profile in adult volunteers, and presatovir treatment reduced viral load and respiratory symptoms in healthy adults challenged with rsv [16] [17] [18] . here the safety, tolerability, and efficacy of presatovir in naturally infected hct recipients with rsv lrti were evaluated. this phase 2, randomized, double-blind, placebo-controlled, 2-group parallel study recruited hct recipients 18-75 years of age from 17 centers in 5 countries (supplemental material, appendix). patients presenting any time post-hct with upper and lower respiratory tract rsv infection documented ≤6 days before start of study treatment and evidence of new abnormalities on chest x-ray obtained ≤48 hours from screening were eligible for inclusion. lower respiratory tract involvement could be documented from induced sputum, bronchoalveolar lavage, or lung biopsy, but not spontaneous sputum. patients with documented concurrent lrti with other respiratory viruses were excluded. full eligibility criteria are provided in supplemental methods. the study followed the international conference on harmonisation good clinical practice guidelines and the principles of the declaration of helsinki and was approved by local ethics committees. written informed consent was obtained from patients or legally responsible representatives. data monitoring committee activities and changes to the study protocol are described in supplemental methods. this trial was registered with clinicaltrials.gov (nct02254421) and eudract (2014-002475-29) before enrollment began. patients were randomly assigned (1:1) to receive presatovir or placebo, stratified centrally by supplemental oxygen use (none to ≤2 l/min vs >2 l/min) and ribavirin use (prescribed at randomization, any route of administration) during the current rsv infection. the randomization schedule used permuted blocks of 2. allocation was concealed by use of presatovir and placebo tablets with identical appearance. study treatment assignment information was provided by an interactive web response system (bracket global, wayne, pa, usa). patients, all study staff, and sponsor were masked to study treatment. patients received presatovir 200 mg (4 × 50 mg tablets) or placebo orally or via nasogastric tube every 4 days (±24 hours) during study visits on days 1, 5, 9, 13, and 17, and were followed through study day 28. patients rsv-positive by local molecular testing on day 22 could participate in an optional extended weekly follow-up through day 56. a detailed schedule of study assessments and procedures is provided in supplemental table 1 . for virology assessments, bilateral intranasal samples were obtained using midturbinate adult flocked swabs (copan diagnostics, murrieta, ca, usa) at each study visit [19, 20] . samples were analyzed using reverse transcription quantitative polymerase chain reaction (rt-qpcr) to determine rsv viral load, rsv sequencing of the f gene to evaluate development of resistance, and a multiplex assay to identify coinfections. all nasal samples were analyzed at central laboratories; further details are provided in supplemental methods. antibody titer and pharmacokinetic methods are described in supplemental methods. clinical assessments included vital signs, weight, and oxygen saturation by pulse oximetry; laboratory safety assessments included complete blood counts and serum electrolyte and liver enzyme measurements. patients were observed without oxygen supplementation at each study visit, and the lowest oxygen saturation during observation was recorded. cardiac safety was assessed via local electrocardiograms and troponin testing on days 1, 17, and 28. additional safety assessments included evaluation of adverse events (aes) and documentation of all concomitant medications, hospitalizations, rehospitalizations, intensive care unit care, invasive and noninvasive mechanical ventilation, and supplemental oxygen use (≥2 l/min). the primary endpoint was time-weighted average change in nasal rsv viral load measured by rt-qpcr (log 10 copies/ml) from day 1 to day 9. key secondary endpoints were number of supplemental oxygen-free days [3] , proportion of patients developing respiratory failure requiring invasive or noninvasive mechanical ventilation, and all-cause mortality through day 28. prespecified exploratory endpoints are described in supplemental methods. safety was assessed from aes and clinical and laboratory parameters. assuming time-weighted average change (standard deviation) in rsv log 10 viral load from day 1 to day 9 of -1.5 (1.75) log 10 copies/ml in placebo-treated patients, 25 patients per treatment group were planned to provide approximately 85% power to detect a ≥1.5 log 10 decrease in the primary endpoint in patients receiving presatovir relative to placebo using a 2-sided α of 0.05. we estimated 85% of patients would be evaluable and planned to enroll 60 patients. the safety population included patients who received ≥1 dose of study drug. the efficacy population included safety population patients with quantifiable rsv viral load on day 1. primary and secondary efficacy endpoints were analyzed in the efficacy population and post hoc in subgroups defined by supplemental oxygen use, ribavirin use, duration of rsv symptoms, graft-vshost disease (gvhd), lymphocyte count, and time from hct to rsv infection on day 1. the primary analysis tested superiority of presatovir vs placebo using parametric analysis of covariance using baseline viral load and randomization stratification factors as covariates with a 2-sided α of 0.05 (supplemental methods). number of supplemental oxygen-free days was analyzed using a negative binomial model with stratification factors as covariates and an offset parameter to account for on-study duration. patients who died prior to day 28 or received supplemental oxygen on all days of the study period were assigned a value of 0 supplemental oxygen-free days. the proportion of patients developing respiratory failure of any cause requiring invasive or noninvasive mechanical ventilation through day 28 and allcause mortality through day 28 were analyzed using cochran-mantel-haenszel tests adjusting for the stratification factors at the 2-sided 0.05-level, with 2-sided 95% exact confidence interval (ci) based on the clopper-pearson method for each treatment group. where number of events was small, fisher exact test was used. a sequential testing procedure was used to control the type i error rate of 0.05 across the primary and secondary endpoints [21] . from january 31, 2015, to march 20, 2017, 71 patients were screened for eligibility and 11 were excluded, mostly due to lack of new radiographical abnormalities or inability to confirm lower respiratory tract rsv infection ( figure 1 ). sixty patients were randomized, of whom 31 were assigned to presatovir and 29 to placebo; 1 patient randomized to presatovir withdrew consent before receiving study drug. notable protocol deviations are described in supplemental results. patient demographics and baseline characteristics were generally balanced between study groups (table 1) . overall, the majority of patients (50/59; 84.7%) underwent allogeneic hct and had chronic or acute gvhd (34/59, 57.6%). at start of study treatment, 53 (89.8%) patients were hospitalized for a median of 3 days (range, 0-133 days). twenty-one (35.6%) patients required >2 l/min of oxygen supplementation, and 23 (39.0%) patients were prescribed ribavirin (any formulation). rsv lrti was confirmed from induced sputum in 41 (69.5%) patients and by bronchoalveolar lavage in 18 (30.5%) patients. median time from onset of rsv infection symptoms to start of study treatment was 5 days (range, 1-26 days). infection was due to rsv a in 29 (49.2%) patients and rsv b in 28 (47.5%) patients; 2 (3.4%) patients (1 presatovir, 1 placebo) had missing day 1 rsv viral load data and were excluded from the efficacy population (n = 57). median intranasal rsv viral load on day 1 was 6.36 log 10 copies/ml (range, 2.5-8.23 log 10 copies/ml). nine patients (3 presatovir, 6 placebo) prematurely discontinued study treatment, and 4 patients discontinued study participation before day 28 (1 presatovir, 3 placebo) ( figure 1 ). twenty-seven (90.0%) of 30 patients in the presatovir group and 23/29 (79.3%) patients in the placebo group completed treatment to day 17 ( figure 1 ). figure 2a -b shows median absolute rsv viral load and change from baseline at each study visit. despite adequate plasma concentrations (supplemental results and supplemental table 3 ), presatovir treatment did not significantly reduce time-weighted average change in log 10 rsv viral load from day 1 to day 9 (−1.12 [1.226] log 10 copies/ml versus −1.09 [1.028] log 10 copies/ ml; treatment difference, −0.02 log 10 copies/ml; 95% ci, −.62, .57; p = .94) compared with placebo ( table 2) . during the 28-day study period, 14/29 (48.3%) presatovirtreated patients and 12/28 (42.9%) placebo-treated patients required supplemental oxygen. median (range) number of supplemental oxygen-free days was similar between presatovirtreated (26 [0-33] days) and placebo-treated (28 [0-30] days) patients (p = .84) ( table 2 ). three presatovir-treated patients (10.3%) and 3 placebo-treated patients (10.7%) developed respiratory failure requiring mechanical ventilation through study day 28 (p = 1.0). no presatovir-treated patients and 2 placebotreated patients (7.1%) died through day 28 (p = .24) ( table 2) ; 1 death was due to respiratory failure. exploratory efficacy outcomes are described in supplemental results. primary and secondary efficacy endpoints did not differ appreciably between patients treated with presatovir relative to placebo in subgroups defined by absolute lymphocyte count on day 1, presence of gvhd, time from onset of rsv symptoms to study treatment, and timing of rsv infection after hct (supplemental tables 6-9 ). optional extended viral monitoring and serologic responses to rsv infection are presented in the supplemental results. sequencing of rsv f gene detected postbaseline amino acid substitutions at resistance-associated positions in 6/29 (20.7%) of presatovir-treated patients and 0/28 placebo-treated patients. these substitutions were detected a median of 25 (range, 7-56) days after start of treatment (supplemental table 10 ). twenty-four presatovir-treated patients (80.0%) and 23 placebo-treated patients (79.3%) experienced ≥1 ae, whereas 7 presatovir-treated patients (23.3%) and 7 placebo-treated patients (24.1%) experienced serious aes (saes). adverse events ≥grade 3 occurred in 7 presatovir-treated patients (23.3%) and 9 placebo-treated patients (31.0%). individual aes occurred in ≤10% of presatovir-treated patients (table 3) . numerically more frequent aes in patients treated with presatovir versus placebo were pneumonia, increased alanine aminotransferase, hypokalemia, nausea, acute sinusitis, and epistaxis (3 patients each, 10%), and increased aspartate aminotransferase, dry mouth, and increased alkaline phosphatase (2 patients each, 6.7%; table 3 ). except for sae pneumonia in 3 presatovirtreated patients (10%), grade 3 or 4 aes and saes occurred in 1 patient each and were numerically less frequent overall in patients treated with presatovir versus placebo (supplemental tables 11-12 ). there were no significant imbalances in electrocardiogram and troponin results during the study. no patients treated with presatovir and 2 patients treated with placebo (6.9%) died during the 28-day study period; 1 death was due to respiratory failure and 1 to progressive acute leukemia. another 2 patients (6.9%) who received placebo died after day 28, 1 of respiratory failure, and 1 of invasive fusariosis. this is the first placebo-controlled clinical trial, to our knowledge, evaluating treatment of rsv lrti with a new antiviral agent in hct recipients. presatovir had a favorable safety profile and was well tolerated but did not decrease timeweighted average change in nasal rsv viral load from day 1 to day 9, number of days with supplemental oxygen use, or frequency of respiratory failure or mortality relative to placebo. in contrast, presatovir treatment significantly reduced viral load, clinical signs, and symptoms of experimental rsv infection in healthy volunteers treated upon detection of rsv replication [16] . potential explanations for this discrepancy have important implications for design of clinical trials evaluating antiviral treatments for rsv infection in hct recipients and other patient populations. in the past 5 years, treatment with a fusion inhibitor or nucleoside polymerase inhibitor significantly reduced rsv viral load, signs, and symptoms in 3 challenge studies in healthy human volunteers [16, 22, 23] . however, clinical trials of presatovir conducted in multiple different patient populations, including this study and a companion urti study (chemaly et al [24] , this issue), indicate difficulties remain in translating challenge study results to successful clinical trials in patients with natural infection [25, 26] . one partial explanation is the challenge model's inconsistent representation of the natural infection setting. challenge study volunteers were inoculated intranasally with rsv, then monitored for nasal rsv replication with twice-daily nasal washes that were immediately evaluated with molecular assays for rsv [16, 22, 23] . antiviral treatment was initiated 6-24 hours after rsv detection, generally several days before peak viral load and prior to manifestation of significant clinical signs and symptoms [16, 22, 23] . in the present study, patients with naturally acquired rsv lrti received presatovir later in the disease course compared with challenge study subjects (median [range], 5 days after symptom onset); delay was also observed in the urti trial (median [range], 4 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] days) and other studies of presatovir in natural rsv infection [25, 26] . because clinical signs and symptoms tend to correlate with nasal viral load [16, 22] , these patients presumably presented near or more likely after peak nasal viral load, potentially beyond the therapeutic window for presatovir even in immunocompromised patients. host immune-mediated clearance of the virus at this stage may also mask treatment-induced reduction in viral load. thus, treatment delay may explain lack of presatovir efficacy in the current study. the mechanism of action of presatovir may also have limited efficacy in this study. because rsv is capable of cell-to-cell spread, inhibition of viral fusion may not halt propagation of established infection along the respiratory tract. therefore, fusion inhibitors, such as presatovir, may need to be administered, whereas virus-cell fusion still represents the main mode of viral spread to appreciably reduce nasal viral load. emergence of f gene protein amino acid substitutions associated with fusion inhibitor resistance was also relatively frequent (21%) in this immunocompromised population. polymerase inhibitors can terminate intracellular rsv replication and may have wider data are n (%) unless otherwise specified. abbreviations: cmv, cytomegalovirus; gvhd, graft-vs-host disease; hct, hematopoietic cell transplant; lrt, lower respiratory tract; max, maximum; min, minimum; rsv, respiratory syncytial virus. a on the day of the first dose of study drug, 11 patients receiving presatovir and 10 patients receiving placebo were being treated with ribavirin. current or intended use of ribavirin on day of randomization was used to stratify randomization. b these patients were excluded from the efficacy population. c for these values, n = 29 for the presatovir arm and n = 28 for the placebo arm. [27] . however, no polymerase inhibitor has demonstrated clinical efficacy in a natural infection setting to date. furthermore, nasal viral load is questionable as a primary endpoint for proofof-concept studies in naturally infected patients with lrti because upper respiratory tract samples, although more convenient to obtain, may not reflect viral activity in the lower respiratory tract, particularly in immunocompromised patients. alternative approaches are needed for noninvasive measurement of viral disease dynamics and antiviral activity in lower airway and alveolar tissue. the findings of this trial call into question whether appearance of new radiological opacities with documentation of rsv in the upper or lower respiratory tract accurately classifies patients with rsv infection. this approach may be satisfactory for retrospective studies but inadequate as an enrollment criterion for prospective clinical trials. radiographic findings in adults with rsv lrti confirmed from a lower respiratory tract specimen are not well characterized, and radiographic abnormalities in these patients may be caused by other viruses, bacteria, or fungi-particularly in immunocompromised patients-or even noninfectious processes. as rsv chiefly affects airway epithelium [28] , rsv lrti could manifest without radiographic findings, and present only as lower airway symptoms (eg, wheezing or obstructive spirometry pattern). furthermore, the degree of lung injury in patients with rsv lrti may not be reversible by antiviral treatment alone. these issues will need to be considered in future clinical trials. the lack of clinical benefit in this study may also relate to the selected clinical endpoints, which occurred at lower-thananticipated rates that decreased power to detect a treatment effect. although all subjects enrolled in the current study met waghmare et al's criteria for proven or probable lrti, median number of supplemental oxygen-free days through day 28 was much higher (26 and 28 days for presatovir-treated and placebo-treated patients, respectively, vs 17 days), and fewer patients required >2 l/min supplemental oxygen at baseline (35.6% versus 57%) relative to patients who presented with or developed lrti in the waghmare study [3] . furthermore, lymphopenia is a major risk factor for rsv infection and subsequent poor outcomes in hct recipients [2, 4, 29] , but only 4/47 patients with available data in the current study were lymphopenic (<200 cells/mm 3 ) at baseline, possibly because rsv infection occurred relatively late after hct (median, 485 vs 129 days in the waghmare study). these differences could be due to limited enrollment of patients perceived as fragile because of the requirement for lower respiratory sampling to confirm rsv lrti. enrichment of the study population for immunosuppressed patients should be considered in future clinical trials of therapies for rsv infection in hct recipients. in summary, presatovir treatment was generally well tolerated in hct recipients with naturally acquired rsv lrti but did not achieve virologic or clinical endpoints. the tendency of adults with naturally acquired rsv infection to seek treatment only after several days of symptoms, when the treatment window may have closed for fusion inhibitors in particular, is a challenge for clinical trials of rsv-specific antiviral therapies. the numerically lower rate of pulmonary complications in the presatovir urti trial suggests that early treatment, before lrti develops, is key for success in future studies. acute kidney injury 0 2 (6.9) atrial fibrillation 0 2 (6.9) alkaline phosphatase increased 2 (6.7) 0 dizziness 0 2 (6.9) fatigue 0 2 (6.9) neutropenia 0 2 (6.9) data are shown as n (%). respiratory syncytial virus infection in adults respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease supplemental oxygen-free days in hematopoietic cell transplant 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self-collection in respiratory virus infection diagnostic testing gatekeeping strategies for clinical trials that do not require all primary effects to be significant activity of oral als-008176 in a respiratory syncytial virus challenge study safety and efficacy of oral rv521 in a human respiratory syncytial virus (rsv) phase 2a challenge study a phase 2, randomized, double-blind, placebo-controlled trial of presatovir for the treatment of respiratory syncytial virus upper respiratory tract infection in hematopoietic-cell transplant recipients a phase 2b, randomized controlled trial of presatovir, an oral rsv fusion inhibitor, for the treatment of respiratory syncytial virus (rsv) in lung transplant (lt) recipients a phase 2b, randomized, double-blind, placebo-controlled trial of presatovir (gs-5806), a novel oral rsv fusion inhibitor, for the treatment of respiratory syncytial virus (rsv) in hospitalized adults late therapeutic intervention with a respiratory syncytial virus l-protein polymerase inhibitor, pc786, on respiratory syncytial virus infection in human airway epithelium severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center financial support. this work was supported by gilead sciences, inc. potential conflicts of interest. f. m. m. reports research grants paid to his institution and consulting fees for clinical trial design from gilead sciences, inc., during the conduct of the study; and research support paid to his institution and consulting fees for clinical trial design from chimerix and glaxosmithkline, research grant support paid to his institution from ansun, and personal fees from roche molecular diagnostics and visterra outside the current study. r. f. c. reports research grants paid to his institution and personal fees from gilead sciences, inc., during the conduct of the study; research grants paid to his institution from ablynx, aicuris, ansun, chimerix, merck, oxford immunotec, novartis, pulmotec, shire, and xenex; and consultant fees/honoraria from ablynx, achaogen, adma biologics, ansun, astellas, chimerix, clinigen, janssen, oxford immunotec, merck/ merck sharp and dohme (msd), pulmotect, shionogi, shire, and xenex outside the current study. k. m. m. reports grants as a clinical trial investigator from gilead sciences, inc., during the conduct of the study; clinical research grants from ansun, astellas, merck, rebiotix, scynexis, and shire; and consultant fees/honoraria from chimerix, glaxosmithkline, merck, and scynexis outside the current study. d. g. l. reports research grants and consultant fees or speaker honoraria from astellas, msd, and pfizer outside the current study. h. h. h. reports nothing to disclose. c. b. s. reports grants paid to her institution from gilead sciences, inc., during the conduct of the study and from abbott, ablynx, chimerix, glaxosmithkline, merck, schering, shire, and viiv outside the current study. a. b. reports funding to her institution from gilead sciences, inc., during the conduct of the study; and research grants from pfizer and sos oxygène and consultant/speaker fees from ablynx; gilead sciences, inc.; merck; pfizer; shire; therakos; and zambon outside the current study. s. s. reports grants paid to his institution from gilead sciences, inc., during the conduct of the study; grants paid to his institution from astellas, ansun, cidara, emergent biosolutions, merck, scynexis, shiongi, shire, and t2 microsystems outside the current study; and direct payments as a member of a data safety monitoring board from merck outside the current study. p. l. reports research grants or fees/ honoraria paid to his institution from astellas; gilead sciences, inc.; merck; oxford immunotec; and shire; and consultant fees/honoraria from ablynx and aicuris outside the current study. a. w. reports clinical trial support from gilead sciences, inc., during the conduct of the study. e. b. reports speaker fees from boehringer ingelheim; gilead sciences, inc.; novartis; pfizer; and roche outside the current study. y. j. k. reports grants from gilead sciences, inc., during the conduct of the study. m. m., d. p. p., y. g., p. g., and t. r. w. are employees of gilead sciences, inc., and may hold stock. r. j. is a former employee of and holds stock in gilead sciences, inc. m. b. reports clinical trial support and consulting fees from gilead sciences, inc., during the conduct of the study; and grants and personal fees from ablynx; ansun; chimerix; glaxosmithkline; merck; shire; and vir bio; and personal fees from bavarian nordic; humabs, inc.; janssen; modema therapeutics; pulmocide; and pulmotect outside the current study. j. w. c. is an employee and stockholder of janssen biopharma and a former employee and current stockholder of gilead sciences, inc. s. s. d. reports grants paid to his institution from gilead sciences, inc., during the conduct of the study; and grants to his institution and personal fees for consulting, advisory board, and speaker bureau participation from merck; personal fees for advisory board participation from clinigen and janssen; and grants paid to his institution from ablynx, aicuris, ansun, glaxosmithkline, oxford immunotec, and shire outside the current study. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-299720-f0ny4ur5 authors: kim, seung woo; park, jung wan; jung, hee-dong; yang, jeong-sun; park, yong-shik; lee, changhwan; kim, kyung min; lee, keon-joo; kwon, donghyok; hur, young joo; choi, boyoul; ki, moran title: risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea date: 2017-03-01 journal: clin infect dis doi: 10.1093/cid/ciw768 sha: doc_id: 299720 cord_uid: f0ny4ur5 background. transmission heterogeneity was observed during the 2015 korean outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection. only 22 of 186 cases transmitted the infection, and 5 super-spreading events caused 150 transmissions. we investigated the risk factors for mers-cov transmission. methods. epidemiological reports were used to classify patients as nonspreaders, spreaders, or those associated with a super-spreading event (5 or more transmissions). logistic regression analyses were used to evaluate the factors for mers-cov transmission. results. compared to nonspreaders, spreaders exhibited a longer interval from symptom onset to isolation (7 days vs 3 days) and more frequent pre-isolation pneumonia diagnoses (68.2% vs 17.1%). spreaders also exhibited higher values for pre-isolation contacts (149 vs 17.5), pre-isolation hospitalization (68.2% vs 16.5%), and emergency room (er) visits (50% vs 7.3%). spreaders exhibited lower cycle thresholds for the upe and orf1a genes (22.7 vs 27.2 and 23.7 vs 27.9, respectively). in multivariate analysis, transmission was independently associated with the cycle threshold (odds ratio [or], 0.84; 95% confidence interval [ci], 0.75–0.96) and pre-isolation hospitalization or er visits (or, 6.82; 95% ci, 2.06–22.84). the super-spreading events exhibited higher values for pre-isolation contacts (777 vs 78), pre-isolation er visits (100% vs 35.3%), and doctor shopping (100% vs 47.1%) compared to non-super-spreading events. conclusions. these findings indicate that transmission is determined by host infectivity and the number of contacts, whereas super-spreading events were determined by the number of contacts and hospital visits. these relationships highlight the importance of rapidly enforcing infection control measures to prevent outbreaks. transmission heterogeneity was a significant characteristic of the 2015 south korean outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection [1] . transmission heterogeneity describes a state in which most transmissions are related to a few patients and most patients do not transmit the disease. numerous other infectious diseases exhibit transmission heterogeneity [2] , and this concept is important for understanding epidemics. the course of an epidemic is influenced by the basic reproduction number (r 0 = the average number of cases that 1 case produces in a susceptible population) and transmission heterogeneity [3] . as r 0 represents an average quantity, it is often insufficient to explain individual variation, and as transmission heterogeneity reflects individual variation, it can help predict the likelihood of super-spreading events. even in instances with a low r 0 , a disease with high transmission heterogeneity (eg, severe acute respiratory syndrome [sars]) can cause super-spreading events [2] , such as the super-spreading that occurred during the 2003 sars outbreak [2, 4] . transmission heterogeneity was observed during early mers-cov outbreaks [1] and became prominent during the 2015 south korean outbreak. among the 186 confirmed korean cases of mers-cov infection, >80% of the transmissions were epidemiologically associated with 5 patients [5] , and almost 90% of the cases caused no transmission. furthermore, a recent study revealed that mers has greater transmission heterogeneity compared to sars [6] . therefore, to successfully control mers-cov infection, it is essential to identify high-risk patients and perform targeted infection control [2] . however, these patients are difficult to identify, as an individual's infectiousness is affected by complex interactions between the pathogen, host, and environment. several researchers have attempted to identify risk factors for super-spreading events during the sars outbreak [3, 4, 7] , although there is little information regarding the high-risk group(s) from the mers-cov outbreak. the recent south korean mers-cov outbreak was triggered by a single imported case, and epidemiological tracing was performed for all laboratory-confirmed cases and their close contacts [5, [8] [9] [10] [11] [12] [13] . thus, it is possible to precisely reconstruct the transmission chain and identify patients who transmitted mers-cov infection. therefore, we analyzed the epidemiological characteristics that were associated with mers-cov transmission and super-spreading events. cases of mers-cov infection were confirmed using real-time reverse-transcription polymerase chain reaction (rt-pcr) assays, regardless of their clinical manifestations. the epidemiological reports were analyzed by epidemic intelligence service officers who participated in the mers-cov outbreak investigation. when a case was exposed to multiple confirmed cases, the transmission was attributed to the case with the highest likelihood of transmission, and any conflicts were resolved through the consensus of the epidemic intelligence service officers. spreaders were defined as confirmed cases of mers-cov infection that were epidemiologically suspected of transmitting mers-cov to 1 or more persons. super-spreading events were arbitrarily defined as transmission of mers-cov infection to 5 or more cases. the patient who triggered the outbreak was defined as patient zero. cases that were infected by patient zero were defined as first-generation cases; cases that were infected by first-generation cases were defined as second-generation cases; and cases that were infected by second-generation cases were defined as third-generation cases [14] . isolation was defined as separating symptomatic patients from others to prevent spreading, and quarantine was defined as separating or restricting the movement of healthy individuals who may have been exposed to the infection within the maximum incubation period. the transmission date was defined as the date of contact between the spreader and suspected secondary case during the spreader's infectious period. in cases with an exposure duration of longer than 1 day, the transmission date was defined as the day with the highest likelihood of transmission or as the median day during the exposure period in cases with consistent contact throughout the exposure. the date of sampling was the day on which the first positive respiratory specimen was collected. close contacts were defined using the guidelines on middle east respiratory syndrome [15] , which include persons who stayed in a room or ward with a confirmed case, who directly contacted respiratory secretions from confirmed cases, or who stayed within 2 m from the confirmed cases without wearing appropriate personal protective equipment. pre-isolation pneumonia diagnoses were based on radiographic evidence. doctor shopping was defined as visiting multiple healthcare facilities without an official interhospital transfer after developing mers-cov symptoms [16] . epidemiological reports from the outbreak were evaluated to collect data regarding basic demographic characteristics, medical history, mers-cov exposure, symptoms and their onset date(s), sampling date(s), contact history, and post-exposure infection control. the reports were drafted during the outbreak based on direct interviews with the confirmed cases and follow-up epidemiological investigations that were performed to identify the exposure route and close contacts. hospital information systems were reviewed to identify patients who stayed in the hospital during the exposure period and healthcare providers who contacted the patient(s). persons who contacted confirmed cases outside healthcare facilities were also traced. data from closed circuit television, credit card transactions, and health insurance services were also reviewed [5] . the numbers of close contacts were calculated based on the number of quarantines during the outbreak. all data were collected as part of the public health response and in accordance with the infectious disease control and prevention act [17] . clinical specimens were collected in sterile containers and immediately transferred to qualified facilities. sputum samples were mixed with 0-1× phosphate-buffered saline and vortexed vigorously to reduce their viscosity. viral rna was extracted from the clinical specimens using a qiagen viral rna mini kit (qiagen, hilden, germany). all laboratory diagnoses of mers-cov were confirmed using the world health organization guidelines [18] and results from real-time rt-pcr assays that target upstream of the mers-cov envelope protein gene (upe) and the open reading frame 1a gene (orf1a) [19] . cycle threshold (ct) values for the upe and orf1a genes were obtained during testing. we analyzed the ct value from the first positive mers-cov specimen (or the specimen obtained immediately after a positive screening test). categorical variables were compared using the χ 2 test and fisher exact test, and the mann-whitney test was used for continuous variables. the variables' associations with mers-cov transmission were evaluated using multiple logistic regression analyses, and covariates were selected based on a p value of < .1 in the univariate analyses. a p value of < .05 was considered statistically significant. all analyses were performed using r software (version 3.2.2; r foundation, vienna, austria). we identified 186 cases of confirmed mers-cov infection. patient zero infected 28 first-generation cases. among the 28 cases, 8 were responsible for transmission to 121 second-generation cases. among the 121 cases, 12 infected 30 third-generation cases. one patient with an unclear source of infection (case 119) transmitted the infection to another patient. four patients exhibited unclear sources of transmission (cases 43, 178, 184, and 185). each confirmed case transmitted the infection to 0-83 secondary cases ( figure 1 ). there were 164 nonspreaders and 22 spreaders (1 or more transmission). of the spreaders, 5 cases transmitted the infection to 5 or more cases (super-spreading event). after excluding the 5 cases with unclear infection sources, we identified 180 transmissions generated by 22 spreaders. a total of 150 transmission events (83.3%) were epidemiologically linked to the 5 super-spreading events. twenty-five transmission events (13.9%) occurred within 3 days after symptom onset, 136 transmissions (75.6%) occurred 4-7 days after symptom onset, and 19 transmissions (10.6%) occurred >7 days after symptom onset. a total of 170 transmission events (94.4%) occurred on the day of or after a radiographically confirmed diagnosis of pneumonia. a total of 173 transmissions (96.1%) occurred before appropriate in-hospital isolation. seven transmissions (3.9%) occurred between confirmed cases and healthcare personnel after in-hospital isolation: 4 (cases164, 169, 181, and 183) were doctors or nurses who managed confirmed cases, 1 (case148) participated in cardiopulmonary resuscitation of a confirmed case, 1 (case 162) involved portable radiography for a confirmed case, and 1 (case 179) rode in an ambulance with a confirmed case during a hospital transfer. ; p = .009). the intervals from symptom onset to diagnosis or obtaining a respiratory specimen were also significantly longer among spreaders (to diagnosis: 9 [5.5-10] days vs 5 [3] [4] [5] [6] [7] [8] days; p = .008 and to sampling: 8 [5-9.3 ] days vs 4 [2] [3] [4] [5] [6] days; p < .001). furthermore, the interval from symptom onset to isolation was longer among spreaders (7 [4.5-9] days vs 3 [1] [2] [3] [4] [5] [6] days; p = .002). spreaders exhibited a significantly higher proportion of pre-isolation pneumonia diagnoses (68.2% vs 17.1%; p < .001) and a longer interval from the pneumonia diagnosis to isolation (4 [3] [4] [5] [6] [7] days vs 1 [0-3] days; p = .008). the overall number of contacts was significantly larger among spreaders compared to nonspreaders (149 [22.3-640.5] vs 17.5 [2-92.5]; p = .004). compared to nonspreaders, spreaders exhibited a significantly higher proportion for pre-isolation hospitalization (68.2% vs 16.5%; p < .001), visiting outpatient clinics (59.1% vs 33.5%; p = .019), and visiting emergency rooms (ers; 50% vs 7.3%; p < .001). we used logistic regression analyses to evaluate the risk factors for transmission (table 2 ). in the univariate analyses, transmission was associated with underlying respiratory disease, ct value, interval from symptom onset to diagnosis, number of contacts, and pre-isolation hospitalization or er visits. in the multivariate analyses, transmission was independently associated with a low ct value for upe (odds ratio [or], 0.84; 95% confidence interval [ci], 0.75-0.96) and pre-isolation hospitalization or er visits (or, 6.82; 95% ci, 2.06-22.84). we compared the epidemiological characteristics of the 5 spreaders with 5 or more transmissions and the 17 spreaders with 4 or fewer transmissions (table 3) . both groups exhibited similar host factors and contact durations. however, spreaders with 5 or more transmissions exhibited higher values for we evaluated the epidemiological characteristics of patients who transmitted mers-cov during the recent south korean outbreak. among the 186 confirmed mers-cov cases, only 22 cases transmitted the infection to other individuals. these spreaders had higher host infectivity and wider and prolonged contacts compared to nonspreaders. the risk factors for super-spreading events included a larger number of contacts and a pre-isolation er visit. doctor shopping was marginally associated with a super-spreading event. however, both spreaders with 5 or more transmissions and spreaders with 4 or fewer transmissions exhibited similar levels of host infectivity. it appears that both host infectivity and the number of contacts influenced mers-cov transmission, whereas super-spreading events were mostly associated with a greater likelihood of encountering other people under diverse environmental conditions. during the 2015 outbreak, approximately 75% of the transmissions occurred during days 4-7 after symptom onset, and this may be a period when the risk of transmission is particularly high. furthermore, this high-risk period was temporally associated with other epidemiological factors. first, the period overlapped with the confirmed cases' visits to healthcare facilities, as hospitalization and er visits peaked during days 4-7 after symptom onset. it is well known that mers-cov outbreaks generally occur in the healthcare setting [1, 5, 13, 20] , and the high-risk period may be associated with healthcare-seeking behaviors. second, the high-risk period was several days (1-4 days) after the radiographic diagnoses of pneumonia, which generally occurred on days 3-4 after symptom onset. although the significance of pre-isolation pneumonia has not been discussed previously, a radiographic diagnosis of pneumonia may influence transmission in 2 ways. first, it may directly increase the chance of transmission by actively generating lower respiratory tract secretions and a productive cough. second, it may be an indirect index of disease severity and hospital visiting status. in our study, cases with pre-isolation pneumonia had lower ct values and more frequent pre-isolation hospital visits. the epidemiological significance of the high-risk period could also be observed when we compared the spreaders and nonspreaders. the spreaders were typically isolated after the high-risk period (median, 7 days after symptom onset and 4 days after a diagnosis of pneumonia), whereas nonspreaders were typically isolated before this period (median, 3 days after symptom onset and 1 day after a diagnosis of pneumonia). similar results were observed in a study of the sars outbreak, which revealed that late admission to healthcare facilities (especially >4 days after symptom onset) was associated with super-spreading events [21] . thus, infection prevention measures should target isolation before this critical period (ie, within 4-7 days after symptom onset and within 1 day after the detection of pneumonia). interestingly, the average duration from symptom onset to isolation dropped to <4 days during the first week of june 2015, and reports of new cases have rapidly decreased since that time. among the host factors that were associated with transmission, only the ct value was statistically significant in the multivariate analyses. the ct value is a semiquantitative continuous variable that is inversely proportional to the viral load. ct values are associated with the severity of mers-cov infection [22] , although its relationship with transmission has rarely been studied. in the present study, spreaders had significantly lower ct values compared to nonspreaders, which suggests that ct values might reliably predict transmission. moreover, the cases with very low ct values (ct <23) tended to transmit the infection in uncommon circumstances. in both the present study and previous studies, mers-cov transmission usually occurred in the hospital setting [1, 11, 13, 23] . in contrast, cases with very low ct values transmitted the infection in more diverse settings in the present outbreak (eg, their household, in an ambulance, in an outpatient clinic, or to healthcare personnel after in-hospital isolation). these findings suggest that cases with very low ct values can potentially transmit the infection in unexpected conditions. however, our data regarding the ct values have several limitations. first, various amounts of phosphate-buffered saline were added to dilute the respiratory specimens, and this there was no multicollinearity between the independent variables (all variables: r score of <0.5). abbreviations: ci, confidence interval; or, odds ratio. may have affected the ct values. second, the ct value is influenced by the specimen type and the interval between symptom onset and sample collection [22, 24] , but various different types of specimens were collected at different time points in the present study. however, we only evaluated 5 nonsputum specimens, and there was no linear correlation between the ct values and the interval from onset to sampling. our comparison of the spreaders with 5 or more transmissions and spreaders with 4 or fewer transmissions revealed that the spreaders with 5 or more transmissions had an approximately 10-fold higher number of contacts. furthermore, there were no significant differences in host infectivity. these findings may suggest that the underlying likelihood of transmission has the greatest influence on super-spreading events rather than an intrinsic difference in host infectivity. a similar finding was observed in a previous study of the sars super-spreading event [4] , with those super-spreaders having 11-74 contacts compared to 1-4 contacts for the spreaders with 1-2 transmissions. our study also revealed that a pre-isolation er visit and doctor shopping were associated with super-spreading events. in addition, super-spreading events were associated with the number of healthcare facilities that each patient visited for hospitalization or er treatment but not with the number of hospitals visited for outpatient treatment. in south korea, patients who seek hospitalization without prior arrangements tend to visit the er, and a history of 2 or more er visits strongly suggests that the patient had been doctor shopping during hospitalization. specific environmental conditions have been suggested to increase the likelihood of a super-spreading event [3] , and doctor shopping may increase the likelihood of encountering these conditions. for example, when a confirmed case changes hospital during hospitalization without an official interhospital transfer, multiple environments are exposed to the infected case (an ambulance, an er, and a ward). thus, doctor shopping can greatly increase the likelihood of encountering conditions that are suitable for a super-spreading event. in the present outbreak, 4 of the 5 super spreaders (cases 1, 14, 16, and 76) transmitted the infection at 2 or more hospitals, as they had visited multiple healthcare facilities. therefore, it is very important to have an early suspicion of mers-cov infection and minimize doctor shopping during the early stage of an outbreak. our study has several limitations. first, some of the confirmed cases had multiple potential sources of infection, and we attributed the transmission to the case with the highest epidemiological probability. the source of infection was clear in >95% of the transmissions, and we excluded 3 cases that had contact with multiple cases and an unclear source of transmission. however, as the analyses of the epidemiological data are ongoing, the list of spreaders may change if new epidemiological evidence is uncovered. second, we did not have access to genomic sequencing data, which might have provided information regarding the relatedness of transmitted strains. third, transmission may be affected by other epidemiological factors, including aerosol-generating procedures, differences in environmental conditions, and variations in crowdedness [3, 13, 25] . however, these factors were not included in the present analysis. fourth, serological testing was not performed for every close contact, and additional asymptomatic cases may have been present. however, the seropositive rate was 0.7% in a recent serological study of close contacts [26] . thus, the absence of serological testing likely did not significantly influence our results. we evaluated the epidemiological risk factors for mers-cov transmission during the recent south korean outbreak. superspreading events were not related to intrinsic host characteristics and were attributable to the likelihood of transmission. therefore, strict er triage and minimizing doctor shopping during an outbreak's early stage may help prevent super-spreading events. hospital outbreak of middle east respiratory syndrome coronavirus superspreading and the effect of individual variation on disease emergence why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? superspreading sars events middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study super-spreaders in infectious diseases mers outbreak in korea: hospital-to-hospital transmission epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital epidemiological investigation on the 119th confirmed mers-cov case with the indefinite mode of transmission in pyeongtaek outbreak the first case of the 2015 korean middle east respiratory syndrome outbreak mers epidemiological investigation to detect potential mode of transmission in the 178th mers confirmed case in pyeongtaek mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study ministry of health and welfare. guidelines on middle east respiratory syndrome doctor shopping: a phenomenon of many themes korea ministry of government legislation infectious disease control and prevention act world health organization. laboratory testing for middle east respiratory syndrome coronavirus. interim recommendations complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/002_05_2015, isolated in south korea mers-cov outbreak in jeddah-a link to health care facilities predicting super spreading events during the 2003 severe acute respiratory syndrome epidemics in hong kong and singapore association of higher mers-cov virus load with severe disease and death, saudi arabia an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity acknowledgements. the authors thank sung soon kim and jeong-gu nam (division of respiratory viruses, center of infectious disease, korea centers for disease control and prevention) for laboratory assistance and technical support in the production of the manuscript. in addition, we thank all administrative and laboratory staff of the korea centers for disease control and prevention who participated in the mers-cov outbreak control effort.author contributions. s. w. k. performed the literature search, study design, data collection, analysis, interpretation, and writing. m. k. contributed to the study design, data interpretation, and writing. j. w. p., y. s. p., c. l., k. m. k., k. j. l., and d. k. contributed to the data collection and interpretation. h. d. j. and j. s. y. contributed to the data collection, mers pcr testing, data interpretation, and analysis. y. j. h. and b. y. c. contributed to the study design, data interpretation, and revising the manuscript. s. w. k. and m. k. revised the manuscript. all authors contributed to writing and approved the final manuscript.potential conflicts of interest. authors certify no potential conflicts of interest. the authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-294558-cqa58db8 authors: wang, yubo; tong, jin; qin, yalan; xie, ting; li, jianghua; li, jianrong; xiang, jianhua; cui, yong; higgs, elizabeth s; xiang, jianglin; he, yong title: characterization of an asymptomatic cohort of sars-cov-2 infected individuals outside of wuhan, china date: 2020-05-22 journal: clin infect dis doi: 10.1093/cid/ciaa629 sha: doc_id: 294558 cord_uid: cqa58db8 background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2, resulting in the coronavirus disease covid-19) is highly transmissible among people. asymptomatic infections are also an important source of infection. here, we aimed to further clarify the epidemiologic and clinical characteristics of asymptomatic sars-cov-2 infections. methods: we identified close contacts of confirmed covid-19 cases in northeast chongqing who were rt-pcr+ yet remained asymptomatic throughout their infections. we stratified this cohort by normal versus abnormal findings on chest ct, and compared the strata regarding comorbidities, demographics, laboratory findings, viral transmission and other factors. results: between january and march, 2020, we identified and hospitalized 279 rt-pcr+ contacts of covid-19 patients. of these, 63 (23%) remained asymptomatic until discharge; 29 had abnormal and 34 had normal chest ct findings. the mean cohort age was 39.3 years, and 87.3% had no comorbidities. mean time to diagnosis after close contact with a covid-19 index patient was 16.0 days (range 1 to 29), and 13.4 days and 18.7 days for those with abnormal and normal ct findings, respectively (p < 0.05). nine subjects (14.3%) transmitted the virus to others; 4 and 5 were in the abnormal and normal ct strata, respectively. the median length of nucleic acid turning negative in asymptomatic covid-19 patients was 13 days, compared to 10.4 days in those with normal chest ct (p < 0.05). conclusions: a portion of these asymptomatic individuals, with and without abnormal chest ct scans, were capable of transmitting the virus to others. given the frequency and potential infectiousness of asymptomatic infections, testing of traced contacts is essential. studies of the impact of treatment on asymptomatic rt-pcr+ individuals on disease progression and transmission should be undertaken. in december 2019 a novel coronavirus, which was later named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), caused a large outbreak of infectious disease, designated covid-19. it spread from wuhan, hubei province, to the whole of china and continues to pose a great threat to public health [1] . compared to sars-cov or mers-cov, it appears to be transmitted more easily [2] [3] [4] . sars-cov-2 can be transmitted through a variety of modes, including droplets, close contact, aerosol and potentially also fecal-oral transmission [5, 6] . people of all ages are generally susceptible to the virus. symptomatic covid-19 patients and asymptomatic cases are both a source of infection and patients in the incubation period can transmit sars-cov-2 to other persons [7] [8] [9] [10] . patients with symptoms are more likely to be detected. among patients with sars-cov-2, fever has been the most common symptom, followed by dry cough, dyspnoea, myalgia, headache and diarrhoea [11] [12] [13] . in severe covid-19 cases, symptoms can progress to acute respiratory distress syndrome, septic shock and metabolic acidosis [14] . the clinical presentation of covid-19 therefore ranges from asymptomatic to severe respiratory illness [15] . the presentation of symptoms provides an opportunity for case detection and isolation, facilitating the interruption of transmission. because most sars-cov patients had severe symptoms, they were relatively simple to identify and isolate, thus interrupting the chain of transmission. unfortunately, covid-19 has an incubation period from infection to onset of symptoms that lasts an average of 10 days (with a reported range of 2-14 days)[11, 16, 17] . the virus is also able to spread from one person to another before any symptom onset [14] . it is reported that about 1.2% of the patients have no any actual clinical manifestations during the entire course of the disease [18] . asymptomatic cases, however, particularly those with no history of contact with a known sars-cov-2, are hard to identify. there are several case reports of sars-cov-2 transmission from asymptomatic individuals [8, 9, 13, 19] , a c c e p t e d m a n u s c r i p t including a report of presumed transmission by asymptomatic carriers with normal chest computed tomography (ct) [20] . together, these traits make control of sars-cov-2 very difficult. however, the exact profile of these asymptomatic cases and their role within the wider epidemic are unclear. it is important to understand the proportion of asymptomatic or mildly ill cases and their role in driving the epidemic [21] . since many sars-cov-2 infections are asymptomatic, subclinical or very mild the hallmarks and course of asymptomatic infection warrants for further investigation [22] [23] [24] . our goal was to document the characteristics of asymptomatic infections and identify factors associated with asymptomatic infection, enabling the formulation of corresponding strategies and control measures. we conducted a retrospective study focusing on the characteristics and length of rt-pcr positivity and associated hospitalization of confirmed asymptomatic cases of sars-cov-2 in northeast region of chongqing almost in the chongqing three gorges center hospital and wanzhou district hospital since january 2020. active contact tracing in china includes nasopharyngeal swab diagnostic testing of all contacts regardless of symptoms. in accord with chinese guidelines for the investigation and management of close contacts of covid-19 cases, close contacts were defined as people who had not taken effective protection while in close contact with suspected or confirmed cases 2 days prior to symptom onset or, if the case was asymptomatic, 2 days before sampling [25] . we screened close contacts from january through march 2020 in northeast chongqing by real time reverse transcription polymerase chain reaction (rt-pcr) of nasopharyngeal swabs. only a c c e p t e d m a n u s c r i p t patients with an rt-pcr-confirmed infection and without any apparent symptoms, including (but not limited to) cough, fever, short of breathless and muscle soreness were enrolled in this study. these individuals were identified through close symptomatic contacts in both the clinic and the community. in accord with china's prevention and control policy, all rt-pcr positive cases were hospitalized and treated whether they have symptoms or not. we monitored asymptomatic individuals daily during hospitalization to document the development of any signs and symptoms. we recategorized patients who were asymptomatic at baseline to symptomatic if they reported any symptoms. case definitions of confirmed human infection with sars-cov-2 are in accordance with the interim guidance from the world health organization. following a positive sars-cov-2 nucleic acid test, a chest ct scan was done [26] . individuals were then divided to two groups by chest ct scan: a group with changes visible on the radiographic imaging, called asymptomatic covid-19 patients, and a group without any detectable imaging changes, called asymptomatic with normal chest ct. during hospitalization, all participants, regardless of group, had follow-up chest ct scans every 4 to 5 days until discharge. individuals initially allocated to the asymptomatic with normal chest ct group were immediately reallocated to the asymptomatic covid-19 patient group if a new abnormal finding was detected on any chest ct during hospitalization. with the approval of the ethics committee, we collected both epidemiological data and medical reports for these two groups. epidemiological data collection was achieved by interviewing each patient and their family members, including the dates and times of close contact with (working together, living or gathering) or to exposure individuals from the affected area (not only wuhan) with confirmed or suspected sars-cov-2 infection. all the data were checked by two researchers. if there existed anything ambiguous, we consulted the attending physician as soon as possible. a c c e p t e d m a n u s c r i p t diagnosis was made by nasopharyngeal swab and rt-pcr to confirm sars-cov-2 infection. the virus detection protocol was repeated on subsequent days until a negative nucleic acid was obtained on two consecutive occasions. other laboratory tests were conducted at admission. table 1 ). all of these comorbidities were reported to be very mild by the patients' clinicians. regarding potential sources of infection, less than a third (n=17, 27%) of cases had made short trips to hubei province and none had been to huanan seafood market. a c c e p t e d m a n u s c r i p t on admission, only 4 of the 63 (6.3%) cases showed leucopenia (white blood cell count <4×10 9 /l) and 4 (6.3%) showed leukocytosis (white blood cell count >10×10 9 /l). all the cases had a neutrophil count within the normal range. eight (12.7%) cases had low level lymphocyte count (<1.0×10 9 /l), including six (20.7%) asymptomatic covid-19 patients and two (5.9%) asymptomatic with normal chest ct. thirty-five of the 63 cases were given procalcitonin tests, with a mean of 0.029 ng/ml (range from 0.02 to 0.039) indicating a level within the normal range. almost half of the 63 cases were tested for cytokines such as il-6, il-10 and il-17, liver and kidney function, with all showing normal results. in all of these laboratory tests, the two imaging groups showed no significant difference ( table 2) . fitness for discharge was based on two consecutive negative rt-pcr tests of oropharyngeal swabs and a normal chest ct scan. all patients were discharged, and no one died. the median time between the initial positive rt-pcr test and discharge was significantly longer in asymptomatic covid-19 patients than in asymptomatic patients with normal chest ct, 13.0 and 10.4 days, respectively (p<0.05; figure 2 ). the number of sars-cov-2 infections is still rising rapidly in many parts of the world, and asymptomatic infections likely play a large role in transmission [22] . to our surprise, 23% of those identified with sars-cov-2 infection, including those with abnormal chest ct scans on admission, were completely asymptomatic throughout their infections-a much higher proportion than previously reported [18] . the high proportion may be related to the extensive and strict close a c c e p t e d m a n u s c r i p t contact screening policy adopted locally resulting in early detection and treatment of cases. this finding suggests that there are actually more asymptomatic covid-19 cases than previously thought, highlighting asymptomatic cases as a noteworthy source of infection. a recent study reported that the viral load in asymptomatic patients is similar to that in symptomatic patients, further underscoring the transmission potential of these asymptomatic patients [27] . together these results suggest that strict isolation and screening should be carried out in all asymptomatic close contacts. ct scans can assist in the detection of asymptomatic pneumonia, but cannot identify asymptomatic cases with normal chest ct. if nucleic acid testing were reserved only for suspected patients with obvious symptoms, many contacts with mild symptoms or asymptomatic would be missed-as would their contacts. since there are no obvious symptoms, asymptomatic cases typically remain undiagnosed for a relatively long time. in our study the mean latency between close contact and diagnosis was 16.0 days, with a maximum of 29 days. this suggests that rt-pcr screening should be undertaken even if an individual shows no symptoms more than 14 days after close contact with an infected person. in asymptomatic patients, patients whose ct scans show signs of pneumonia are typically diagnosed earlier than those without such signs. because the pathogenesis of covid-19 is not well understood, all nucleic acid positive cases received antiviral treatment to prevent progression of the disease. although the efficacy of antiviral therapy is still unknown, it is possible that treatment prevented the progression of disease in this cohort. asymptomatic covid-19 patients with abnormal chest cts showed improvement over time, suggesting that these patients may have benefitted from the antiviral therapy. even with antiviral therapy the average time for viral nucleic acid testing assays to return to negative in the asymptomatic covid-19 patients after treatment was 13 days, which is not shorter than time reported for mild symptomatic patients [12] . however, cases with abnormal chest ct took significantly longer to become rt-a c c e p t e d m a n u s c r i p t pcr negative than those without chest ct abnormalities. these characteristics indicate that asymptomatic covid-19 cases are important recessive sources of infection. thus, diagnosing all sars-cov-2 infections cases including the very mild, subclinical or asymptomatic as soon as possible and immediately isolating them is likely to be critical to cutting off the source of infection. although they have no symptoms or even no abnormal chest image, they spread the virus, causing infection and morbidity [20] . we found that 18 of the 63 asymptomatic cases (28.6%) had infection associated with familial clustering, indicating that asymptomatic infections can be identified through screening family members of index case. the high proportion may be related to the extent and length of close contact, as well as the relative ease of tracking and screening family members [8, 9] ; however, it is sometimes difficult to tell who transmitted the virus, the asymptomatic case or the infected family member [8] . it also suggests that occult transmission of sars-cov-2 may exist. only 14% of the asymptomatic cohort infected others, occurring equally between those with and without chest ct abnormalities. but it is certain that there are still a few asymptomatic cases that can cause transmission [28] . serum antibody tests may provide a low cost and rapid method for screening and could come to play an important role in the auxiliary diagnosis of sars-cov-2 infection[29]. as a group, the asymptomatic cases in our study were younger and had fewer comorbidities compared to severe cases [11, 12] . research shows that the adaptive immune response against the virus in people with asymptomatic infection is stronger than in those with symptomatic infection [30, 31] . asymptomatic individuals without imaging abnormalities are younger than those with pneumonia. from our results, it seems that young individuals with normal immune function and without comorbidities are more likely to become asymptomatically infected. all patients in the study, with and without abnormal chest cts, steadily improved and were discharged smoothly after testing negative by rt-pcr. however, we do not know whether these asymptomatic cases, particularly those who had developed this study has several limitations. first, only 63 asymptomatic cases were included; as such it may be that we found no other associated epidemiologic factors due to sample size. second, we used a qualitative rt pcr test and could not quantitate viral load; thus, asymptomatic cases with low viral load may have been missed. third, a precise incubation period and transmissibility are difficult to document due to the concealed nature of asymptomatic infection. finally, our study does not provide a mechanistic explanation for asymptomatic status; a study linking asymptomatic status with differences in individual immu1nity, virus serotypes, viral load or other factors would contribute useful insights. in conclusion, here we provide an initial assessment of epidemiologic characteristics of asymptomatic infections of sars-cov-2. early identification of sars-cov-2 cases with subsequent isolation and treatment may contribute to decreased transmission and mortality. the authors declare no conflict of interest. m a n u s c r i p t table 1 personal and clinical characteristics of 63 asymptomatic patients with sars-cov-2 a c c e p t e d m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t world health organization. novel coronavirus (covid-19) situation. 2020 transmission dynamics and control of severe acute respiratory syndrome different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures modeling the spread of middle east respiratory syndrome coronavirus in saudi arabia a novel coronavirus outbreak of global health concern aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 new coronavirus pneumonia prevention and control program asymptomatic cases in a family cluster with sars-13 a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin novel coronavirus pneumonia emergency response epidemiology t clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing, china. science china life sciences 2020 presumed asymptomatic carrier transmission of covid-19 covert coronavirus infections could be seeding new outbreaks asymptomatic coronavirus infection: mers-cov and sars-cov-2 (covid-19). travel medicine and infectious disease advances on presymptomatic or asymptomatic carrier transmission of covid-19 a c c e p t e d m a n u s c r i p t key: cord-307770-1igydu3y authors: rawson, timothy m; moore, luke s p; zhu, nina; ranganathan, nishanthy; skolimowska, keira; gilchrist, mark; satta, giovanni; cooke, graham; holmes, alison title: bacterial and fungal co-infection in individuals with coronavirus: a rapid review to support covid-19 antimicrobial prescribing date: 2020-05-02 journal: clin infect dis doi: 10.1093/cid/ciaa530 sha: doc_id: 307770 cord_uid: 1igydu3y background: to explore and describe the current literature surrounding bacterial/fungal co-infection in patients with coronavirus infection. methods: medline, embase, and web of science were searched using broad based search criteria relating to coronavirus and bacterial co-infection. articles presenting clinical data for patients with coronavirus infection (defined as sars-1, mers, sars-cov-2, and other coronavirus) and bacterial/fungal co-infection reported in english, mandarin, or italian were included. data describing bacterial/fungal co-infections, treatments, and outcomes were extracted. secondary analysis of studies reporting antimicrobial prescribing in sars-cov-2 even in the absence of co-infection was performed. results: 1007 abstracts were identified. eighteen full texts reported bacterial/fungal co-infection were included. most studies did not identify or report bacterial/fungal coinfection (85/140;61%). 9/18 (50%) studies reported on covid-19, 5/18 (28%) sars-1, 1/18 (6%) mers, and 3/18 (17%) other coronavirus. for covid-19, 62/806 (8%) patients were reported as experiencing bacterial/fungal co-infection during hospital admission. secondary analysis demonstrated wide use of broad-spectrum antibacterials, despite a paucity of evidence for bacterial coinfection. on secondary analysis, 1450/2010 (72%) of patients reported received antimicrobial therapy. no antimicrobial stewardship interventions were described. for non-covid-19 cases bacterial/fungal co-infection was reported in 89/815 (11%) of patients. broad-spectrum antibiotic use was reported. conclusions: despite frequent prescription of broad-spectrum empirical antimicrobials in patients with coronavirus associated respiratory infections, there is a paucity of data to support the association with respiratory bacterial/fungal co-infection. generation of prospective evidence to support development of antimicrobial policy and appropriate stewardship interventions specific for the covid-19 pandemic are urgently required. the emergence and subsequent pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus has required major adjustments to healthcare systems and frameworks. [1] [2] [3] as part of the response, infection control and antimicrobial stewardship programs have had to rapidly adapt in real-time in the face of an evolving body of evidence. [4] [5] [6] antimicrobials have several potential roles in the management of covid-19. experimental therapies for the treatment of sars-cov-2 are being explored, for example hydroxychloroquine and azithromycin. [7] antimicrobial therapy has a role in the treatment of suspected or confirmed bacterial or fungal (bacterial/fungal) respiratory co-infection. this may be empiric or targeted in patients presenting to hospital or for the management of nosocomial infection acquired during admission to hospital, such as hospital acquired pneumonia or ventilator associated pneumonia. patients may also be suffering from secondary co-infections, not linked to their respiratory presentation, for example urinary tract or blood stream infection. in terms of antimicrobial prescribing bacterial/fungal co-infection of the respiratory tract; some patients presenting to hospital with sars-cov-2 infection have a clinical phenotype that is not dissimilar from atypical bacterial pneumonia. [1, 2, 8] furthermore, sars-cov-2 infection may also be difficult to distinguish from hospital acquired and ventilator associated pneumonia in hospital inpatients. [1, 2, 8] patients often present febrile with respiratory symptoms, such as a dry cough, associated with bilateral chest x-ray changes. [1, 2, 8] therefore, it is not unreasonable to treat empirically with antimicrobials for bacterial/fungal pneumonia in unwell patients. a c c e p t e d m a n u s c r i p t some national guidelines and cases series have suggested the use of broadspectrum antibiotics [9, 10] or the benefit of atypical antibiotic cover. [11] it is anticipated that during the epidemic an increased number of patients will require commencement on empirical antimicrobial therapy. therefore, it is important that antimicrobial stewardship programs focus on supporting the optimal selection of empirical therapies and the rapid de-escalation of treatment once sars-cov-2 infection is confirmed. given the suggested use of broad-spectrum agents and macrolides; [9] [10] [11] this is important to prevent unintended consequences of antimicrobial therapy including toxicity (such as qt prolongation), [12] antibiotic associated diarrhoea, and the propagation of antimicrobial resistance through increased usage of antimicrobials within healthcare systems. [13] we performed a review of the medical literature to explore commonly reported bacterial/fungal co-infections in patients admitted to hospital with coronavirus lower respiratory tract infections. given the lack of data surrounding sars-cov-2 we also opted to include other coronavirus infections. whilst acknowledging that evidence may differ between coronavirus infections, we wanted to explore whether similar observations have been made between these infections. we opted to include, severe acute respiratory syndrome (sars-1), middle eastern respiratory a c c e p t e d m a n u s c r i p t this review was performed following prisma guidelines [14] using an online tool for evidence synthesis (covidence; australia). the review was conducted to identify a c c e p t e d m a n u s c r i p t in total, 1007 abstracts were identified for consideration. three duplicates were excluded and 1004 abstracts were deemed irrelevant at the screening phase. of the 140 texts that were reviewed for eligibility, a further 122 were excluded. eighty-five full text articles excluded (85/122; 70%) either did not report on bacterial co-infection or did not identify any. the remaining 37/122 (30%) articles were excluded as they did not meet inclusion criteria on full text review. eighteen full texts were included in the final report. [2, 8, 10, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] synthesis of results table 1 summarises the current evidence of bacterial/fungal co-infection in patients admitted to hospital with coronavirus. nine of eighteen (50%) studies reported covid-19, 5/18 (28%) sars-1, 3/18 (17%), 1/18 (6%) mers. of the covid-19 studies, 7/9 (78%) reports were from china with 2/9 (22%) from the usa. of non-covid-19 studies, 2/9 (22%) were from china, 2/9 (22%) hong kong, 1/9 (11%) taiwan, 1/9 (11%) singapore, 1/9 (11%) saudi arabia, 1/9 (11%) canada, and 1/9 (11%) south korea. studies reporting on covid-19 [2, [17] [18] [19] 21, 22, 25, 26, 30] reported 62/806 (8%) of bacterial/fungal co-infection. most studies failed to differentiate the setting where sampling was performed (critical versus non-critical care). the largest series reporting bacterial/fungal co-infection was reported by goyal and colleagues in the usa. [22] in this study, the authors report 19/338 (6%) rate of bacteraemia during hospital admission. it is not clear whether these patients were in critical or nonselection of empiric antimicrobial therapy for respiratory bacterial/fungal co-infection and recommendations for duration of treatment require several considerations. as the pre-test probability of sars-cov-2 positive presentations increase, the role of empirical atypical coverage needs to be considered. there have been concerns associated with the potential of sudden cardiac arrest secondary to qt prolongation that is associated with many of the agents we use for atypical infection. [12] the mainstay of treatment for atypical organisms are the macrolides, tetracyclines, and quinolones. some of these can prolong qt and therefore the potential benefits of such treatment must be carefully balanced against risks. [12] macrolides have also been associated with potential antiviral effect in combination with hydroxychloroquine, but also have a potential synergistic effect on qt prolongation. [11] current evidence reported from mers cohorts does not suggest any added benefit from the use of macrolides in the treatment of ards associated with coronavirus infection. [27] furthermore, very few atypical bacterial co-infections have been identified in reports of covid-19 cases to date. therefore, the potential unintended consequences of prolonged macrolide use must be weighed against potential likelihood of atypical bacterial co-infection within covid-19 cohorts. a c c e p t e d m a n u s c r i p t a further concern with the rapid expansion of critical care capacity to manage sars-cov-2 is the potential increased rate of nosocomial infection within the hospital environment. [41] whilst many studies reported failed to separate reporting on critical and non-critical care settings, a large proportion of reported bacterial co-infections within coronavirus literature appear to be healthcare associated, including centralline associated blood stream infections, and ventilator associated pneumonia. [8, [24] [25] [26] 29] with observed strain being placed on healthcare systems currently during the upstroke of the sars-cov-2 pandemic; guidelines must focus on maintenance of good infection control, antimicrobial stewardship, and robust surveillance for hcais and antimicrobial resistance. ensuring access to core antimicrobials must also be a primary goal. potential stewardship interventions to support reduced antimicrobial prescribing during the covid-19 pandemic urgently require consideration. [41] traditional markers used to support antimicrobial decisions, such as vital signs, blood tests like white cell count and c-reactive protein, and imaging tend to be abnormal in sars-cov-2 infection. [1] [2] [3] this makes decision making surrounding the requirement for empiric antibacterial cover challenging. furthermore, with fears surrounding prolonged patient contact and aerosol generation, the number of patients undergoing routine microbiological investigation may be reduced. [41] one potential solution to support antimicrobial prescribing in covid-19 is the use of bacterial specific biomarkers, such as procalcitonin. [42] procalcitonin has been demonstrated to support differentiation between bacterial and viral infection and supports early cessation of antibiotics in confirmed bacterial infection with no effect on patient mortality. [42, 43] procalcitonin use has been reported in the covid-19 literature and may be an important tool to support reducing antimicrobial a c c e p t e d m a n u s c r i p t use. [8, 17, 19, 22, 25, 30, 33] furthermore, the use of clinical decision support systems may facilitate better use of data in supporting decision making, especially when linked with artificial intelligence. [44] in addition, infection specialties who are normally responsible for co-ordinating stewardship programs must continue to provide support to clinical teams managing covid-19 patients to ensure that regular review and cessation of antimicrobial therapy is considered based on the limited clinical evidence available within these patients. [41] supporting appropriate microbiological sampling prior to commencement of antimicrobial therapy should also be encouraged within this patient cohort to ensure that the clinician has as much data as possible to support decision making. with medication shortages, including key antimicrobials, being a concern across areas currently affected by the pandemic, [45, 46] judicious use of antimicrobials will be vital to ensure access to therapy by those with confirmed bacterial infection. with a growing body of evidence supporting short-course antimicrobial therapy, [47] guidelines and stewardship programs during this time should reflect this. evidence also supports the safety of early oral versus intravenous antibiotics for a range of infections including bone and joint infection, infective endocarditis, and lower respiratory tract infection. [48] [49] [50] [51] with a need to ensure that bed capacity is maintained, a focus on developing guidance on optimal pharmacokineticpharmacodynamic strategies for common infections requiring antimicrobial should be considered to support early oral antibiotic switch and treatment de-escalation in patients with short-and long-term infections. [52, 53] a c c e p t e d m a n u s c r i p t this review had several limitations that must be considered. the rapidly evolving nature of the covid-19 pandemic means that data is continuously evolving. this study included coronavirus infections from predominantly asia, which may limit the generalisability of the findings. furthermore, the studies described often did not uniformly report or undertake examination for bacterial co-infection, which may have under or over-estimated the rates of respiratory bacterial co-infection. our decision to exclude studies reporting no observed bacterial co-infections may also have overa c c e p t e d m a n u s c r i p t tmr & ah developed the concept and methodology for the review. tmr, lspm, nz, and gs undertook data extraction and reviewing. all authors contributed significantly to data interpretation. tmr drafted the initial manuscript. all authors contributed significantly to the revision of the manuscript and finalisation for submission. m a n u s c r i p t patient days a novel coronavirus from patients with pneumonia in china articles clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan , china : a retrospective cohort study the novel coronavirus originating in wuhan, china: challenges for global health governance the toughest triage -allocating ventilators in a pandemic facing covid-19 in italy -ethics, logistics, and therapeutics on the epidemic's front line covid-19 and the stiff upper lip -the pandemic response in the united kingdom journal pre-proof hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical features of 69 cases with coronavirus disease vademecum per la cura delle persone con malattia da covi-19 versione 2.0, 13 marzo 2020 2 s i m i t società italiana di malattie infettive e tropicali sezione regione lombardia gruppo collaborativo-terapia covid-19 lombardia coordinamento redazionale emanuele focà malattie infettive pathogenic characteristics of hospitalized severe acute respiratory infections in hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial antimicrobials and qt prolongation understanding the mechanisms and drivers of antimicrobial resistance preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews (prisma-scr) checklist section item prisma-scr checklist item reported on page # title title 1 identify the report as a scoping review organism distribution and drug resistance in 7 cases of severe acute respiratory syndrome death patients with secondary bacteria infection clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study clinical features of patients infected with 2019 novel coronavirus , china characteristics and outcomes of 21 critically ill patients with covid-19 in washington state severity of coronavirus respiratory tract infections in adults admitted to acute care in toronto, ontario analysis of clinical features of 29 patients with clinical characteristics of covid-19 in new york city elucidation of bacterial pneumonia-causing pathogens in patients with respiratory viral infection severe acute respiratory syndrome in surgical patients: a diagnostic dilemma epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study eleven faces of coronavirus disease 2019 macrolides in critically ill patients with middle east respiratory syndrome severe acute respiratory syndrome in taiwan: analysis of epidemiological characteristics in 29 cases increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china case report isolated candida infection of the lung covid-19 in critically ill patients in the seattle region -case series clinical characteristics of coronavirus disease 2019 in china first case of 2019 novel coronavirus in the united states rates of co-infection between sars-cov-2 and other respiratory pathogens a locally transmitted case of sars-cov-2 infection in taiwan risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore sars-cov-2 infection (covid-19) in febrile infants without respiratory distress clinical features and short-term outcomes of 102 patients with corona virus disease covid-19 and the potential long term impact on antimicrobial resistance procalcitonin-guided antibiotic treatment in patients with positive blood cultures: a patient-level meta-analysis of randomized trials efficacy and safety of procalcitonin guidance in reducing the duration of antibiotic treatment in critically ill patients: a randomised, controlled, open-label trial a real-world evaluation of a case-based reasoning algorithm to support antimicrobial prescribing decisions in acute care ema announces measures to manage drug shortages due to covid-19 short course antibiotic therapy-replacing constantine units with "shorter is better oral versus i.v. antibiotics for communityacquired pneumonia in children: a cost-minimisation analysis oral versus intravenous antibiotics for community acquired lower respiratory tract infection in a general hospital: open, randomised controlled trial partial oral versus intravenous antibiotic treatment of endocarditis oral versus intravenous antibiotics for bone and joint infection how to optimize antibiotic pharmacokinetic/pharmacodynamics for gram-negative infections in critically ill patients pharmacokinetics and pharmacodynamics of antibacterial agents the authors would like to thank members of imperial college nhs healthcare trust who participated in the study. the authors would also like to acknowledge 1) the national institute for health research health protection research unit (nihr hpru) in healthcare associated infections and antimicrobial resistance at imperial college london in partnership with public health england (phe), in collaboration with, imperial healthcare partners, university of cambridge and university of warwick and 2) the department for health and social care funded centre for antimicrobial optimisation (camo) at imperial college london. the views expressed in this publication are those of the author(s) and not necessarily those of the nhs, the national institute for health research, the department of health and social care or public health england. professor alison holmes is a national institute for health research (nihr) senior investigator. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request where not presented in the manuscript or figure. this report is independent research funded by the centre for antimicrobial optimisation at imperial college london. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-307497-wtfvoifb authors: abu-raya, bahaa title: predictors of refractory coronavirus disease (covid-19) pneumonia date: 2020-04-09 journal: clin infect dis doi: 10.1093/cid/ciaa409 sha: doc_id: 307497 cord_uid: wtfvoifb nan dear editor-the pandemic of coronavirus disease (covid-19) is rapidly spreading and clinicians across the globe are facing it with limited scientific evidence to support their management. there is a critical lack of predictive factors that can guide clinicians in identifying patients at risk for prolonged, and thus potentially more severe, covid-19 that needs early and aggressive therapeutic approach. this fact has urged mo and colleagues to retrospectively characterize patients with refractory covid-19 as compared with patients with non-refractory covid-19 (termed "general covid-19") 1 . the authors reported that patients classified as refractory covid-19 were more likely to be older, of male sex, have underlying comorbidities, be afebrile, have shortness of breath and anorexia, and to have laboratory abnormalities and radiological changes. in addition, refractory covid-19 patients were more likely to receive oxygen, mechanical ventilation, expectorant, adjunctive treatment including corticosteroids, antiviral drugs and immune enhancers when compared to general covid-19 patients. the authors have further attempted to explore predictive factors for refractory covid-19 using a multivariate analysis showing that male sex, anorexia on admission and being afebrile are independent risk factors for the development of a refractory disease. furthermore, out of this analysis the authors have found that patients with refractory covid-19 were more likely to receive oxygen, expectorant, corticosteroids, lopinavir/ritonavir and immune enhancer. in their paper, the authors compared categorical and continuous variables between the two groups of patients and these variables that differed significantly between the two groups were included in the multivariate regression analysis. in addition to comparing the frequencies by χ2 test and continuous data by nonparametric comparative test, it is well-accepted to perform a univariate regression analysis assessing the different variables and their association with the outcome of interest (i.e. refractory vs. general covid19) and report their odds ratio (or) with a measure of variance (e.g. 95% ci). based on such a univariate regression analysis, authors can determine the variables to be included in the multivariate logistic regression model to assess independent variables associated with the outcome and their estimated adjusted ors 2,3 . in their paper, the authors included the treatment introduced at the time of patient admission and/or hospitalization in the multivariate analysis aiming to identify independent risk factors for refractory covid-19. one potential concern is that patients who are sicker at time of admission or during hospitalization are more likely to need oxygen and receive expectorant, given the unusual and unprecedented circumstances our clinical community is experiencing, integrating the aforementioned information in the article by mo and colleagues will provide scientific evidence to help physicians and healthcare providers across the world to tackle the escalating covid-19 pandemic as best as we can. clinical characteristics of refractory covid-19 pneumonia in wuhan, china an easter outbreak of salmonella typhimurium dt 104a associated with traditional pork salami in italy burden of children hospitalized with pertussis in canada in the acellular pertussis vaccine era key: cord-307660-onz6vfre authors: titanji, boghuma k; farley, monica m; mehta, ashish; connor-schuler, randi; moanna, abeer; cribbs, sushma k; o’shea, jesse; desilva, kathryn; chan, bonnie; edwards, alex; gavegnano, christina; schinazi, raymond f; marconi, vincent c title: use of baricitinib in patients with moderate and severe covid-19 date: 2020-06-29 journal: clin infect dis doi: 10.1093/cid/ciaa879 sha: doc_id: 307660 cord_uid: onz6vfre cytokine storm and hyperinflammation are associated with increased mortality in covid-19. in this small uncontrolled cohort of patients with moderate-severe covid-19, treatment with baricitinib plus hydroxychloroquine was associated with recovery in 11 of 15 patients. baricitinib for the treatment of covid-19 should be further investigated in randomized controlled clinical trials. coronavirus infectious disease-19 (covid-19), caused by severe acute respiratory virus syndrome coronavirus-2 (sars-cov-2) has led to over 8 million confirmed infections worldwide with an estimated global mortality of 5.6% as of june 17 th 2020 1 . virus-driven hyperinflammation and cytokine storm syndrome are important features of severe covid-19 2 . a multi-center, retrospective study of 150 patients with severe covid-19 showed a strong association between elevated ferritin and interleukin (il)-6 levels and adverse clinical outcomes 3 . recognizing and treating cytokine storm may present a viable approach to reducing the mortality from covid-19. the janus kinase (jak) 1/2 inhibitor, baricitinib, is an attractive candidate due to its properties as a potent anti-inflammatory agent and its hypothesized off-target antiviral effects against sars-cov-2 4, 5 . in this brief report of 15 patients, we present the clinical use of baricitinib for the treatment of patients with moderatesevere covid-19. this study was conducted at the atlanta veterans affairs medical center (vamc) with approvals from the emory university institutional review board and atlanta vamc office of research and development. data were abstracted from the electronic health records between march 1, 2020 and april 18, 2020. patients had laboratory confirmed covid-19, diagnosed by quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) testing of oropharyngeal, nasopharyngeal or tracheal aspirate samples and were treated at the discretion of the medical team with a combination of hydroxychloroquine and baricitinib if they fulfilled at least one of the following criteria: 1) evidence of pneumonia on lung imaging and requiring supplemental oxygen on admission or development of a new oxygen requirement during the course of their hospitalization 2) moderate disease requiring hospitalization (e.g., severe diarrhea requiring volume resuscitation, encephalopathy, evidence of end-a c c e p t e d m a n u s c r i p t organ damage); 3) elevated or rising inflammatory markers during hospitalization. statistical analyses were performed using graphpad and r software. moderate covid-19 patients met any of the following criteria: fever, confusion, severe diarrhea, dyspnea, evidence of pneumonia on lung imaging, requiring hospitalization for ongoing medical care but not icu-level care. severe covid-19 patients met any of the following criteria; respiratory rate (rr) >30 times/min, oxygen saturation by pulse oximetry 93% at rest or pao2/fio2<300 mmhg. however, 20% of patients progress to moderate-severe disease and require hospitalization and peak viral loads occur approximately one week after the initial onset of symptoms 2 . disease severity is marked by hyperinflammation and cytokine storm syndromes with increased levels of type ii (gamma) which drives il-6 expression through the jak-stat pathway 2 . in animal models of sars and mers, the initial (type 1) interferon response has beneficial effects in the early phases of disease, but may become damaging in the latter phases (type ii) 8 . this suggests that patients most likely to benefit from treatment with jak1/2 inhibitors are those with evidence of disease progression and hyperinflammation characterized by rising levels of inflammatory markers, progressive pulmonary disease, and overall clinical deterioration. in addition, at a 2-4 mg daily dosage, baricitinib primarily inhibits type ii interferon and less likely type i interferon a c c e p t e d m a n u s c r i p t responses. baricitinib is fda approved for the treatment of ra 10 . long-term use has been associated with increased risk for infections and thrombo-embolic events 11 . these adverse events are less likely to occur with a limited course of therapy as received by patients in this cohort. however, it is unclear whether the thrombotic events noted in 3 of the 15 patients were related to baricitinib therapy, were general nosocomial complications, or occurred as part of the pathogenesis of covid-19 disease. this study has several limitations. it is a small non-randomized cohort and does not include controls. thus, it is unclear if these patients would have improved without baricitinib treatment. all patients were concomitantly treated with hydroxychloroquine, which has in-vitro antiviral activity against sars-cov-2 as well as anti-inflammatory properties 12 world health organization. coronavirus disease 2019 -situation report number 148 covid-19: consider cytokine storm syndromes and immunosuppression clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china baricitinib as potential treatment for 2019-ncov acute respiratory disease covid-19: combining antiviral and anti-inflammatory treatments baricitinib therapy in covid-19: a pilot study on safety and clinical impact mechanism of baricitinib supports artificial intelligence-predicted testing in covid-19 patients ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes estimates of the severity of coronavirus disease 2019: a model-based analysis baricitinib for the treatment of rheumatoid arthritis and systemic lupus erythematosus: a 2019 update thu0211 meta-analysis of serious infections with baricitinib, tofacitinib and biologic dmards in rheumatoid arthritis in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) vincent marconi and raymond f schinazi are partially funded by -emory university center for aids research (ai050409). raymond f schinazi and christina gavegnano are funded in part by nih grant 5-r01-mh116695. dr. vincent c. marconi has consulted or received research support from viiv, gilead, lilly and bayer. dr. raymond schinazi served as an unpaid consultant for eli lilly whose drugs are being evaluated in the research described in this paper. in addition, dr. schinazi owns shares in eli lilly and gilead, and has been issued patents 20190134039, 10022378, and 9662332. the terms of this arrangement have been reviewed and approved by emory university in accordance with its conflict of interest policies. all other authors do not have any conflicts to declare. a c c e p t e d m a n u s c r i p t key: cord-303968-ikr6eeov authors: perinel, sophie; launay, manon; botelho-nevers, élisabeth; diconne, éric; louf-durier, aurore; lachand, raphaël; murgier, martin; page, dominique; vermesch, régine; thierry, guillaume; delavenne, xavier title: towards optimization of hydroxychloroquine dosing in intensive care unit covid-19 patients date: 2020-04-07 journal: clin infect dis doi: 10.1093/cid/ciaa394 sha: doc_id: 303968 cord_uid: ikr6eeov hydroxychloroquine (hcq) appears to be a promising treatment for covid-19. however, all ongoing clinical trials with hcq use different dosing regimens, resulting on various concentrations pk studies are therefore needed to define the optimal dosing regimen. since december 2019, an outbreak of covid-19 due to sars-cov-2 virus has spread from china. on march 11, 2020, the who described the global covid-19 situation as a pandemic. as of march 30, 2020, no treatment has demonstrated clinical efficacy against covid-19. however, several treatment strategies are being considered and evaluated in numerous clinical trials. among these strategies, the use of hydroxychloroquine (hcq) appears to be a promising option, although only limited evidence is available at the present time. above all, hcq has the advantage of being widely available to a large number of patients. hcq is a well-known drug, effective in the treatment of malaria and autoimmune diseases. more recently, an in vitro antiviral effect has been demonstrated on sars-cov-2. recent work by wang et al. showed that a chloroquine concentration of 0.36 mg/l decreased viral load by 50% in a cell model [1] . apart from the ongoing clinical evaluation of the efficacy of hcq, little information is available concerning the modalities of administration of this drug for intensive care unit (icu) patients, especially in the context of covid-19. hcq can be responsible for adverse events and probably an increased incidence of adverse events in the case of inappropriate dosing regimens. one of the most serious adverse events in this population is cardiac toxicity, characterized by prolongation of the qt interval, which can lead to arrhythmia in patients at risk. hcq also has very particular pharmacokinetic (pk) properties that require certain precautions. it exhibits strong tissue tropism, particularly for the kidney and liver, with a long half-life (several weeks). the risk of overdose is higher in the icu population with impaired renal and/or hepatic function than in other populations. to date, hcq pk parameters have been estimated from studies in patients with rheumatoid arthritis or lupus [2] or healthy volunteers [3, 4] . however, physiological changes in infused, ventilated patients with multiple organ failure may modify hcq pk parameters. for these reasons, we conducted a prospective study to evaluate the pk properties of hcq in icu covid-19 patients. this prospective cohort study was conducted at saint etienne university hospital (france) between 03/13/2020 and 03/23/2020. the study was approved by the institutional review board [irbn462020/chuste]. all consecutive patients with laboratory-confirmed sars-cov-2 infection treated by hcq in the critical care unit were included. m a n u s c r i p t treatment patients received 200 mg of oral hcq, three times daily, as suggested by a recent study [9] . blood samples for determination of drug levels were drawn as part of routine care, with the decision to perform therapeutic drug monitoring based on medical guidance. hcq trough levels >1 mg/l and <2 mg/l were considered to be therapeutic [5, 6] . the medical team received all results in real time to allow for dose adjustments, as necessary. blood samples were drawn from an arterial catheter and transferred to ethylenediamine tetraacetic acid-containing tubes. hcq blood levels were analyzed using a validated liquid chromatography-mass spectrometry method. briefly, blood samples (200 µl) were spiked with 200 µl hydroxychloroquine-d4 prepared (0.2 mg/l) in 15% trichloroacetic acid, and 200 µl zinc sulfate was used for liquid-liquid extraction. the lower limit of quantification was 0.010 mg/l. to more clearly understand hcq pk and the effect of the dosing regimen, a simulation based on a pk population study in patients with rheumatoid arthritis was performed [2] . from the variance-covariance matrix of the estimated pk parameters, monte carlo simulations were performed using mlxplore (lixsoft). a total of 200 patients were generated receiving different dosing regimen based on ongoing clinical trials. thirteen patients were included in this prospective pk study. the median age of patients was 68 years [38 -82 years]. most patients were male (85%). median body weight was 82.7 kg [63 -117 kg] and 46% were considered to be obese (body mass index > 30 kg/m 2 ). median renal function estimated by the ckd-epi formula was 79.6 ml.min -1 [12 -118]; 30.7% of subjects presented moderate or severe renal failure. twelve patients were mechanically ventilated. one patient was treated by ecmo and another patient was treated by renal replacement therapy. m a n u s c r i p t a total of 161 blood levels were recorded and used for the analysis and 6 samples were below the limit of quantification of the assay (figure 1 ). only 8/13 patients (61%) achieved the supposed minimum therapeutic level of 1 mg/l and 2/13 patients exceeded a concentration of 2 mg/l. the mean time to reach the minimum therapeutic level was 2.7 days [1-4.5 days]. four patients underwent dose de-escalation and subsequently received 200 mg of hcq twice daily. hcq was withdrawn in two patients: due to qt interval prolongation (381 to 510 ms and 432 to 550 ms) on day 2 and 3 with hcq blood levels of 0.03 mg/l and 1.74 mg/l, respectively. the various dosing regimens used in currently recruiting clinical trials were simulated in order to determine the variability of hcq pk parameters ( figure 2 ). treatment a achieved target levels on day 3. treatment b achieved target levels but resulted in blood levels exceeding 2 mg/l on day 3. treatment c rapidly achieved target levels (1 st dose), but blood levels exceeded 2 mg/l after 8 hours. treatment d achieved target levels after 2.5 days and a level of 2 mg/l after 4 days finally, we proposed treatment d to rapidly achieve target levels without exceeding 2 m/l. to our knowledge, this is the first study to describe hcq pk in icu patients. in this study, we demonstrated that the 200 mg three times daily dosing regimen is inappropriate to reach a supposed target blood level of 1 -2 mg/l in this population. using this dosing regimen, the mean time to reach therapeutic levels was more than 2 days and only 61% of patients reached target levels with this dosing regimen. this result is not surprising in view of the pk properties of hcq [7] . hcq presented marked pk variability with a very long half-life (5 to 40 days), particularly due to large distribution into blood and tissues. steady-state concentrations are therefore achieved within weeks and vary from individual to individual with the same dosing regimen. however, icu patients present certain characteristics that can affect the pk of drugs. for example, the presence of ecmo may affect the already altered pk of icu patients by further increasing the volume of distribution, causing changes in clearance, and causing adsorption or absorption into the circuit [8] . in the one patient treated by ecmo in this study, hcq blood levels increased more slowly than in the other patients. the therapeutic level of hcq in covid-19 patients has not yet been established. some in vitro and in silico studies have reported a virustatic effect of chloroquine and hcq and estimated the therapeutic blood level from ec50, ranging from 0.3 to 2.1 mg/l [1, 5] . the toxic hcq concentration has not been established, although a number of arguments suggest that a concentration of 2 mg/l should not be exceeded to avoid ocular toxicity. however, the most dreaded adverse effect for covid-19 patients is cardiac toxicity. to our knowledge, the m a n u s c r i p t relationship between cardiac toxicity and hcq blood levels has not been determined, but it can be assumed that excessive hcq exposure is likely to be harmful. in this study, two patients experienced cardiac toxicity at variable hcq blood concentrations. no guidelines for administration of hcq are currently available. using physiologicallybased pharmacokinetic models, yao et al. suggested a dosage of 400 mg twice daily for 1 day, followed by 200 mg twice daily for another 4 days [5] . this regimen could constitute an appropriate option, although the results of our study suggest that 800 mg once daily on the first day can more rapidly reach therapeutic levels in icu patients. all of the 9 clinical trials concerning the therapeutic use of hcq in covid-19 registered in clinicaltrials.gov (on march 26) are using different dosing regimens. based on our simulations, we demonstrate that some of these dosing regimens will fail to reach therapeutic levels, while others will probably induce levels higher than 2 mg/l. this work strongly suggests that the hcq dosing regimen should be optimized on the basis of pk data available in special populations. there is an urgent need for health agencies to clarify the standard dosing regimen of hcq in order to have comparable data across clinical trials, and to avoid dubious efficacy or toxicity results due to pk profiles. based on this prospective study, we demonstrate that pk studies are needed to define the optimal dosing regimen for icu covid-19 patients. based on our simulations, we propose a loading dose of 800 mg once daily on day 1, followed by 200 mg twice daily for 7 days. therapeutic drug monitoring should be used to personalize the optimal dosing regimen. further pk and pd (virological) studies are also warranted. m a n u s c r i p t red dots represent hcq blood levels for a dosing regimen of 200 mg three times daily, triangles represent hcq blood levels after discontinuation of treatment, circles represent hcq blood levels for a dosing regimen of 200 mg twice daily. the green shaded zone represents the 90% simulation interval obtained with the model of carmichael et al for hcq 200 mg three times daily [2] . the brown line represents treatment a: 400 mg once daily for 5 days (nct04261517). the blue line represents treatment b: 400 mg twice daily for 7 days (nct04316377). the pink line represents treatment c: 800 mg loading dose followed by 600 mg 8 hours later and then 600 mg once daily for 4 days (nct04308668). the red line represents treatment d: 200 mg three times daily for 7 days [9] . the green line represents the recommended treatment e: 800 mg loading dose followed by 200 mg twice daily for 6 days; the green shaded zone represents its 90% simulation interval. m a n u s c r i p t remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro population pharmacokinetics of hydroxychloroquine in patients with rheumatoid arthritis bioavailability of hydroxychloroquine tablets in healthy volunteers pharmacokinetics and bioequivalence study of hydroxychloroquine sulfate tablets in chinese healthy volunteers by lc-ms/ms. rheumatol ther in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine blood levels in systemic lupus erythematosus: clarifying dosing controversies and improving adherence steady-state pharmacokinetics of hydroxychloroquine in patients with cutaneous lupus erythematosus pharmacokinetics and extracorporeal membrane oxygenation in adults: a literature review hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial m a n u s c r i p t key: cord-321603-lbbsnriv authors: rao, mohan; rashid, fairuz a; sabri, fashihah s a h; jamil, nur nadia; zain, rozainanee; hashim, rohaidah; amran, fairuz; kok, huey tean; samad, md anuar abd; ahmad, norazah title: comparing nasopharyngeal swab and early morning saliva for the identification of sars-cov-2 date: 2020-08-06 journal: clin infect dis doi: 10.1093/cid/ciaa1156 sha: doc_id: 321603 cord_uid: lbbsnriv background: the ideal sars-cov-2 testing method would be accurate and also be patient-performed to reduce exposure to healthcare workers. the aim of this study was to compare patient-performed testing based on a morning saliva sample with the current standard testing method, healthcare worker-collected sampling via a nasopharyngeal swab (nps). methods: this was a prospective single center study which recruited 217 asymptomatic adult male participants in a covid-19 quarantine center who had tested positive for sars-cov-2 8-10 days prior isolation. paired nps and saliva specimens were collected and processed within 5 hours of sample collection. real time reverse transcriptase polymerase chain reaction (rt-pcr) targeting envelope (e) and rna-dependent rna polymerase (rdrp) genes was performed and the results were compared. results: overall, 160 of the 217 (74%) participants tested positive for covid-19 based on saliva, nps, or both testing methods. the detection rate for sars-cov-2 was higher in saliva compared to nps testing (93.1%, 149/160 vs 52.5%, 84/160, p<0.001). the concordance between the two tests was 45.6% (virus was detected in both saliva and nps in 73/160), while 47.5% were discordant (87/160 tested positive for one while negative for the other). the ct values for e and rdrp genes were significantly lower in saliva specimens compared to np swab specimens. conclusions: our findings demonstrate that saliva is a better alternative specimen for detection of sars-cov-2. taking into consideration, the simplicity of specimen collection, shortage of ppe and the transmissibility of the virus, saliva could enable self-collection for an accurate sars-cov-2 surveillance testing. a pandemic of sars-cov-2 is spreading across the world and human to human spread has been confirmed (1, 2) . the number of infected sars-cov-2 cases in malaysia has been rising dramatically since the beginning of the spread despite mitigation measures. healthcare worker are at the frontline of the covid-19 outbreak response, making them vulnerable to the infection. up until april 23, 2020, 325 healthcare workers have been confirmed to have contracted sars-cov-2. although none of their source of infection has been linked to the management or treatment of sars-cov-2 patient, source of infection among 30% these healthcare workers are yet to be discovered (3) . the virus has been detected in various clinical specimen such as bronchoalveolarlavage (4), sputum (5) , saliva (6) , throat (7) , stool, nasopharyngeal (nps), oropharyngeal (ops) swabs and blood (8) . the current standard sampling techniques such as nps and ops used for surveillance and serial monitoring of infected patients are exposing healthcare workers to sars-cov-2 virus and other unknown pathogens via aerosols from swabbing and jeopardizing physical distancing. at the same time, the collection of these specimen types causes discomfort and minor injuries such as bleeding and ulceration of mucosal layer, especially in patients with predisposing factors. saliva specimens have demonstrated high concordance rate of greater than 90% with nps in the detection of coronavirus. in addition to that, some studies have used a c c e p t e d m a n u s c r i p t 4 saliva specimen in surveillance (screening coronavirus) and serial monitoring of viral load (9). (6) has demonstrated the present of coronavirus in saliva but not in nasopharyngeal aspirate. moreover, saliva specimens can be obtained noninvasively, simply by spitting into a sterile container. saliva collection is regarded as a safer non-invasive specimen alternative to nps or ops. other than minimizing exposure of the healthcare workers to hazards, it is self-collected, requires no special chilled media for transportation of samples and less specimen degradation from delay in processing. thus, this would maintain physical distancing and minimizing the chance of exposing front-liners to the virus. the exploration on comparability of saliva versus nps will assist in sampling protocol, the management of patients and reduce hazards exposure among healthcare workers. therefore, we assessed the comparability between saliva and nps specimens for sars-cov-2 detection via rt-pcr. this prospective single center diagnostic study was conducted among 217 individuals who were tested positive for sars-cov-2 via nps at a covid-19 quarantine center, maeps. these selected individuals were on day 8-10 of isolation during the sampling. the inclusion criteria were those participants above 18 years old and able to obey commands. individuals with respiratory aid were excluded. assent and written informed consent were obtained from study participants. all participants were male recruits and asymptomatic at the time of sampling. the ct-values were edging the upper limit (fig 2) . obtaining an optimal clinical specimen for detection of sars-cov-2 is central in controlling the global pandemic. less invasive, good safety measure, amicable, cost effective, timely and brief are among the major principles of clinical specimen collection during global pandemic (11, 12) . in this study, we showed a comparable (14) . however, the cohen's  value in this study is weaker than in a previous study (15) . the lower agreement of these two sampling methods could have several reasons. it can be attributed to larger size of discordant results between sampling methods. at the same time, the methodology (study design) and disease prevalence could ascribe the lower cohen's  value in this study (16) . the overall detection rate from saliva samples was higher and significant (p<0.05) than the reference standard for sars-cov-2 testing via rt-pcr assay. this finding reflects that salivary gland and tongue are possibly the major sites for sars-cov-2 viral replication and shedding as these tissues express ace2 receptor for the viral attachment (17) . it is also possible that the virus migrates from the nasopharynx or lower respiratory tract to the oral cavity as described in previous study (18) . besides, it can be hypothesized that the inoculum size of nasopharyngeal swab may not be sufficient for viral transfer. but this hypothesis is yet to be investigated. a few studies have compared the viral load between nasopharyngeal swab and saliva specimens. (19) has demonstrated that viral loads in saliva is higher than a c c e p t e d m a n u s c r i p t 9 nasopharyngeal swab. whereas (20, 21) have demonstrated that saliva is less sensitive in comparison to nasopharyngeal swab. meanwhile, (22, 23) demonstrated equivalent viral load between nasopharyngeal swab and saliva among symptomatic patients but lower in saliva of asymptomatic individuals. contrary to that, our results showed significant difference in detection of sars-cov-2 between these two sampling methods among asymptomatic individuals. saliva had a higher detection rate and lower ct-value (high viral load) than nasopharyngeal swab among the concordant results. however, we speculate that these different findings between studies are possibly due to distinct sampling techniques, detection kits and study population. nevertheless, we had 72 individuals with their saliva specimen tested positive for sars-cov-2 while they were test negative for nasopharyngeal swab. this could be due to the property of saliva that acts as wide resource for genomic information by preventing rna decomposition (24) . it is noteworthy that these inconsistent results may be related to timing of sampling, methodology of sample processing and severity of disease (25) . nonetheless, this finding has raised concerns on management of the individuals and the transmissibility of sars-cov-2 via saliva or other body fluid as demonstrated by (26) . earlier studies have demonstrated that bronchoalveolar lavage fluid and sputum specimens have the highest detection rate for sars-cov-2 testing (27) . however, these specimens are not easy to obtain as 80% of infected individuals commonly present with a dry cough at the onset of illness and they are not suitable for surveillance as large proportion of the population is asymptomatic (28, 29) . a c c e p t e d m a n u s c r i p t for peadiatrics population regardless of illness manifestations. moreover, saliva collection is non-invasive, patient friendly and applicable for surveillance testing. in the face of shortages of both swabs and personal protective equipment as described by (30) , saliva is an alternative diagnostic specimen for the detection of sars-cov-2. self-collecting saliva can negate the need for direct healthcare worker-patient interaction, reduce the overall nosocomial infection risk, reduce the waiting time in a busy clinical setting and cost efficient by easing the supply demands on swabs, personal protective equipment and manpower. in addition to that, the use of saliva instead of nasopharyngeal swab also enhance recruitment of individual for community surveillance studies (31) . taking into consideration, the simplicity of specimen collection, shortage of ppe and the transmissibility of the virus, saliva could enable self-collection for accurate large-scale sars-cov-2 surveillance testing. firstly, we only recruited adult patients. further evaluation should be conducted in the paediatrics population. secondly, the spectrum of the disease ranges from asymptomatic to a severely ill patients but our study only focused on homogenously composed of asymptomatic individuals. therefore, performance of the saliva test for the detection of sars-cov-2 among symptomatic remains unknown. the saliva sample collections were not screened microscopically to assess saliva quality. thirdly, saliva was collected prior nasopharyngeal swab sampling. this is because, longer duration required for collection of 2mls of saliva. for that reason, we believe a c c e p t e d m a n u s c r i p t 11 that walk-in patients' saliva may not meet the specified standards for saliva collection. further study is required to assess the detection of sars-cov-2 in random saliva collection. and lastly, the ct-value in this study portrays a trend in viral load but not the viral copies per ml. this is due to lack of a reliable quantified positive control in our laboratory. a c c e p t e d m a n u s c r i p t we are grateful to the institute for medical research, kuala lumpur and maeps centre for their support and facilities. the authors would like to thank the director general of health malaysia for allowing us to publish our findings. this study obtained approval from the national health institute human research ethics committee (kkm/nihsec/p20-1045). although this study did not involve any types of intervention in diagnosis and treatment, written informed consent was considered necessary. it involves the use of anonymized patient medical data. only demographic data were obtained. therefore, no breach of privacy or confidentiality of patient. this study reviewed human patients only and no animals were involved in any aspect of the study. this study was supported by a grant from the ministry of health, malaysia (nmrr-20-860-54884). all the author declared that there is no conflict of interest in conducting this study. all authors have submitted the icmje form for disclosure of potential conflicts of a c c e p t e d m a n u s c r i p t m a n u s c r i p t novel coronavirus: from discovery to clinical diagnostics initial cluster of novel coronavirus (2019-ncov) infections in wuhan, china is consistent with substantial human-to-human transmission 325 medical workers test positive for covid-19 a novel coronavirus from patients with pneumonia in china novel coronavirus pneumonia outbreak in 2019: computed tomographic findings in two cases consistent detection of 2019 novel coronavirus in saliva the first 2019 novel coronavirus case in nepal molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes the jamovi project. jamovi -stats. open. now world health organization w. guidelines for the collection of clinical specimens during field investigation of outbreaks who/cds/csr/edc/2000.4. world heal organ general principles of specimen collection and transport interim guidelines for clinical specimens for covid-19 | cdc the measurement of observer agreement for categorical data saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study a reappraisal of the kappa coefficient saliva: potential diagnostic value and transmission of 2019-ncov saliva is a reliable tool to detect sars-cov-2 saliva is more sensitive for sars-cov-2 detection in covid-19 patients than nasopharyngeal swabs. medrxiv saliva as a non-invasive specimen for detection of sars-cov-2 saliva is less sensitive than nasopharyngeal swabs for covid-19 detection in the community setting the natural sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) science behind human saliva comparison of sars-cov-2 detection in nasopharyngeal swab and saliva the presence of sars-cov-2 rna in the feces of covid-19 patients detection of sars-cov-2 in different types of clinical specimens effectiveness of airport screening at detecting travellers infected with novel coronavirus (2019-ncov) clinical characteristics of 145 patients with corona virus disease 2019 (covid-19 critical supply shortages -the need for ventilators and personal protective equipment during the covid-19 pandemic key: cord-303603-4mi64bgm authors: martínez-urbistondo, diego; costa segovia, ramón; suárez del villar carrero, rafael; risco risco, carlos; villares fernández, paula title: early combination of tocilizumab and corticosteroids: an upgrade in anti-inflammatory therapy for severe coronavirus disease (covid) date: 2020-07-04 journal: clin infect dis doi: 10.1093/cid/ciaa910 sha: doc_id: 303603 cord_uid: 4mi64bgm nan to the editor-the coronavirus disease (covid) pandemic has been a threatening challenge to health systems worldwide [1] . in this context, the use of anti-inflammatory therapy to treat severe covid patients has been controversial during the whole pandemics. general recommendations were against the use of corticosteroids [2] , although some hope was placed on monoclonal anti interleukin 6 receptor antibodies in blocking the so-called cytokine storm produced by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in selected patients [3, 4] . recently published papers provide high-quality evidence that supports the use of corticosteroids in the early stage of severe covid [5] . nevertheless, a high rate of treatment failure was observed in both treatment subgroups (34.9% vs 54.3%). in our experience, the early administration of corticosteroids associated to tocilizumab, according to the previously published recommendations by the spanish ministry of health [6] , may solve this high failure rate. in the hm sanchinarro hospital, a 200 bed teaching institution, we consecutively and ambispectively collected the outcome of 136 patients who received tocilizumab plus corticosteroids to treat severe covid, defined as a spo2/fio2 <325 with bilateral pneumonia and a clinical diagnosis of infection by sars-cov-2. patients were divided in 2 subgroups: those who received tocilizumab after 24 hours of admission and before spo2/fio2 decreased to ≤250 (early tocilizumab administration [et], n = 38) and those who received the combination directly at admission or when spo2/fio2 was <250 (standard therapy [st], n = 98). statistical analysis was performed to predict a composite outcome of intensive care unit (icu) admission and in-hospital mortality. mean age was 68 years (95% confidence interval [ci], 46-94), with a 72.8% of male individuals. 47 patients reached the composite outcome. mean comorbidity burden was of 3.35 (95% ci, 2.96-3.75) points according to the charlson comorbidity index (cci). in the raw analysis, a 6.25% of patients in the et group developed the composite outcome, whereas 45.3% of the patients in the st group were admitted to the icu and/or died during admission. a χ 2 analysis was performed to compare early and standard groups of the fadel et al cohort with patients who received early and late combined anti-inflammatory therapy in our cohort. no differences were found in the late therapy groups (45.3% vs 54.3%, p = .54), although statistically different results were found between early therapy subgroups (6.25% vs 34.9%, p < .01). generalized linear and cox regression models, adjusted by age, sex, interaction between age and sex, cci, time from onset to admission, dose of corticosteroids and tocilizumab and c-reactive protein and d-dimer levels at anti-inflammatory therapy administration, showed benefit in the use of et in covid patients (relative risk, 0.18, p = .01, and hazard ratio, 0.13, p = .01, respectively). cox model results stratified by time to anti-inflammatory therapy administration are shown in (figure 1 ). the results of our cohort may reinforce the early administration of anti-inflammatory therapy published by fadel et al. in addition, the promising results of the combination of corticosteroids and tocilizumab highlight the need of further investigations in the anti-inflammatory treatment of severe covid. world health organization. coronavirus disease 2019 (covid-19) situation reports clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology impact of low dose tocilizumab on mortality rate in patients with covid-19 related pneumonia early short course corticosteroids in hospitalized patients with covid-19 drugs and sanitary products spanish agency, available treatments for management of sars-cov2 respiratory infection key: cord-314489-e5r5s5ee authors: katsidzira, leolin; gwaunza, lenon; hakim, james g title: the sars-cov-2 epidemic in zimbabwe: quo vadis? date: 2020-05-11 journal: clin infect dis doi: 10.1093/cid/ciaa552 sha: doc_id: 314489 cord_uid: e5r5s5ee the trajectory, and impact of the sars-cov-2 pandemic in sub-saharan africa is unclear, but it is seemingly varied between different countries, with most reporting low numbers. we use the situation in zimbabwe to build an argument that the epidemic is likely to be attenuated in some countries with similar socio-economic and cultural structures. however, even an attenuated epidemic may overwhelm weak health systems, emphasising the importance of prevention. these prevention strategies should be tailored to the unique social and cultural networks of individual countries which may facilitate the spread of sars-cov 2. it is also equally important to maintain services for the major infectious diseases in the region such as tuberculosis and malaria. a breakdown of treatment and prevention services for these conditions may even overshadow the projected morbidity and mortality from covid-19. m a n u s c r i p t 4 the emergence of sars-cov-2 has shaken the globe in fundamental ways, and sub-saharan africa has been no exception. however, the magnitude and trajectory of the epidemic in the region is unclear, and the reflex response has ranged from nonchalant to assuming the worst-case scenario. the region was spared from the first two coronavirus epidemics of the 21 st century, sars and mers, initially raising expectations of a similar scenario unfolding with sars-cov-2 [1] . however, emerging information about the increased vulnerability of underprivileged groups to covid-19 disease in the united states has heightened fears that africa will be disproportionately affected by this pandemic [2] . using zimbabwe as an example, we argue that the magnitude, and impact of the epidemic in most of sub-saharan africa is likely to be smaller than anticipated, with a reduced morbidity and mortality. however, the nature, and impact of such an attenuated epidemic remains unclear, and may still overwhelm weak health systems. in zimbabwe, the threat of covid-19 burst into national consciousness after the death of the second case to be diagnosed in the country. he was a young prominent media personality, and the son of a well-known politician and businessman, and had recently returned from new york. his demise gained intense media coverage, and highlighted the structural deficiencies in the zimbabwean health system in dealing with a highly contagious disease, particularly one which may require critical care [3, 4] . this case strongly influenced the subsequent response to covid-19 by both the government, and the private healthcare industry in zimbabwe, and played a pivotal role in raising public awareness. subsequently, government reserved 425 hospital beds and 5 ventilators for covid-19 at one of the tertiary care hospitals in the capital, harare, and is upgrading and refurbishing infectious diseases hospitals around the country. another set of, five this highlights the need to balance the response to covid-19 with on-going public health needs [5] . it is not inconceivable that we may see an increase in mortality from non-covid-19 related conditions, and this increase may even dwarf covid-19 related deaths, as was previously observed after the ebola outbreak in west africa [6] . thus, it is essential to maintain a sense of proportion, particularly in sub-saharan africa, where access to healthcare is a challenge even during normal times. third, zimbabwe has been relatively isolated from global air travel. in 2018, only 8 foreign airlines were flying into the country, mostly from regional destinations [10] . there is a link between the volume of international flights, and the magnitude of the sars-cov-2 epidemic in sub-saharan africa [7, 11] . it is no coincidence that south africa, with the most advanced economy in the region, and multiple international flights daily, has the highest number of sars-cov-2 cases in the region [7, 12] . countries such as zimbabwe, which are less integrated with the global economy, may have been inadvertently less exposed to the sars-cov-2 pandemic. even after arrival in zimbabwe, travellers are often ferried to their homes or hotels in private vehicles, limiting exposure to the public that may occur with efficient mass transport systems. this situation has an inherent social distancing, and may simplify contact tracing. finally, zimbabwe and most of sub-saharan africa have a predominantly young population. according to the 2012 census, 89% of the zimbabwean population was a c c e p t e d m a n u s c r i p t 8 younger than 50 years and only 2.8% were older than 75 years [13] . older age has been consistently associated with heightened mortality from covid-19 [14] [15] [16] [17] . consequently, differences in the population age structure can lead to dramatic differences in mortality for covid-19 disease [18] thus, it is reasonable to anticipate a much lower mortality from covid-19 in sub-saharan africa, compared to europe and north america, where there is a much larger proportion of older people. nonetheless, it is important to protect the small, elderly population in sub-saharan africa, and this may be easier to implement. there is limited use of institutional care for the elderly such as nursing homes, or retirement villages [19] . this may reduce the risk of nosocomial sars-cov-2 outbreaks in a vulnerable, captive population since the majority of elderly people live in the rural areas, their risk of infection can be reduced by restricting movement between urban and rural areas. a potential source of higher than anticipated mortality from covid-19 disease in sub-saharan africa is the high burden of hiv infection [5] . it is also possible that individuals with hiv infection may have increased susceptibility to sars-cov-2 infection. however, there is no robust data on the interaction between hiv and covid-19, although initial evidence from a small case series suggests that the impact could be less than initially feared [20] . moreover, considerable progress has it remains unclear whether complete lockdowns are the most ideal method to limit the spread of sars-cov-2 in sub-saharan africa [22] . a complete lockdown has its m a n u s c r i p t 9 own economic ramifications, and the sars-cov-2 pandemic in itself has led to the first recession in a quarter of a century in the region [23] . however, the degree of the adverse impact of lockdowns on the economy will differ between different countries. in zimbabwe, there is likely to be disproportionate effect at household level, as most people now depend on the informal sector, in a country with limited formal social safety nets [24] . moreover, enforcement of lockdowns is potentially unequal, and may take a punitive form in the poor neighbourhoods, that paradoxically, are less likely to have the initial imported, and imported-associated sars-cov-2 infections. flattening the curve may not have a significant impact if the epidemic is small, and the existing healthcare infrastructure is already overwhelmed by the large burden of communicable and non-communicable diseases. however, the net effect of lockdowns will only become clearer retrospectively, and data from countries such south africa becomes available. south africa has the largest epidemic in the region, and has implemented a strict lockdown, and is generating high quality epidemiological data. for now, policymakers have to make decisions based on imperfect information, which is continuously changing as our understanding of the virus incrementally increases. in the meantime, incorporating other approaches into the on-going measures may also help in limiting the spread on sars-cov-2. one such approach that can be undertaken in sub-saharan africa is to restrict the movement of people between different suburbs, and between urban and rural areas, while allowing some level of economic activity. active surveillance, and testing for both imported and community cases with stringent contact tracing and isolation should continue. it is instructive that this approach has identified most of the reported cases in zimbabwe. localised lockdowns may also be considered for specific foci of infections. international travel a c c e p t e d m a n u s c r i p t 10 should continue to be curtailed, and more effective screening strategies at the ports of entry must be developed and implemented when it resumes. in zimbabwe there is a dichotomous health system, one public, catering for the majority, and the other private sector, catering for the minority who are on medical insurance or can afford to pay. given the aforementioned profile of the typical case in the country, it is important to ensure that prevention strategies are also implemented stringently at the private health facilities, which should be capacitated where necessary. the protection of healthcare workers should be a key priority, and some of them serve both the public and private sectors, and are a potential bridge of infection. all the cases seen in zimbabwe so far have either been identified at private hospitals, or from screening returning travellers, or from contact tracing. as of 2 may 2020, there has not been a case, presenting initially to a public healthcare facility. in conclusion, it is conceivable that the impact of covid-19 in zimbabwe could be attenuated in comparison to what has happened in economies with mass transport systems, high volume air travel and over-crowded social gatherings. this however, is on the proviso that there is adequate insulation of the community from returning travellers and a robust implementation of hygienic practices and social distancing. finally, the covid-19 epidemic is an opportunity for countries in sub-saharan africa to invest and innovate in the delivery of better health care including critical care infrastructure and to redirect dollars being lost in medical tourism inwardly [25] . strategy and technology to prevent hospitalacquired infections: lessons from sars, ebola, and mers in asia and west africa covid-19 and african americans zimbabwean broadcaster zororo makamba died 'alone and scared makamba family fumes over zororo's death the late arrival of covid-19 in africa -mitigating pan-continental spread effects of response to 2014-2015 ebola outbreak on deaths from malaria, hiv/aids, and tuberculosis passengers' destinations from china: low risk of novel coronavirus (2019-ncov) transmission into africa and south america income inequality trends in sub-saharan africa: divergence, determinants and consequences zimbabwe national statistical agency and icf international zim's airline industry needs serious attention covid-19 pandemic in west africa preparedness and vulnerability of african countries against importations of covid-19: a modelling study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis risk factors for severity and mortality in adult covid-19 inpatients in wuhan clinical characteristics and outcomes of older patients with coronavirus disease 2019 (covid-19) in wuhan, china (2019): a single-centered, retrospective study demographic science aids in understanding the spread and fatality rates of covid-19 the key actors maintaining elders in functional autonomy in bobo-dioulasso (burkina faso) covid-19 in patients with hiv: clinical case series. the lancet hiv. 21. unaids. country factsheet: zimbabwe covid-19: africa records over 10 000 cases as lockdowns take hold covid-19 (coronavirus) drives sub-saharan africa toward first recession in 25 years shadow economies around the world: what did we learn over the last 20 years? essential care of critical illness must not be forgotten in the covid-19 pandemic a c c e p t e d m a n u s c r i p t 11 funding: no funding was required for this work. a c c e p t e d m a n u s c r i p t 12 key: cord-313693-qmkrn7pr authors: wong, bonnie c. k.; lee, nelson; li, yuguo; chan, paul k. s.; qiu, hong; luo, zhiwen; lai, raymond w. m.; ngai, karry l. k.; hui, david s. c.; choi, k. w.; yu, ignatius t. s. title: possible role of aerosol transmission in a hospital outbreak of influenza date: 2010-11-15 journal: clin infect dis doi: 10.1086/656743 sha: doc_id: 313693 cord_uid: qmkrn7pr background. we examined the role of aerosol transmission of influenza in an acute ward setting. methods. we investigated a seasonal influenza a outbreak that occurred in our general medical ward (with open bay ward layout) in 2008. clinical and epidemiological information was collected in real time during the outbreak. spatiotemporal analysis was performed to estimate the infection risk among patients. airflow measurements were conducted, and concentrations of hypothetical virus-laden aerosols at different ward locations were estimated using computational fluid dynamics modeling. results. nine inpatients were infected with an identical strain of influenza a/h3n2 virus. with reference to the index patient's location, the attack rate was 20.0% and 22.2% in the “same” and “adjacent” bays, respectively, but 0% in the “distant” bay (p=.04). temporally, the risk of being infected was highest on the day when noninvasive ventilation was used in the index patient; multivariate logistic regression revealed an odds ratio of 14.9 (95% confidence interval, 1.7–131.3; p=.015). a simultaneous, directional indoor airflow blown from the “same” bay toward the “adjacent” bay was found; it was inadvertently created by an unopposed air jet from a separate air purifier placed next to the index patient's bed. computational fluid dynamics modeling revealed that the dispersal pattern of aerosols originated from the index patient coincided with the bed locations of affected patients. conclusions. our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. source and engineering controls, such as avoiding aerosol generation and improving ventilation design, may warrant consideration to prevent nosocomial outbreaks. nosocomial transmission of influenza is frequently reported [1] [2] [3] . it typically occurs during seasonal peaks, and may involve almost all types of healthcare facilities [1, 2] . its consequences are considerable: it may result in significant disease and even fatality among hospitalized patients, because these patients often are older and/or have multiple comorbidities [1] [2] [3] . health care professionals are frequently involved, and the affected hospital units may require temporary closure with service suspension [2] [3] [4] [5] [6] . lack of preexisting immunity toward the recently emerged pandemic influenza h1n1 virus and its potential to cause serious disease in both young and older adults have further raised the importance of hospital infection control [7] [8] [9] . however, how influenza is transmitted in the health care setting and what control measures are effective have remained largely unclear [2, [8] [9] [10] [11] [12] . under natural conditions, influenza virus is transmitted predominantly via droplets and direct contact [13] . thus, adequate spacing, hand washing, and droplet precautions, including the use of face masks, are likely effective in preventing transmission [8, 9, 11] . however, in indoor health care settings, because of the special clinical and environmental conditions, aerosol transmission of diseases might become possible, as described for other viral and bacterial infections [1, 3, [13] [14] [15] [16] [17] . emerging evidence suggests that influenza infection can also be transmitted via the aerosol route, as shown in animal models and in experimental studies involving human subjects [3, 13, 18, 19] . in this study, we report an influenza outbreak that occurred in an acute medical ward. epidemiological, airflow and computational fluid dynamics analyses were performed. the possible role of aerosol transmission of influenza was examined. the implications of the results on hospital infection control strategies will be discussed. at prince of wales hospital (pwh; hong kong), an outbreak of influenza a occurred in an adult general medical ward in april 2008. in hong kong, peak influenza activities occur in both spring (january-april) and summer (july-september) [20] . pwh is a 1350-bed acute care general hospital operated by the hospital authority of hong kong that serves an urban population of 1.5 million. each year, 1200 adult cases of confirmed influenza are being treated at pwh [5, 21, 22] . a major nosocomial outbreak of severe acute respiratory syndrome (sars) occurred at pwh in 2003 [15, 23] ; since then, all patients hospitalized with acute febrile respiratory illnesses are put on droplet precautions; if influenza is confirmed, the patients will be isolated or cohorted in designated wards [21, 22] . as a hospital policy, all health care personnel working in medical wards are required to wear surgical face masks, and an on-duty nursing officer is responsible for monitoring compliance [5] . outbreak investigation. nosocomial outbreaks of infectious diseases, once identified, would be investigated by the outbreak management team (which consisted of physicians, microbiologists, infection control practitioners, nurses, and hospital epidemiologists) as part of the management protocol [23] . affected cases would be reviewed daily, and clinicoepidemiological information collected real time as the outbreak evolved. these data were studied and discussed in daily meetings throughout the whole outbreak period. control measures, including ward closure, patient isolation, and use of antiviral prophylaxis, would then be recommended. virological investigations for influenza have been described elsewhere [21, 22] . in brief, nasopharyngeal aspirate specimens were collected from symptomatic individuals and subjected to immunofluorescence assay and virus culture for diagnosis [21, 22] . in addition, for all confirmed cases, viral rna was directly extracted for sequencing of the whole length of the hemaggultinin gene, as described elsewhere [24] ; the nucleotide sequences in individual cases were then compared. finally, a hemagglutination inhibition assay was performed (using the virus isolate obtained from the index patient as antigen source) to detect antibody rise in the paired serum samples collected from affected individuals [25] . spatiotemporal analysis of epidemiological data. the epidemic curve for the outbreak was produced. the floor plan of the affected wards was studied; in pwh, the design of general medical wards followed an open bay ward layout [15] . for the analysis, all patients who had ever stayed with the index patient during the period of his presence in the ward (from admission to transfer or isolation) and all health care workers (hcws) who had worked in the same ward during the period were included. attack rates of influenza among inpatients were calculated each day on the basis of their locating "bay" [15] . univariate relationships between risk of acquiring influenza infection and spatiotemporal variables were analyzed using the x 2 test or the fisher exact test. variables with a p value !.1 were entered into a multivariate (stepwise) logistic regression model to identify independent factors associated with infection. odds ratio (ors) and 95% confidence intervals (cis) were reported for explanatory variables. in all analyses, a p value of !.05 was considered to indicate statistical significance. all probabilities were 2-tailed. statistical analysis was performed using pasw statistics software, version 17.0 (spss). airflow measurements and fluid dynamics analyses. information on the ward's ventilation systems was collected during the outbreak investigation. these included the location and size of air supply diffusers, return grills, and the highefficiency particulate absorbing (hepa) air purifier units that were present in each ward bay. airflow rate through each supply diffuser, return grill, air purifier, and exhaust fan was measured in detail, as described elsewhere [15] . dispersion of the hypothetical virus-laden aerosols, originated from the index patient's bed through the entire ward, was analyzed by computational fluid dynamics (cfd) method. the hypothetical virus-laden aerosols were modeled as gaseous and passive tracers, which have been shown to be able to model well the dispersion of droplet nuclei that were less than 5-10 microns in diameter. the commercial cfd software, fluent, version 6.2, was used [15, 26] . the predicted concentration fields were compared with the locations of affected patients found in the outbreak. the 'outbreak management team' was being alerted of a possible outbreak in a general medical ward on 4 april 2008, when 7 inpatients were found to have developed fever and respiratory symptoms. the ward was immediately closed to new admissions, and transfer to other hospitals or institutes was suspended; sick patients were isolated. for all hcws and the remaining patients, strict droplet precautions were implemented, and the individuals were required to wear surgical masks at all times. hand hygiene was reinforced. postexposure prophylaxis with oseltamivir was offered, and both groups were monitored for development of symptoms. discharged patients were put under continuous home medical surveillance for 1 week. the ward returned to normal function on 12 april, when the outbreak was declared over. an epidemiological investigation was started on 4 april. at the end of the outbreak, a total of 9 inpatients (patients a-i) were found to have symptoms that fulfilled the case definition of influenza-like illness (figures 1 and 2) . no visitor was known to be affected. the symptom onset dates of the first (index) figure 1 . epidemic curve of the influenza outbreak. patients were shown according to their symptom onset date (fever or new respiratory symptoms); the order does not necessarily reflect the order in which they acquired infection. the arrow indicates the time when the index patient (patient a) commenced bi-level positive airway pressure (bipap) ventilation support. prior to that, he was receiving supplemental oxygen therapy via nasal cannula. the bipap ventilation lasted for 116 h; he was subsequently transferred to the intensive care unit. patient i started to receive oseltamivir prophylaxis on 4 april (the ward was closed and sick patients were isolated); however, he soon became unwell and developed fever on 10 april, despite receipt of prophylaxis. staff 1 and 2 had symptoms; however, the results of serological tests for recent influenza infection were negative (table 1 ). case and the last case were 27 march and 10 april, respectively. all 9 inpatients tested positive for influenza a by immunofluorescence assay and culture of nasopharyngeal aspirate specimens. all isolates belonged to the h3n2 virus subtype (influenza a/brisbane/10/2007) and were 100% identical, as determined by nucleotide sequence analysis (table 1) . nasopharyngeal aspiration was not performed for the 2 hcws who had reported symptoms; however, serological test results did not suggest recent influenza infection ( table 1) . the index patient (patient a) ( figure 1 ) was a 68-year-old man who had underlying chronic obstructive pulmonary disease. he was admitted on 27 march with unresolved pulmonary shadows, despite having completed a course of antibiotic treatment, and increased dyspnea, which initially required suppleall secondary cases were detected to have fever and/or new onset of respiratory symptoms. once influenza was confirmed, the patients started to receive oseltamivir treatment (75 mg twice per day for 5 days) within 24 h after symptom onset. the clinical courses of 7 patients remained uncomplicated, and the patients were subsequently discharged. the remaining patient (patient c) (figure 1 ) was a 73-year-old man who had originally been admitted for pseudomonas pneumonia that had complicated advanced bronchiectasis; although afebrile, he developed lower respiratory tract complication after influenza infection and died of progressive respiratory failure. spatiotemporal analysis. the outbreak ward was a male general medical ward consisting of 30 beds arranged in 3 major bays (2 adjacent rear bays b and c separated by a ∼2-m wide corridor and 1 front bay a) (figure 2 ). the distance between adjacent beds was ∼1 m. the index patient's bed was located at the wall end of bay c, right next to a hepa air purifier. in total, 59 patients and 29 hcws had stayed or worked on the ward during the period from 27 march to 1 april. the overall attack rate among patients was 13.6% (8 of 59 subjects). we found that patients who had stayed in the "adjacent" bay b (attack rate, 22.2%) were affected to a degree similar to those who stayed in the "same" bay c with the index patient (attack rate, 20.0%); however, no patients in the front, "distant" bay a or side room were affected (attack rate, 0%; p p .041, by the fisher exact test). when analyzed according to date, presence in rear bays was associated with attack rates of 15.8%, 15.8%, 26.3%, and 26.3% on the dates 27-30 march, respectively. the risk of being infected was highest on 31 march (30.4%; p p .005) and 1 april (30.0%; p p .016), which coincided with the time of bipap ventilation use in the index patient. in a final multivariate logistic regression model, staying in the rear bays on 31 march was independently associated with a higher risk of acquiring influenza (or, 14.9; 95% ci, 1.7-131.3; p p .015). the attack rate among hcws was not analyzed in detail, because only 2 were reported to have had symptoms, and neither had laboratory-confirmed infection. they reported close contact with patients staying at all 3 bays while performing their routine duties. airflow measurements and analysis. air conditioning in the outbreak ward was provided by fan coil unit systems with a 4-way diffuser at the ceiling level in each of the 3 bays. the return air grills were located at the ceiling of the corridor. a hepa air purifier was placed at the wall end of each bay ( figures 2 and 3) ; it functioned by drawing in air from a lower level, and after filtration, injecting the air back into the ward at an upper level. during the outbreak, the fan setting of the hepa air purifiers was found to set to low in bays a and b and to medium in bay c inadvertently, with injection velocity measuring 1.47 m/s, 1.44 m/s, and 1.90 m/s respectively. airflow measurements revealed that, under this situation, there was an imbalance in the air supply to and return from different bays, and the net flow toward the corridor was 70-100 l/s ( figure 3 ). the injecting air velocities from the hepa air purifiers were substantially higher than that of the diffusers and thus dominated the overall airflow pattern. because of the higher air injection velocity from the air purifier in bay c compared with that in the adjacent bay b, air from bay c was expected to be "pushed" into the corridor and toward bay b (figure 4 ). cfd simulations were performed, and the distributions of hypothetical virus-laden aerosols originated from the index patient are shown in figure 5 . the normalized concentration of the hypothetical virus-laden aerosols was found to be the highest in bay c ("same"), followed by bay b ("adjacent"), and the lowest in bay a ("distant"). the estimated spatial distribution figure 5 . the spatial distribution of normalized concentration of hypothetical virus-laden aerosols (modeled as gaseous tracer) in the outbreak ward at a height of 1.1 m. the flow rates used in this model were those described in figure 3 . all high-efficiency particulate absorbing (hepa) filters were assumed to function with 100% filtration of the modeled droplet nuclei. the 3 hepa air purifiers are shown as black boxes, the 4 diffusers are shown by a square with an x, and the 4 returns are shown as a small rectangular filled box. affected patients are represented by white ovals (the index patient is marked as a red oval). was found to correspond to the locations of affected patients in the outbreak. large droplets would not have accounted for such distribution because of their fast deposition onto surfaces [27, 28] . we report a nosocomial outbreak of seasonal influenza in an acute ward setting. it was temporally related to the use of an aerosol-generating device in the index patient. this had occurred together with an imbalanced indoor airflow; and the spatial distribution of cases was found to follow the directional airflow and coincided with a cfd-estimated aerosol dispersal pattern. our findings suggest a possible role of aerosol transmission in this outbreak. we have previously reported aerosol transmission of sarsassociated coronavirus in a similar ward setting [15] . in that study, cfd modeling demonstrated a close relationship between concentration of virus-laden aerosols and the risk of acquiring infection in various ward locations. we studied this outbreak using similar approach, and our findings provide further evidence to support the hypothesis that, under suitable clinical and environmental conditions, aerosol transmission of influenza virus can occur [2, 3] . in this outbreak, we postulate that infectious aerosols were generated continuously through the use of noninvasive ventilation for the index patient (which projected for at least 1 m sagittally to bed end) [29] [30] [31] for 16 h and these aerosols were blown toward the adjacent bay by an imbalanced indoor airflow created by an air purifier's outflow jet, which was located at patient's bed end. we believe that droplets and contact routes of transmission cannot entirely explain the outbreak because (1) the epidemic curve suggested a point-source mode of infection, instead of successive propagation (especially for the aggregation of symptom onset on 2 and 3 april); (2) the index patient and most other inpatients were immobile during their illnesses and, therefore, direct contacts should have been minimal; (3) patients 12 m away were affected; and (4) locations of affected patients coincided with dispersal pattern of aerosols and the directional airflow. in this outbreak, patients in the "adjacent" and "same" bay were similarly affected, but the "distant" bay was spared. a more random distribution of infected cases would be expected if hcws had acted as vectors, because they were responsible for the care of patients in all bays (duties were assigned on the basis of function and not location) [15] . similarly, close patient-patient contact, visits to a contaminated common area, and transmission through a wandering, sick visitor are unlikely explanations in our setting [15] . finally, high air velocities (110 m/s), which can carry even large droplets beyond 2 m, were not found in our ward setting, and they would have been deposited too fast onto surfaces to account for the observed dispersal pattern [27, 28] . we hold the view that the predominant modes of influenza transmission are via droplets and direct contact [2, 3, 8, 13] . however, accumulating evidences suggest that the aerosol route may have a contributing role [3, 13, 18, 19] . in animal models, influenza virus (eg, h3n2 or h1n1) has been shown to transmit efficiently through air, whereas fomite or contact spread is relatively inefficient [13, 18, 19, [32] [33] [34] . in clinical studies, virus-laden particles less than 5-6 mm (ie, within the respirable aerosol fraction) have been detected in exhaled breaths of patients with influenza and in the air sampled from an acute healthcare setting during seasonal peak [19, 35, 36] . in contrast to natural coughing or sneezing, artificially generated respiratory particles are often much smaller in size ( less than 5-6 mm), can penetrate more readily into the lower respiratory tract, and can cause infection with a smaller dose [3, 13, 18, 19] . it has been shown that certain clinical procedures (eg, endotracheal intubation, cardiopulmonary resuscitation, noninvasive ventilation, and receipt of high-flow oxygen) can generate a large amount of respiratory aerosols [8, 9, 13, 16, 18, [29] [30] [31] , and transmission of respiratory infection related to some of these procedures-despite implementation of droplet precautions-has been reported [37] [38] [39] . since the outbreak, we have used a hierarchy of control measures to prevent influenza transmission in our hospital, such as administrative and source controls, engineering controls, and use of personal protective equipment [8, 9] . the policy of isolating or cohorting patients with suspected or confirmed influenza is reinforced; application of an aerosol-generating procedure is allowed only in adequately ventilated single rooms before influenza can be excluded; all patients with respiratory infections are required to wear face masks, which are freely provided, until symptoms subside; air-conditioning units and their settings are regularly checked to avoid airflow imbalance; and hcws are advised to use n-95 respirators, face shields, gloves, and gowns while performing aerosol-generating procedures and to receive annual influenza vaccines [2, 5, 8, 9] . we have adopted these measures on all medical wards, because there might be similar unsuspected or "invisible" patients with influenza acting as infection sources. we did not encounter another influenza outbreak in open wards during the subsequent 24 month period, which included the first wave of the influenza h1n1 pandemic [7, 20] . although each institute's infrastructure may be different, our findings suggest that the strategies of source and engineering controls might be important considerations to prevent nosocomial influenza transmission. our study is limited by its descriptive nature. we could not analyze the impact of influenza vaccination on the size of outbreak, because such information was unavailable from many patients; however, the general vaccine uptake rate in our community was reported to be very low [40, 41] . also, we could not entirely eliminate the role of sick hcws in transmitting infection, albeit no case of influenza was eventually confirmed among them. however, because all hcws were required to wear surgical face mask during work and to report any influ-enza-like illnesses through a daily reporting system (the presence of which would immediately exempt them from duty), the chance of hcws cross-infecting patients should be rather small [5] . furthermore, the spatial distribution of cases could not be easily explained by hcw-to-patient or patient-to-patient transmission, as discussed above [15] . our findings indicate the need to evaluate the infection risks of aerosol-generating procedures or devices, especially when applied to the disease state [8, 9, 29, 30, 37] ; the effectiveness of various source and administrative control strategies [2, 3, 8, 9, 39] ; the ventilation systems in different healthcare settings; and the impact of airflow and humidity on nosocomial influenza transmission with an architectural aerodynamics approach [13, 17, 42, 43] . in conclusion, our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. source and engineering controls, such as avoiding aerosol generation and improving ward ventilation design, may warrant consideration to prevent nosocomial outbreaks. hospital-acquired influenza: a synthesis using the outbreak reports and intervention studies of nosocomial infection (orion) statement influenza in the acute hospital setting transmission of influenza: implications for control in health care settings disruption of services in an internal medicine unit due to a nosocomial influenza outbreak preventing healthcare workers from acquiring influenza a nosocomial outbreak of influenza during a period without influenza epidemic activity world health organization. pandemic (h1n1) infection prevention and control in health care for confirmed or suspected cases of pandemic (h1n1) 2009 and influenza-like illnesses interim guidance on infection control measures for 2009 h1n1 influenza in healthcare settings, including protection of healthcare personnel physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review surgical mask vs n95 respirator aerosol transmission of influenza • cid 2010:51 (15 november) • 1183 for preventing influenza among health care workers: a randomized trial novel h1n1 influenza and respiratory protection for health care workers transmission of influenza a in human beings evidence of airborne transmission of the severe acute respiratory syndrome virus temporal-spatial analysis of severe acute respiratory syndrome among hospital inpatients detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units role of ventilation in airborne transmission of infectious agents in the built environment-a multidisciplinary systematic review review of aerosol transmission of influenza a virus aerosol transmission of influenza a virus: a review of new studies outcomes of adults hospitalized with severe influenza factors associated with early hospital discharge of adult influenza patients a major outbreak of severe acute respiratory syndrome in hong kong seasonality of influenza a(h3n2) virus: a hong kong perspective lack of cross-immune reactivity against influenza h5n1 from seasonal influenza vaccine in humans fluent 6.2 user's guide removal of exhaled particles by ventilation and deposition in a multibed airborne infection isolation room how far droplets can move in indoor environments-revisiting wells evaporation-falling curve of droplets exhaled air dispersion distances during noninvasive ventilation via different respironics face masks noninvasive positive-pressure ventilation: an experimental model to assess air and particle dispersion exhaled air dispersion during oxygen delivery via a simple oxygen mask the guinea pig as a transmission model for human influenza viruses transmission of influenza virus via aerosols and fomites in the guinea pig model transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice measurement of airborne influenza virus in a hospital emergency department influenza virus in human exhaled breath: an observational study investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto, canada possible sars coronavirus transmission during cardiopulmonary resuscitation why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? cross-sectional and longitudinal factors predicting influenza vaccination in hong kong chinese elderly aged 65 and above willingness of hong kong healthcare workers to accept pre-pandemic influenza vaccination at different who alert levels: two questionnaire surveys influenza virus transmission is dependent on relative humidity and temperature factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises we thank mr. li liu and ms caroline xiaolei gao for their participation in the field measurement in the outbreak ward.potential conflicts of interest. all authors: no conflicts. key: cord-312928-ef8hqs4s authors: chavanet, pascal title: viral upper respiratory tract infection and otitis media complication in young children date: 2008-03-15 journal: clin infect dis doi: 10.1086/528686 sha: doc_id: 312928 cord_uid: ef8hqs4s nan viral infections in children are very common and raise huge problems of management. the burden of these infections is difficult to quantify because of the lack of pathognomonic features. there is a clear need to know the characteristics of these infections to give directions for care and possibly to provide specific treatments, prevention, and prophylaxis. in this issue of clinical infectious diseases, chonmaitree et al. [1] conducted a large, longitudinal observational study of children with upper respiratory infection and examined for otitis complicationeither acute otitis media or otitis media with effusion. they found that 63% of children were virus positive and concluded that the rate of otitis media complication was 61% (37% for acute otitis media and 24% for otitis media with effusion), that these risks decrease as the age increases, and that adenovirus, coronavirus, and respiratory syncitial virus represent more than two-thirds of otitis media complications. these findings confirm the findings of previous works but with differences. the main problem with this kind of study is the accuracy and sensitivity of viral investigations. indeed, in this study, the rate of respiratory syncitial virus infection was found to be low, probably as a result of the method used (conventional assays, type of molecular technique, rt-pcr vs. microarray, etc). furthermore, human metapneumovirus and bocavirus were not studied [2, 3] . thus, extensive (one-third of the children in the study were not virus positive) and "stabilized" viral investigations are still needed to capture the real facts, to create a precise hierarchy of all of the different pathogens involved [4] . however, the findings of chonamaitree et al. [1] also confirm that respiratory syncitial virus and influenza virus are 2 of the main causes of complications of upper respiratory infection. in this study, children aged !1 year and those who attended day care centers had a greater risk of acquiring upper respiratory tract infection and otitis media, compared with older children and those who were cared for at home. this finding is well known; however, the expectation that ill children can be kept out of day care centers or that home care can be provided for children until at least 1 year of age raises very difficult socioeconomic barriers. it should be noted that chonamaitree et al. [1] did not mention the rate of antibiotic treatment [5] or the number of hospitalizations [6] . management of viral infection in children is still complicated by genetics. in fact, it was recently reported that suscep-tibility to respiratory syncitial virus bronchiolitis, for example, is related in part to a genetic profile of innate immunity [7] . this article is important, especially because the rate of complications of otitis in upper respiratory tract infection was longitudinally studied. however, the results have to be integrated with other investigations that use different diagnostic tests. viral upper respiratory tract infection and otitis media complication in young children prevalence of respiratory viruses, including newly identified viruses, in hospitalised children in austria epidemiological and clinical features of hmpv, rsv and rvs infections in young children respiratory virus infections. paediatr antibiotics for acute otitis media: a meta-analysis with individual patient data rates of hospitalisation for influenza, respiratory syncytial virus and human metapneumovirus among infants and young children genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes potential conflicts of interest. p.c.: no conflicts. key: cord-313427-6y4zvrmn authors: mani, nandita s; budak, jehan z; lan, kristine f; bryson-cahn, chloe; zelikoff, allison; barker, gwendolyn e c; grant, carolyn w; hart, kristi; barbee, carrie j; sandoval, marissa d; dostal, christine l; corcorran, maria; ungerleider, hal m; gates, jeff o; olin, svaya v; bryan, andrew; hoffman, noah g; marquis, sara r; harvey, michelle l; nasenbeny, keri; mertens, kathleen; chew, lisa d; greninger, alexander l; jerome, keith r; pottinger, paul s; dellit, timothy h; liu, catherine; pergam, steven a; neme, santiago; lynch, john b; kim, h nina; cohen, seth a title: prevalence of covid-19 infection and outcomes among symptomatic healthcare workers in seattle, washington date: 2020-06-16 journal: clin infect dis doi: 10.1093/cid/ciaa761 sha: doc_id: 313427 cord_uid: 6y4zvrmn background: healthcare workers (hcw) serving on the front lines of the coronavirus disease 2019 (covid-19) pandemic have been at increased risk for infection due to sars-cov-2 in some settings. healthcare-acquired infection has been reported in similar epidemics, but there are limited data on the prevalence of covid-19 among hcws and their associated clinical outcomes in the united states. methods: we established two high-throughput employee testing centers in seattle, washington with drive-through and walk-through options for symptomatic employees in the university of washington medicine system and its affiliated organizations. using data from these testing centers, we report the prevalence of sars-cov-2 infection among symptomatic employees and describe the clinical characteristics and outcomes among employees with covid-19. results: between march 12 and april 23, a total of 3,477 symptomatic employees were tested for covid-19 at two employee testing centers; 185 (5.3%) employees tested positive for covid-19. the prevalence of sars-cov-2 was similar when comparing frontline hcws (5.2%) to non-frontline staff (5.5%). among 174 positive employees reached for follow-up at least 14 days after diagnosis, 6 reported covid-related hospitalization; all recovered. conclusions: during the study period, we observed that the prevalence of positive sars-cov-2 tests among symptomatic hcws was comparable to that of symptomatic non-frontline staff. reliable and rapid access to testing for employees is essential to preserve the health, safety, and availability of the healthcare workforce during this pandemic and to facilitate the rapid return of sars-cov-2 negative employees to work. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus that causes coronavirus disease 2019 , was declared a pandemic by the world health organization on march 11, 2020. it continues to disrupt life for millions around the globe. 1 the first case of covid-19 in the united states (u.s.) was diagnosed in washington state on january 20, 2020 in a returning traveler from wuhan, china. 2 subsequently, the greater seattle area became the first recognized epicenter of the covid-19 outbreak in the u.s. with over 22,000 confirmed cases and 1,100 deaths statewide as of june 4, 2020. 3 during the 2003 severe acute respiratory syndrome (sars) outbreak, healthcare workers (hcws) were disproportionately affected, accounting for 21% of all cases, due to documented nosocomial transmission. 4-6 sars-cov-2 has also caused significant morbidity and mortality among hcws globally, particularly in china and italy early in the outbreak. 7-10 as of april 9, a total of 9,282 hcws in the u.s. were confirmed to have covid-19, as reported to the centers for disease control (cdc). 11 in the setting of widespread community transmission, hcws are at risk for community acquisition as well as potential healthcare-acquired infection, making it difficult to discern their route of exposure. multiple factors have been reported to contribute to the risk of infections in hcws, including lack of awareness during the early weeks of the outbreak, inadequate personal protective equipment (ppe) supply and training, insufficient rapid diagnostic testing for covid-19, long work hours in high-risk environments, and ongoing community spread and household exposures. [12] [13] [14] a c c e p t e d m a n u s c r i p t early and high-throughput testing for sars-cov-2 among symptomatic employees is essential to prevent nosocomial transmission of covid-19 to patients, minimize clusters among hcws, and maintain staffing during the pandemic. 15 to that end, on march 6, 2020, we implemented a drive-through testing center for employees across the university of washington (uw) medicine healthcare system. on march 14, 2020, a second testing center opened to increase testing capacity. data on sars-cov-2 infections among hcws in the u.s. and associated strategies to optimize their safety are urgently needed in order to prepare healthcare systems, assess the efficacy of infection prevention policies, and better understand the risk of covid-19 transmission to hcws. 16 here we describe the approach to establishing high-throughput employee testing centers, the prevalence of infections among symptomatic frontline versus non-frontline staff, and clinical outcomes associated with covid-19 in these employees. a questionnaire was created to collect and manage data through research electronic data capture (redcap), a secure web-based software platform hosted at uw. 17 the questionnaire served as the main portal for employees to request testing and was posted on a university website on march 12, 2020. all employees who requested testing had to complete this survey. employees were eligible for testing if they had any new symptoms concerning for sars-cov-2 infection, including but not limited to fever, cough, shortness of breath, sore throat, fatigue, headache, anosmia, muscle aches, and diarrhea (supplementary materials). in addition to a symptom screen, basic demographic variables, and primary site of work, employees were asked whether they had face-to-face contact with patients, which was used to prioritize testing and determine if they were frontline hcws. employees were asked if they lived communally, which was not further defined. asymptomatic employees were not offered testing at the testing center outside of outbreak investigations. staff were advised to remain home from work while awaiting test results. in accordance with cdc and wa department of health guidelines at the time, uw medicine sick policy required employees infected with covid-19 to remain at home for at least 7 days from symptom onset and until they were asymptomatic for 72 hours, whichever was longer. repeat testing was not required or routinely performed before returning to work. employees who tested negative for sars-cov-2 were permitted to return to work after 24 hours of complete symptom resolution. a c c e p t e d m a n u s c r i p t at this institution, ppe protocols for acute care patients with confirmed or suspected covid-19 consist of standard/droplet/contact precautions (surgical mask, eye protection, gown, and gloves) while patients requiring icu level care are placed in standard/airborne/contact precautions (powered air purifying or n95 respirator, eye protection, gown, and gloves) due to the potential for frequent aerosol-generating procedures. all staff members donning and doffing ppe when caring for these patients are monitored by a trained observer to minimize breaches in precautions and hcw self-contamination. an optional extended use masking policy for staff was implemented on april 1, 2020, and universal masking was implemented on april 27, 2020. staff at each testing site were trained to perform nasopharyngeal swabs. a flexible or standard synthetic fiber nasopharyngeal swab was inserted into the nostril for 2-3 seconds and rotated 360 degrees for 10-15 seconds. this technique was repeated in the contralateral nostril using the same swab. the swab was then placed into a sterile vial containing universal transport media; samples were either taken to the laboratory within the hour or refrigerated until they could be transported for processing. pcr testing was performed at the university of washington virology lab as previously described. 18, 19 inconclusive results were initially reflexed to the wa state department of health laboratory for confirmatory testing until march 30th, after which inconclusive results were a c c e p t e d m a n u s c r i p t interpreted presumptively as positive and were no longer sent for confirmatory testing. during the study period, the turnaround time for laboratory results ranged from 6 to 10 hours. employees were able to access their results through a secure electronic medical record portal or through a quick response (qr) code reader. in addition, all employees were contacted by phone to notify them of their test result. employees with a positive or inconclusive result received a second phone call from trained healthcare providers who asked questions regarding infection prevention practices and gathered data on whether any exposures occurred at work or in the community. another follow-up call was made after 14 days to all positive employees to ascertain whether the employee required hospitalization due to covid-19, what level of hospital acuity had been required, and to assess the duration of their leave before returning to work. up to three telephone call attempts were made for each employee, followed by an e-mail. all symptomatic employees who self-initiated and completed testing from march 12 to april 23 were included in the analysis. frontline hcws were defined as those who answered "yes" to whether they had face-to-face contact with patients. the primary outcome of interest was the prevalence of sars-cov-2 infection among symptomatic employees seeking testing, examined across time in an epidemic curve, stratified by frontline hcws versus non-frontline employees, and with 95% confidence intervals (ci) for these rates. at one campus, asymptomatic screening of exposed staff was performed as part of three specific outbreak investigations during which there was concern for ongoing staff-tostaff transmission on the unit. asymptomatic staff tested in the context of these clusters were a c c e p t e d m a n u s c r i p t not included in the primary analysis, as much of this evaluation was performed on the hospital unit rather than in the employee testing center. a secondary analysis was performed to calculate the prevalence of infection with a 95% ci of all hcws, including those who underwent asymptomatic testing, to help estimate the impact of these investigations on the overall employee burden of covid-19. for the summary of baseline characteristics, symptoms, and clinical conditions, continuous variables were displayed as median values with simple ranges. categorical variables were summarized as counts of all patients or a subset of evaluated patients with percentages. chisquare testing was done as appropriate for comparison of features between positive and negative cases on a selected rather than wholesale basis to reduce the risk of false discovery rate. the proportion hospitalized (and 95% ci) was calculated as the number of employees reporting hospitalization among all who tested positive and responded to post-testing assessment. all analyses were conducted using r version 3.6.3. this study was approved by the uw institutional review board (irb). informed consent was waived for retrospective review of deidentified employee data. a total of 3,477 symptomatic employees were tested for covid-19 from march 12, 2020 to april 23, 2020 with an average of 83 employees tested daily across both sites. employees were scheduled in 5-minute intervals, and median turn-around time from survey submission to scheduled appointment time was 11 hours. during the study period, 185 staff members tested a c c e p t e d m a n u s c r i p t positive for covid-19, yielding an overall prevalence of 5.3% (95% ci 4.41, 6.07). the cumulative incidence of positive sars-cov-2 tests increased over time as the epidemic spread in the seattle area ( figure 1 ). the median age of positive and negative employees was 40 and 39, respectively (table 1) table 2 . positive cases were more likely to report fever (p <0.001), myalgias/arthralgias (p <0.001), and anosmia (p <0.001) than those who tested negative. additionally, though we intended to test only symptomatic individuals, a small number of asymptomatic employees were able to get tested. out of 185 positive employees, 5 reported none of the symptoms listed in table 2 . among all symptomatic employees, 2309 (66.4%) were identified as frontline hcws. demographic characteristics for frontline hcws were similar to those of all employees (supplementary materials). the prevalence of sars-cov-2 infection among frontline and nonfrontline staff was 5.2% (95% ci 4.33, 6.15) and 5.5% (95% ci 4.17, 6.78), and epidemiologic a c c e p t e d m a n u s c r i p t curves were similar for these two groups over time (figure 1 ). among staff who underwent asymptomatic screening as part of outbreak investigations, 9 of 151 (6.0%) tested positive. secondary analysis combining these staff with all symptomatic employees revealed a sars-cov-2 prevalence among frontline hcws of 5.3% (95% ci 4.42, 6.21) and among all employees, 5.3% (95% ci 4.45, 6.08). on follow-up assessment after at least 14 days from covid-19 diagnosis, we were able to contact 174 (94.1%) of 185 employees who tested positive, and six (3.2%) reported hospitalization related to covid-19 (95% ci 0.01, 0.07). of these 6 employees, the median age was 49.5 years; 3 identified as male, and 3 identified as female. one employee required icu admission; all employees recovered and were discharged from the hospital. of the 174 employees reached for follow-up phone call, 151 had already returned to work, and the median duration of their leave was 14 days (iqr 8.5). rapid access to sars-cov-2 testing is crucial for symptomatic healthcare workers, both to confirm the diagnosis and to ensure a safe and timely return to work. sick policies and testing strategies for hcws are essential pillars of infection prevention and control efforts to prevent nosocomial transmission of infection and to limit critical staff shortages during a time of unprecedented need. accessible testing also serves to strengthen employee trust in their workplace and reduce fear and anxiety surrounding contracting the virus or transmitting to patients and family members 20 . supporting the mental health and emotional well-being of hcws during any pandemic is key to preserving workforce morale, confidence, and availability. 21 m a n u s c r i p t we successfully implemented high throughput drive-through and walk-through employee testing for covid-19 at a large multi-hospital academic medical center that employs approximately 26,000 individuals. during the study period, we found that 5.3% of symptomatic employees were positive for covid-19, compared to 10.3% of patients (n = 17,681) tested for sars-cov-2 within the uw medicine system during the study period. this difference may be attributable to a relatively lower threshold to perform sars-cov-2 tests in hcws, which may have led to higher sampling of minimally symptomatic workers compared to the general public, particularly in the earlier weeks of the epidemic when testing was limited and individuals in the community with mild disease were not yet eligible for testing. notably, there was no significant difference in prevalence of infection between frontline hcws and non-frontline staff. we suspect that early in the local epidemic, community transmission played a significant role in illness among hcws. determination of healthcare-versus community-acquired infection fell outside the scope of this study. our cohort was predominantly young and healthy, which is consistent with national data on hcws with covid-19. 11 we observed a wide range of presenting symptoms among employees. a similar range of symptoms was reported among a smaller cohort of positive hcws in king county, wa, emphasizing the importance of the recently expanded symptom screening criteria beyond fever, cough, and shortness of breath. 22 although this is a large representative sample, our study had limitations. the definition we use for frontline healthcare worker is broad. while a wide range of staff might indicate having "faceto-face contact" with patients, individual risk for exposure may markedly differ. second, as testing was selectively restricted to employees who had symptoms, the proportion of positive tests reported here may not reflect the true prevalence of infection within the overall employee a c c e p t e d m a n u s c r i p t population. testing criteria expanded over the course of the study as laboratory capacity increased, with high priority initially given to employees with more severe symptoms. theoretically, this could have enriched our prevalence estimates during the first week of our study, however, this effect was not seen. anosmia was added as a screening symptom later in the study period, and estimates related to this symptom should be interpreted carefully. in addition, we cannot ascertain whether infection was acquired in the community or in the healthcare setting. statewide measures including stay-at-home orders had concurrent impacts on community transmission and overall incidence in our state. as washington residents increasingly sheltered in place, the study population may not have remained consistent throughout the study period. for example, uw medicine staff with office-based jobs began to telecommute; similarly, clinical rotations were cancelled for all medical students during this time. in contrast, frontline hcws continued to come to work, placing them at ongoing risk for community as well as nosocomial transmission. last, the actual prevalence of sars-cov-2 infection may be higher than reported here, as these data do not include routine screening in asymptomatic or presymptomatic hcws. while 9 staff were identified as positive following asymptomatic unitbased testing in the context of outbreak investigations, we do not know what proportion of these went on to develop symptoms and would have eventually been identified through traditional symptom screening. future studies using serologic tests may be useful to understand the true prevalence in this population. we present a representative sample of 3,477 symptomatic employees of a large healthcare system who underwent nasopharyngeal testing for sars-cov-2 and observed a prevalence m a n u s c r i p t of 5.3% over the first several weeks of the epidemic, compared with 10.3% of all patients tested within the uw medicine system during the same time period. among 185 positive employees, six reported hospitalization. rapid and high throughput testing of hcws for covid-19 is feasible using drive-through and walk-through testing clinic models and facilitated the rapid return of sars-cov-2 negative hcws to work. m a n u s c r i p t we greatly appreciate the time and effort of volunteers within the division of allergy and infectious diseases at the university of washington. we would also like to recognize the dedication of healthcare workers, first responders, and frontline employees in the fight against covid-19. world health organization. who director-general's opening remarks at the media briefing on covid-19 first case of 2019 novel coronavirus in the united states novel coronavirus outbreak (covid-19) world health organization. summary of probable sars cases with onset of illness from 1 protecting healthcare workers during the coronavirus disease 2019 (covid-19) outbreak: lessons from taiwan's severe acute respiratory syndrome response taiwan's traffic control bundle and the elimination of nosocomial severe acute respiratory syndrome among healthcare workers doctors and nurses fighting coronavirus in china die of both infection and fatigue italian doctors call for protecting healthcare workers and boosting community surveillance during covid-19 outbreak death from covid-19 of 23 health care workers in china epidemiology of and risk factors for coronavirus infection in healthcare workers: a living rapid review characteristics of health care personnel with covid-19 -united states protecting chinese healthcare workers while combating the 2019 novel coronavirus reasons for healthcare workers becoming infected with novel coronavirus disease 2019 (covid-19) in china risk factors of healthcare workers with corona virus disease 2019: a retrospective cohort study in a designated hospital of wuhan in china states lack key data on virus cases among medical workers research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support covid-19 in critically ill patients in the seattle region -case series comparison of commercially available and laboratory developed assays for in vitro detection of sars-cov-2 in clinical laboratories understanding and addressing sources of anxiety among health care professionals during the covid-19 pandemic supporting the health care workforce during the covid-19 symptom screening at illness onset of health care personnel with sars-cov-2 infection in king county seattle area used early social distancing, testing, to help begin flattening the coronavirus curve washington state governor's office. inslee announces statewide school closures, expansion of limits on large gatherings a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-325068-j1lfq60o authors: pene, frédéric; merlat, annabelle; vabret, astrid; rozenberg, flore; buzyn, agnès; dreyfus, françois; cariou, alain; freymuth, françois; lebon, pierre title: coronavirus 229e-related pneumonia in immunocompromised patients date: 2003-10-01 journal: clin infect dis doi: 10.1086/377612 sha: doc_id: 325068 cord_uid: j1lfq60o coronaviruses strains 229e and oc43 have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. here we report 2 well-documented cases of pneumonia related to coronavirus 229e, each with a different clinical presentation. diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase—polymerase chain reaction. on the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients. pulmonary complications occur frequently in immunocompromised patients who are treated for hematological malignancies, and they cover a wide range of etiologies, including infections of bacterial, fungal, parasitic, or viral origin; pulmonary edema; diffuse alveolar hemorrhage; drug-or radiation-induced toxicity; graft-versus-host disease; and bronchiolitis obliterans [1] . in addition, an entity named "idiopathic pneumonia syndrome," defined as diffuse lung injury for which the etiology is not identified, is frequently recognized after hematopoietic stem cell transplantation (hsct). a large number of these cases of pneumonia may be related to unrecognized viral infections, and, in the absence of routine screening or reliable diagnostic procedures, this number is probably an underestimate. coronaviruses are positive-sense, single-stranded rna viruses whose particles are irregularly shaped. the outer envelope carries distinctive club-shaped peplomers, giv-ing a crown-like appearance. two coronavirus strains, oc43 and 229e, are known to be involved in human diseases, mainly in the common cold syndrome, but also in pneumonia [2] [3] [4] [5] . coronaviruses recently became a subject of particular interest because a novel strain was identified as the primary agent associated with the worldwide outbreak of severe acute respiratory syndrome (sars) [6] . we report 2 well-documented cases of coronavirus-related pneumonia in immunocompromised patients who were treated for hematological malignancies. our diagnostic strategy combined viral culture of huh7 cells and rt-pcr controlled by hybridization with strain-specific probes. a 33-year-old white man was admitted to the hospital with fever, weight loss, and continuous nonproductive cough. three years previously, this patient was treated for stage ii large b cell non-hodgkin lymphoma, according to the french lnh93 protocol [7] . because of disease progression, he underwent highdose chemotherapy intensification supported by autologous hsct. long-term, complete remission was achieved. relapse occurred 2 years after transplantation and was treated with high-dose sequential salvage therapy combining etoposide, ifosfamide, mitoxantrone, and cytarabine. ten days after the second cycle of chemotherapy, disseminated cutaneous vesicles appeared, suggesting varicella zoster virus (vzv)-associated eruptions. no respiratory symptoms or abnormal chest radiograph findings were present. the patient was successfully treated with intravenous acyclovir (10 mg/kg every 8 h for 21 days). after completion of acyclovir treatment, the patient developed a febrile nonproductive cough without dyspnea. a chest radiograph showed bilateral interstitial syndrome, and this was confirmed by a ct scan, which showed disseminated micronodular opacities. arterial blood gas values while breathing in room air were normal. other laboratory findings were as follows: wbc count, 2100 cells/mm 3 , with 1600 polymorphonuclear cells and 500 lymphocytes; platelet count, 20,000 platelets/mm 3 . liver enzyme levels were normal. sputum samples were negative for legionella species when subjected to direct immunofluorescence, and bacterial culture of the sputum samples showed no growth. results of tests of serum samples for aspergillus galactomannan antigen were also negative. following failure of empirical antibiotic treatment (piperacillin and ciprofloxacin), fiber-optic bronchoalveolar lavage (bal) was performed in the middle lobe. cytological analysis of specimens of the bal fluid showed 290 nucleated cells/mm 3 , with 18% polymorphonuclear cells, 40% macrophages, and 42% lymphocytes (identified as t-cell lymphocytes by flow cytometry analysis [37% cd4 + cells and 50% cd8 + cells]). the findings of extensive microbiological direct examinations and cultures remained negative for bacteria; mycobacteria; fungi; and parasites, such as pneumocystis carinii. the results of immunofluorescent assays for multiple respiratory viruses (respiratory syncitial virus [rsv]; parainfluenza viruses i, ii, and iii; adenovirus; and influenzae viruses a and b) and herpes viruses (herpes simplex virus [hsv], vzv), and immunoperoxydase staining for cytomegalovirus (cmv) were negative in specimens of bal fluid. the result of a pcr test performed on specimens of bal fluid with vzv-specific primers was also negative [8] . viral cultures showed no growth in human amniotic cells or in mrc5 and vero cell lines. in contrast, a cytopathic effect was observed in the human hepatoma huh7 cell line cultured with specimens of the bal fluid, which was unrecognized with influenza and parainfluenza viruses, rsv, adenovirus, measles, enterovirus, or vzv-specific antibodies. electron microscopy findings identified corona-like particles in the culture supernatant medium ( figure 1 ). an rt-pcr test performed with infected and noninfected huh7 cells and using strain-specific primers located in the m gene of human coronaviruses confirmed the diagnosis and typed the virus as 229e strain, but not as oc43 strain (figure 2). rt-pcr products were also characterized as sequences of 229e virus by hybridization with a specific probe in a dna enzyme immunoassay [9] . the presence of coronavirus rna in bal fluid specimens was confirmed by amplification of 229e sequences, whereas the results of rt-pcr performed with oligonucleotide primers derived from the m gene sequence of the oc43 strain remained negative. the results of cytopathic effect seroneutralization testing showed a low initial antibody response but a significant difference between the antibody response of preinfection serum samples (titer, !10) and that of postinfection serum samples (titer, 40). all serological studies for respiratory viruses (adenovirus, myxovirus, paramyxovirus, and rsv), cmv, vzv, epstein-barr virus, hsv, and mycoplasma species revealed no significant increase in serum sample antibody titer, and the results of serological testing for chlamydiae bacteria and aspergillus remained negative. the outcome was spontaneously favorable, and the fever disappeared in 5 days without modification of the antibiotic regimen, which was consistent with an improvement of the chest radiograph findings. case study 2. a 16-year-old female patient was admitted to the intensive care unit (icu) for acute respiratory failure 5 days after allogeneic bone marrow transplantation. she had been treated with mechlorethamine, vincristine, procarbazine, and prednisone (mopp) chemotherapy combination 3 years previously for hodgkin lymphoma with cervical and mediastinal lymphadenopathies. a first relapse with pulmonary and gastric involvement was treated with high-dose chemotherapy intensification (with melphalan, misulban, and cytarabine) supported by autologous hsct. because of a second relapse with pulmonary and gastric involvement, the patient underwent allogeneic bone marrow transplantation with a conditioning regimen combining high-dose cyclophosphamide and fludarabine. after initiation of the conditioning regimen, she presented with bilateral knee arthritis. samples of the joint fluid tested positive for aspergillus fumigatus, and the patient was treated with amphotericin b (1 mg/kg per day). four days after transplantation, rapidly progressive respiratory failure developed, leading to hospitalization in the icu. at admission, physical examination revealed acute respiratory distress with tachypnea (30 breaths/min), bilateral crackles, and oxygen saturation of 98% while breathing with a highconcentration oxygen mask, and shock (heart rate, 140 beats/ min; blood pressure, 105/74 mm hg) with disseminated mottling. arterial blood gases obtained while breathing oxygen (with a high-concentration oxygen mask at a rate of 12 l/min) were as follows: ph, 7.48; partial pressure of carbon dioxide, 3.15 kpa; partial pressure of oxygen, 19.4 kpa; bicarbonates, 16.9 mmol/l. laboratory findings revealed pancytopenia (wbc count, 200 cells/mm 3 ; hemoglobin, 8.5 g/dl; platelet count, 18,000 platelets/mm 3 ), acute renal failure (creatinine level, 177 mmol/l), and inflammatory syndrome (c-reactive protein, 207 mg/l; fibrinogen, 5.77 g/l). a chest radiograph revealed disseminated alveolar and interstitial opacities. the patient's condition deteriorated rapidly, requiring endotracheal intubation and mechanical ventilation. results of a test performed on a tracheobronchial secretion sample were positive for a. fumigatus, leading to treatment with a combination therapy of voriconazole and caspofungin. after an initial improvement in clinical characteristics and radiological findings and bone marrow recovery, the patient's respiratory condition worsened, and bilateral alveolar opacities appeared on a chest radiograph. the results of blood tests for aspergillus galactomannan and cmv pp65 antigens remained negative. specimens obtained by bal performed in the lingular region were hemorrhagic, and the findings of a quantitative cytological examination were noncontributive, because of numerous lysed cells. the findings of extensive research to detect the presence of microorganisms (bacteria, mycobacteria, fungi, parasites, cmv, and rsv) were negative. despite aggressive supportive care with mechanical ventilation, fluid resuscitation, and high-dose norepinephrine infusion, refractory hypoxia rapidly led to fatal multiorgan failure. no autopsy was performed. cultures of bal fluid specimens remained negative for bacteria, mycobacteria, and fungi. the same procedures for viral culture and rt-pcr were applied as in case study 1, described above. the results of inoculation tests performed with huh7 cells were also positive, revealing corona-like particles that were subsequently identified as coronavirus 229e by rt-pcr performed on both culture supernatant and bal fluid specimens. in both cases, 2 major facts led to the diagnosis of coronavirus 229e-associated pneumonia: first, the exclusion of alternative infectious etiologies, and, second, the identification of coronavirus 229e in bal fluid samples by a combination of culture, electron microscopy, and rt-pcr. in the case of the first patient, the main differential diagnosis was vzv-associated pneumonia, which is the most frequent complication of varicella in adults and is usually concomitant to cutaneous vesicles. however, respiratory symptoms only appeared after completion of antiviral treatment and improvement of skin eruptions, and both viral culture and pcr for vzv performed on bal fluid specimens were negative. coronavirus seroconversion retrospectively confirmed the diagnosis. the second patient presented with severe acute respiratory failure that led to fatal multiorgan failure, despite maximal life support. ventilator-associated pneumonia is a frequent complication in patients receiving mechanical ventilation, and it is associated with a high mortality rate, especially among bone marrow recipients. bacterial etiology is largely predominant in ventilator-associated pneumonia, but cmv has been identified as a possible cause in adult patients, and outbreaks of coronavirus-and adenovirus-related pneumonia in pediatric icus have been described [10] [11] [12] . in the case we describe, though negative results of bacteriological cultures cannot exclude a bacterial origin, a secondary identification of coronavirus in bal fluid specimens provided a high probability that a diagnosis of viral origin was accurate. various methods to detect coronaviruses exist, but they lack sensitivity or specificity when used for routine screening of respiratory samples. these methods include identification of corona-like particles, immunofluorescent assays with monoclonal antibodies, and rna hybridization in fluid specimens from nasal washing or bal [13] [14] [15] . detection of seroconversion, based on elisa findings, may be useful, but it does not allow rapid virus-identification and only provides a retrospective diagnosis [16] . we identified coronavirus in both patients because the bal specimens were cultured in the human hepatoma huh7 cell line, which expresses a specific receptor for coronaviruses [17] . recent advances in virological detection methods, such as molecular amplification techniques, offer promising perspectives for detection of coronaviruses [18] . indeed, improved sensitivity and specificity have been obtained with molecular detection methods combining rt-pcr with strain-specific primers derived from the m protein gene, followed by molecular hybridization with specific probes recognizing either coronavirus 229e or oc43 [9] . inversely, the high sensitivity of amplification methods may lead to false-positive results caused by contamination of bal fluid specimens with pharyngeal viruses. therefore, the combination of viral culture and rt-pcr of bal fluid samples appears to be an efficient and reliable diagnostic strategy. two coronavirus strains, 229e and oc43, have previously been related to human diseases with various clinical syndromes, ranging from self-resolving common cold to pneumonia [2] [3] [4] [5] . recently, the worldwide outbreak of sars was linked to a novel coronavirus that had not been previously identified in human beings or animals [6] . the clinical features of sars combine flu-like symptoms, dry cough, and shortness of breath, associated with pulmonary infiltrates visible by chest radiography. in contrast to the common benign symptoms of coronavirus infections in healthy individuals, a large proportion of patients with sars have severe respiratory failure requiring ventilatory support in the icu. the impact of a respiratory virus on individuals is largely determined by their underlying conditions, and particularly by whether they are experiencing immunosuppression [19, 20] . the prevalence of coronavirus pulmonary infections among immunocompromised patients is unknown, and it is probably largely underestimated in the absence of the routine performance of sensitive cell culture, rt-pcr, or electron microscopy on bal fluid specimens. however, rt-pcr results were negative for coronaviruses in the bal samples of 46 hsct recipients with acute pulmonary infiltrates [21] . thus, only 1 case of coronavirus-associated pneumonia was previously described in an immunocompromised patient following autologous bone marrow transplantation, with the diagnosis based on the presence of viral particles in bal fluid specimens [22] . the identification of coronavirus in high-risk immunocompromised patients may lead to early adoption of a specific therapeutic strategy, but, in the absence of proof of the efficacy of antiviral drugs, the treatment remains only symptomatic. in these circumstances, different types of ifns that display antiviral properties against coronaviruses may be evaluated [23, 24] . pulmonary complications of bone marrow transplantation coronavirus infections in working adults. eight-year study with 229 e and oc 43 coronavirus infections in military recruits. three-year study with coronavirus strains oc43 and 229e viruses and bacteria in the etiology of the common cold an outbreak of coronavirus oc43 respiratory infection in normandy, france coronavirus as a possible cause of severe acute respiratory syndrome survival benefit of highdose therapy in poor-risk aggressive non-hodgkin's lymphoma: final analysis of the prospective lnh87-2 protocol-a groupe d'etude des lymphomes de l'adulte study detection of varicella-zoster virus dna by polymerase chain reaction in the cerebrospinal fluid of patients suffering from neurological complications associated with chicken pox or herpes zoster direct diagnosis of human respiratory coronaviruses 229e and oc43 by the polymerase chain reaction cytomegalovirus: an unexpected cause of ventilator-associated pneumonia coronavirusrelated nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study nosocomial adenovirus infection in a paediatric respiratory unit electron microscopic studies of coronavirus diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalized children detection of human coronavirus 229e in nasal washings using rna:rna hybridisation occurrence and frequency of coronavirus infections in humans as determined by enzyme-linked immunosorbent assay mouse hepatitis virus strain jhm infects a human hepatocellular carcinoma cell line comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses 229e and oc43 in cell culture rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients impact of respiratory virus infections on persons with chronic underlying conditions rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia coronavirus pneumonia following autologous bone marrow transplantation for breast cancer the efficacy and tolerance of intranasal interferons: studies at the common cold unit the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm key: cord-313499-8ijbesl8 authors: stowell, sean; guarner, jeannette title: role of serology in the covid-19 pandemic date: 2020-05-01 journal: clin infect dis doi: 10.1093/cid/ciaa510 sha: doc_id: 313499 cord_uid: 8ijbesl8 nan in this issue of clinical infections diseases, f xiang et al present a serologic study of 85 nucleic acid test (nat) sars-cov-2 positive patients and 24 nat negative patients who had symptoms suspicious for covid-19 1 . sixty controls were also evaluated and included healthy healthcare staff and patients with a variety of diseases from bacterial pneumonia to lung cancer. serological testing consisted of an enzyme linked immunosorbent assay designed to detect igm and igg antibodies against the n protein of sars-cov-2. serial examination of covid-19 patients resulted in igm seroreactivity by day 4 post symptom onset, which peaked by day 9. in contrast, igg sharply increased 12 days after symptom onset; all nat positive patients were positive for igg 30 days post symptom onset. in patients with suspected covid-19 who tested nat negative, igm antibodies were detected in 87.5% and igg was present in 70.8%. in contrast, only 3 individuals in the control group tested positive for igg but igm positivity was not observed. the authors calculated a sensitivity for diagnosis of covid-19 using igm of 77.3% and a specificity of 100% while for igg the sensitivity was higher at 83.3% and the specificity was 95%. this article highlights a fundamental challenge clinicians face when patients present with covid-19 symptoms yet are nat negative, sometimes even with repeated nat testing. as laboratory medicine physicians, we understand this dilemma and know that preanalytical variables can frequently underlie inaccurate results for all tests. in the case of covid-19 nat, preanalytical variables can include, but are not limited to, inconsistency in obtaining nasopharyngeal swabs, the different swabs and transport media used, time and temperature of specimen transport, and possible inhibitors to nucleic acid detection in the sample. in addition, there are likely analytical variables that can be explained by inherent differences in the ability of various nat platforms to detect sars-cov-2. importantly, a lack of understanding regarding the frequency and variability of viral shedding in patients with covid-19 also makes it difficult to decipher how these variables may also contribute to false negative results. although nat diagnostic testing continues to become a common approach to diagnosing infectious diseases in general, serology preceded nat testing by many decades and still serves as the primary diagnostic tool for many diseases, including hiv, hepatitis b and c. this is in part due to cost and because nat testing remains less sensitive for some pathogens, as occurs in syphilis. different strategies are used for diagnosis by serological methods, for example, in leptospirosis a fourfold increase in igg in convalescent versus acute serum frequently helps determine the diagnosis. in contrast, to diagnose dengue or american trypanosomiasis, nat is most likely to be informative during acute disease while serology is helpful at later stages. in the case of the 2003-04 sars-cov outbreak, serology was primarily an epidemiological tool that could help determine the number of silent infections, how the disease progressed, defining transmission patterns, and the possible origin of the virus itself 2 . in the case of covid-19, while serological analysis may be helpful in examining exposure, such approaches can be more challenging to interpret in patients with acute infection. significant variability in the kinetics and magnitude of the serological response, especially early in infection, could contribute to false negative results. in addition, while both nat and serological assays can suffer from inadequate analytical sensitivity, a broader range of factors can influence the sensitivity and specificity of serological tests, including a variety of platform design considerations such as the antigen coating density, dilution matrix considerations and the actual serum dilution employed. challenges with false positives can also limit the value of serological tests. cross-reactivity with other strains of coronavirus 2 , in addition to other pathogens altogether, can limit their accuracy. consistent with this, during the 2003-04 sars-cov outbreak, various serological assays demonstrated cross-reactivity in assays that used whole virus in addition to the n protein. as a result, false positive serological results, which can be particularly challenging with igm antibodies, can make it difficult to accurately interpret findings. despite these considerations, the results of this manuscript suggest repeat serological testing may be informative in the acute setting, especially in patients who continue to follow a covid-19-like disease course despite nat negativity. the blood donation industry has used the approach of coupling the potential limitations and strengths of nat and serological assays to detect pathogens in blood. a similar approach could increase the overall likelihood of identifying covid-19 infected patients as shown by f xiang et al 1 . while the results presented in this study provide some insight into how to possibly incorporate serological testing into the workup of acutely ill patients, especially if nat test results are negative, how serology will be employed in screening asymptomatic individuals or patients who never underwent nat testing during acute infection still remains unclear. for example, if someone is found to be serologically positive, should a follow up nat test be performed to determine if the individual is shedding virus? furthermore, the extent to which covid-19 seroreactivity reflects actual immunity also represents an outstanding question. while established seroreactive corollaries with actual immunity exist, such as antibodies against hepatitis b surface antigen, these were developed over long periods of time. furthermore, which populations would most benefit from covid-19 serological testing remains unknown. several populations may be good candidates: 1. patients that have had covid-19 compatible symptoms but have been nat negative (as outlined in the present study 1 ). 2. populations in general to define the degree of community exposure which may help define the r null and mortality rate. 3. frontline healthcare workers to likewise define exposure. additional testing, such as nat testing, may be warranted in serological positive individuals to determine potential risk for infecting others. it is important to reiterate that it is unknown the extent to which antibody positivity translates to actual immunity. 4. convalescent patients that want to donate plasma for patients suffering from infection. further testing to define optimal titer and overall neutralizing activity will be needed. in short, the article of f xiang et al. is a first step to better understand the antibody response against sars-cov-2 and in so doing provides important insight into the possible characteristics and use of serological tests in this ongoing pandemic. however, as other serological tests are developed, the analytical parameters of each of these tests will likely differ, making it difficult to fully extrapolate these findings to additional serological tests as they become available. additional studies will therefore be needed to fully characterize serological platforms that are rapidly becoming available, both with respect to their analytical parameters and the role they may play in diagnosing and screening individuals for covid-19. however, regardless of these considerations, this study provides important insight into the seroreactivity of patients with covid-19 in general that will surely guide additional test development and use of serological testing moving forward. neither author has any potential conflicts to disclose. antibody detection and dynamic characteristics in patients with covid-19 serological assays for emerging coronaviruses: challenges and pitfalls key: cord-296588-q2716lda authors: hanson, kimberly e; caliendo, angela m; arias, cesar a; englund, janet a; lee, mark j; loeb, mark; patel, robin; el alayli, abdallah; kalot, mohamad a; falck-ytter, yngve; lavergne, valery; morgan, rebecca l; murad, m hassan; sultan, shahnaz; bhimraj, adarsh; mustafa, reem a title: infectious diseases society of america guidelines on the diagnosis of covid-19 date: 2020-06-16 journal: clin infect dis doi: 10.1093/cid/ciaa760 sha: doc_id: 296588 cord_uid: q2716lda background: accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (covid-19). direct detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) nucleic acids in respiratory tract specimens informs patient, healthcare institution and public health level decision-making. the numbers of available sars-cov-2 nucleic acid detection tests are rapidly increasing, as is the covid-19 diagnostic literature. thus, the infectious diseases society of america (idsa) recognized a significant need for frequently updated systematic reviews of the literature to inform evidence-based best practice guidance. objective: the idsa’s goal was to develop an evidence-based diagnostic guideline to assists clinicians, clinical laboratorians, patients and policymakers in decisions related to the optimal use of sars-cov-2 nucleic acid amplification tests. in addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss the nuance of test result interpretation in a variety of practice settings, and highlight important unmet research needs in the covid-19 diagnostic testing space. methods: idsa convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of sars-cov-2 molecular diagnostics. grading of recommendations assessment, development and evaluation (grade) methodology was used to assess the certainty of evidence and make testing recommendations. results: the panel agreed on 15 diagnostic recommendations. conclusions: universal access to accurate sars-cov-2 nucleic acid testing is critical for patient care, hospital infection prevention and the public response to the covid-19 pandemic. information on the clinical performance of available tests is rapidly emerging, but the quality of evidence of the current literature is considered low to very low. recognizing these limitations, the idsa panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having covid-19. in addition, testing is recommended for asymptomatic individuals with known or suspected contact with a covid-19 case. testing asymptomatic individuals without known exposure is suggested when the results will impact isolation/quarantine/personal protective equipment (ppe) usage decisions, dictate eligibility for surgery, or inform administration of immunosuppressive therapy. ultimately, prioritization of testing will depend on institutional-specific resources and the needs of different patient populations. a c c e p t e d m a n u s c r i p t background in late december 2019, an outbreak of pneumonia cases of unclear etiology was reported in wuhan city, hubei province, china [1] . unbiased next generation sequencing (ngs) using lower respiratory tract (lrt) specimens collected from affected patients subsequently identified a novel coronavirus as the cause of illness now known as coronavirus disease 2019 . the entire viral genome was shared online within days and phylogenetic analyses established close relationship to human severe acute respiratory syndrome coronavirus (sars-cov) as well as several other sars-like bat coronaviruses [1, 2] . based on genetic similarities, the novel coronavirus was officially named sars-cov-2 [3] . by march 11 th , 2020, the virus had spread to at least 114 countries and killed more than 4,000 people, prompting the world health organization (who) to officially declare a global pandemic [4] . public availability of the sars-cov-2 genome was an essential first step enabling development of accurate molecular diagnostic assays. nucleic acid amplification tests (naats) designed to detect one or more gene sequences specific to sars-cov-2 are essential for confirming covidto date, multiple commercial test manufacturers and clinical laboratories, including academic medical centers, have received eua for a sars-cov-2-specific molecular diagnostic test. the first home-based test collection kit was also recently granted an eua [5] . it is important to recognize, however, that eua guidance differs substantially from the standard fda approval process. in the setting of a public health emergency, the fda only requires test developers to establish acceptable analytical accuracy. clinical test performance (i.e., sensitivity and specificity) has yet to be determined or comprehensively compared across eua platforms. as a result, most of the naat performance data used to inform this guideline was derived from a c c e p t e d m a n u s c r i p t studies evaluating assays not widely used in the u.s. we assumed, therefore, that performance of standard naat methods to be comparable across countries (which may or may not be correct). given increasing test availability combined with a rapidly growing number of naat-focused studies published online or in academic journals, the infectious diseases society of america table 1 . at the time of this review, there was little evidence to inform use of serologic testing. therefore, the first version of the idsa diagnostic guideline focuses only on the use of targeted naat applied directly respiratory tract specimens. it is anticipated that these guidelines will be frequently updated as substantive new information becomes available; subsequent versions will also address sars-cov-2 serology due to the rapidly evolving information and uncertainty of the reliability of serological tests. m a n u s c r i p t this guideline was developed using the grading of recommendations assessment, development and evaluation (grade) approach for evidence assessment. in addition, given the need for rapid response to an urgent public health crisis, the methodological approach was modified according to the gin/mcmaster checklist for development of rapid recommendations [6] . the the conflict of interest (coi) review group included two representatives from idsa who were responsible for reviewing, evaluating and approving all disclosures. all members of the expert panel complied with the coi process for reviewing and managing conflicts of interest, which required disclosure of any financial, intellectual, or other interest that might be construed as constituting an actual, potential, or apparent conflict, regardless of relevancy to the guideline topic. the assessment of disclosed relationships for possible coi was based on the relative weight of the financial relationship (i.e., monetary amount) and the relevance of the relationship (i.e., the degree to which an association might reasonably be interpreted by an independent observer as related to the topic or recommendation of consideration). the coi review group ensured that the majority of the panel and chair was without potential relevant a c c e p t e d m a n u s c r i p t (related to the topic) conflicts. the chair and all members of the technical team were determined to be unconflicted. clinical questions were developed into a population, intervention, comparison, outcomes (pico) format [7] prior to the first panel meeting (table s1) . idsa panel members prioritized questions with available evidence that met the minimum acceptable criteria (i.e., the body of evidence reported on at least test accuracy results can be applied to the population of interest). panel members prioritized patient-oriented outcomes related to sars-cov-2 testing such as requirement for self-quarantine, eligibility for investigational covid-19 treatment, timing of elective surgery or procedures, and management of immunosuppressive therapy. we also considered the impact of sars-cov-2 results on infection prevention and public health practices, including the use of personal protective equipment (ppe) and contact tracing. the national institute of health and care excellence (nice) and the center of disease control (cdc) highly-sensitive search was reviewed by the methodologist in consultation with the technical team information specialist and was determined to have high sensitivity. an additional term, covid, was added to the search strategy used in addition to the terms identified in the pico questions (table s2) . ovid medline and embase were searched from 2019 through april 20, 2020. horizon scans were performed daily during the evidence assessment and recommendation process to locate additional grey literature and manuscript preprints from the following sources litcovid, medrxiv, ssrn, and trip database. reference lists and literature suggested by panelists were reviewed for inclusion. no restrictions were placed on language or study type. two reviewers independently screened titles and abstracts, as well as eligible full-text studies. we included studies reporting data on diagnostic test accuracy (cohort studies, cross sectional a c c e p t e d m a n u s c r i p t studies and case-control studies). when questions compared the performance of different tests (e.g., different testing or sampling methods) or testing strategies, we included studies that provided direct test accuracy data about both tests in the same population. when these direct studies where lacking, we included studies that assessed a single test and compared its results to a reference standard. we did not limit our inclusion to a specific reference standard due to sparsity of data. we also included studies that assessed the prevalence of covid-19 in different populations. reviewers extracted relevant information into a standardized data extraction form. two reviewers completed data extraction independently and in duplicate. disagreements were resolved by discussion to reach consensus and in consultation with expert clinician scientists. data extracted included general study characteristics (authors, publication year, country, study design), diagnostic index test and reference standard, prevalence of covid-19, and parameters to determine test accuracy (i.e., sensitivity and specificity of the index test). accuracy estimates from individual studies were combined quantitatively (pooled) for each test using openmetaanalyst (http://www.cebm.brown.edu/openmeta/). we had planned to conduct a bivariate analysis for pooling sensitivity and specificity for each of the test comparisons to account for variation within and between studies. however, this was not feasible due to the sparsity of available data and lack of information on specificity in most instances, so we either presented data as a range of the extreme sensitivity and specificity presented in the studies or pooled as proportions to facilitate decision making. we had also planned to use the breslow-day test to measure the percentage of total variation across studies due to heterogeneity (i 2 ) but were not able to do that due to the sparsity of data. forest plots were created for each comparison. to calculate the absolute differences in effects for different testing or sampling strategies, we applied the results of the sensitivity and specificity to a range of plausible prevalence in the population. we then calculated true positives, true negatives, false positives, and false a c c e p t e d m a n u s c r i p t negatives. to determine the prevalence for each question, we considered the published literature in consultation with the clinical experts. in general, for questions addressing symptomatic individuals we considered the following prevalence: 10% which is typically seen in symptomatic outpatients who have not reached a hospital facility [8] [9] [10] ; 40% which is typically seen in patients meeting clinical definition for covid-19 who were hospitalized [11, 12] ; and 80% which is typically seen in patients meeting clinical definition for covid-19 who were admitted to intensive care units. for questions addressing asymptomatic individuals who were exposed to covid-19, we considered that the prevalence may range from 10% to 50% based on household clusters, nursing home outbreak, active surveillance of passengers quarantined on a cruise ship or passengers of repatriation flights, hospital employees with close contact with covid-19 positive patients and customers and employees of a restaurant that had a covid-19 outbreak [13] [14] [15] [16] [17] [18] [19] . for questions addressing asymptomatic individuals, we considered that the prevalence may range from <1% in general population who are not in hotspots to 10% in asymptomatic patients in hotspots [8, 20, 21] . we conducted the risk of bias assessment for diagnostic test accuracy studies using the quality assessment of diagnostic accuracy studies (quadas)-2 revised tool (table s3 ) [22] . grade framework was used to assess overall certainty by evaluating the evidence for each outcome on the following domains: risk of bias, imprecision, inconsistency, indirectness, and publication bias [23, 24] . grade summary of findings tables were developed in gradepro guideline development tool [25] . the panel considered core elements of the grade evidence in the decision process, including a c c e p t e d m a n u s c r i p t as per grade methodology, recommendations are labeled as "strong" or "conditional". the words "we recommend" indicate strong recommendations and "we suggest" indicate conditional recommendations. figure 2 provides the suggested interpretation of strong and weak recommendations for patients, clinicians, and healthcare policymakers. rarely, low certainty evidence may lead to strong recommendations. in those instances, we followed generally recommended approaches by the grade working group, which are outlined in five paradigmatic situations (e.g., avoiding a catastrophic harm) [26] . for recommendations pertaining to good practice statements, appropriate identification and wording choices were followed according to the grade working group [27] . a "good practice statement" represents a message perceived by the guideline panel as necessary to health care practice, that is supported by a large body of indirect evidence difficult to summarize, and indicates that implementing this recommendation would clearly result in large net positive consequences. for recommendations where the comparators are not formally stated, the comparison of interest was implicitly referred to as "not using the test". some recommendations acknowledge the current "knowledge gap" and aim at avoiding premature favorable recommendations for test use and to avoid encouraging the rapid diffusion of potentially inaccurate tests. detailed suggestions about the specific research questions that should be addressed are found in table 2 . a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t committee reviewed and approved the guideline prior to dissemination. regular, frequent screening of the literature will take place to determine the need for revisions based on the likelihood that new data will have an impact on the recommendations. if necessary, the expert panel will be reconvened to discuss potential changes. in addition, future searches will include critical appraisal of the sars-cov-2 serology literature. systematic review and horizon scan of the literature identified 2,909 references of which 23 informed the evidence base for these recommendations (figure s1 ). characteristics of the included studies can be found in table s4 . figure 1 summarizes a testing algorithm for covid-19 diagnosis guidelines. however, testing is not widely available in some areas.  this recommendation does not address testing a combination of specimen types due to lack of evidence.  the panel considered symptomatic patients to have at least one of the most common symptoms compatible with covid-19 ( table 1) . thirteen studies informed this recommendation [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] and they provided varying descriptions of specimen type (supplement c). in an effort to maintain consistency in the analysis of evidence, reported specimen types were grouped into nasopharyngeal (np), mid-turbinate (mt), nasal, throat, or saliva. in studies that did not define collection techniques for "nasal", we assumed it to mean anterior nasal and not deep-nasal or a c c e p t e d m a n u s c r i p t nasopharyngeal. saliva collection methods were also inconsistent. saliva studies incorporating a "coughed-up" sample were excluded from the urti and ili analysis under the assumption that they likely included some mixture of pure saliva and sputum. analyses of "tongue" swabs were also excluded. it is important to note as well, that not all specimens were collected from the same patient at the same time, the time of collection from symptom onset was not provided in all studies and various approaches for establishing sars-cov-2 positivity were used to define positive results (i.e., clinical evaluation, detection different gene targets versus nucleic acid sequencing). a total of 11 reports presented data about test accuracy of a specific sample type(s); eight of these [35, 36, 38-41, 43, 44] provided comparative data for two or more sample collection sites; and three others [33, 37, 45] provided data for one site only. studies with comparative data showed a lower sensitivity for oral sampling in comparison to np, mt, or nasal sampling. summary statistics different specimen type are shown in table 3 . two studies [38, 39] a c c e p t e d m a n u s c r i p t types are false negative results, which could promote unchecked sars-cov-2 transmission. one potential benefit of the alternative methods are the less-invasive nature of nasal, mt and throat swabs or saliva as compared to np sampling. in addition, the ppe requirements for healthcare providers collecting non-cough inducing specimen types may be less. lastly, the non-np sampling is amendable to patient self-collection, which has the potential to further reduce healthcare worker exposure to infectious droplets and possible droplet nuclei. additional considerations: indirect evidence from influenza and respiratory syncytial virus studies suggest that alternative nasal cavity collection sampling methods such as anterior nasal and mt swabs provide comparable sensitivity to np swabs [46] . using np swab collection as the reference method will bias evaluation of the comparator method by definition. saliva is an easily obtained specimen and there is significant recent interest in its use for sars-cov-2 detection. at the time of this literature review we identified a single study assessing true saliva as a specimen type. this is a promising specimen type given the simplicity of collection. the panel anticipates multiple additional studies to follow, which will be included in future guideline updates. the panel considered indirect evidence for nasal swabs and mt swabs from other respiratory viruses in the decision to list these specimen types are preferred over saliva. in addition, saliva is complex matrix and clinical laboratories will need to carefully assess rna stability during specimen transport and the efficiency of nucleic acid extraction using their own specific methods. we did not identify any studies assessing combinations of specimen types. although oropharyngeal swabs or saliva can be utilized for the diagnosis of covid-19, the available evidence combined with indirect evidence from other respiratory viruses suggests that collection of anterior nares, mt, or np swabs has higher sensitivity. at the current time, there is little evidence to support use of oropharyngeal swabs or saliva alone. however, future studies of saliva as a specimen type for sars-co-2 detection are anticipated. a c c e p t e d m a n u s c r i p t evaluation of alternative collection devices and methods are critically needed as we are facing shortages in test collection supplies such as swabs, transport media and ppe. while np swab collection is widely used and the primary specimen type for commercial direct sars-cov-2 test platforms, based on current available evidence, clinical practice, and availability of testing resources, the panel believes there are comparable alternative methods for sampling the nasal passages. clinical laboratories will need to validate use of individual specimen types. future studies of saliva should clearly describe collection methods, specimen transport media and processing requirements. moving forward, it will be critical to standardize these processes. 9 (0 to 9) 0 (0 to 9) 0 (0 to 9) 0 (0 to 9) 18 (0 to 216) 0 (0 to 18) explanations: this table is based on applying the sensitivity and specificity estimates to calculate true and false positives and negatives in a hypothetical population of 1000 individuals. *no studies reported on the specificity of oral and np a. the case-control design leads to a serious study population bias. b. some studies compared two or more of the specimen types, but no studies compared all specimen types in the same patient population. studies reported test accuracy results but did not report on patient-important and population-important outcomes based on the results. c. there is serious unexplained heterogeneity.  appropriate specimen collection and transport to the laboratory is critical. general instructions for swab-based sars-cov2 testing are shown in table 4 . additional resources are available on the idsa website.  a clear, step-by-step protocol needs to be presented to patients attempting self-collection. this could be in the form of a short video or printed pamphlet with illustrations.  the majority of self-collection studies were performed in the presence of a healthcare worker.  the available evidence for nasal and mt swabs as alternatives to healthcare personnel collection is based on assessment of symptomatic patients. data on self-collection in asymptomatic individuals is currently unavailable. a c c e p t e d m a n u s c r i p t  the panel considered symptomatic patients to have at least one of the most common symptoms compatible with covid-19 ( table 1) . this recommendation is based on three cohort studies (supplement d). in the first study, test accuracy results were provided for self-collected non-invasive specimens compared to healthcare-collected np swabs as the standard [39] . for self-collection, participants were provided with instructions and asked to self-collect tongue, nasal, and mt swabs, in that order. tongue samples were collected with a nylon flocked swab. nasal samples were collected with a foam swab bilaterally. mid-turbinate samples were collected with a nylon flocked swab bilaterally. after patient sampling was completed, np samples were collected by a healthcare worker using a polyester tipped swab on a skinny wire. in the second study, patients attending dedicated covid-19 collection clinics were offered the option to first self-collect nasal and throat swabs followed by healthcare provider collection of nasal, throat or oropharyngeal swabs [44] ; concordance of results were presented. the third study compared positivity for supervised oral fluid sampling, supervised self-collected deep nasal swabs, unsupervised oral fluid sampling and provider collected np swabs [43] . in this analysis, any positive test, obtained from any of the reported sampling methods including the index test, was considered to be a true positive. although the study reported the results for "oral fluid", it is likely these samples were mixed with sputum. lastly, the panel considered unpublished data submitted to the fda on home collection, which demonstrated good stability of specimens stored in universal transport media (utm) during transport from homes to laboratories and comparable quantities of virus in self-collected compared to healthcare provider collected swabs. summary statistics for self-collected versus health-care worker collected nasal swabs are shown in table 5 . the studies used to inform the recommendation were small and heterogeneous. sources of heterogeneity included variable swab and transport media types as well as use of unilateral versus bilateral nares self-collection. the timing of collection relative to symptom onset is also important but was not well documented in available data. due to the mentioned concerns with a c c e p t e d m a n u s c r i p t the studies and the lack of direct comparisons between different specimen types in the same patient population, the panel agreed that overall certainty of evidence was low. benefits and harms: the panel placed a high value on avoiding the close exposure of healthcare providers to patient droplets and possible droplet nuclei generated during specimen collection. we assumed that self-collected specimens including anterior nasal swabs, mt swabs and saliva (without cough) would reduce provider exposure and could reduce mask or respirator use. the overall sensitivity of testing when samples were collected by patients was comparable to those collected by healthcare providers. other potential benefits of self-collection include increasing the availability of testing outside the healthcare system and increased patient satisfaction with selfcollection. concerns with self-collection include lack of experience or documentation for actual collection methods by patients; inappropriate sample collection and/or handling could then lead to inaccurate results. although data is limited, both healthcare provider collected, and self-collected nasal or mt swabs appear to result in similar rates of detection of sars-cov-2. self-collection of np swabs is unlikely to be an option as a self-collection method. there are advantages of having multiple strategies to collect clinical specimens, particularly in times of ppe shortages when limiting exposure to healthcare personnel or other patients is important, or when testing in specific populations without access to the healthcare system is required. further comparative studies of self-collected non-invasive specimens (i.e., nasal, mid-turbinate, and throat swabs, as well as saliva) compared with m a n u s c r i p t abbreviations: np = nasopharyngeal; op = oropharyngeal; mt = nasal mid-turbinate; ns = anterior nares swab. ^c autions: do not use calcium alginate swabs or swabs with wooden shafts, which may contain substances that interfere with nucleic acid amplification. rayon swabs may not be compatible with all molecular platforms. clinical laboratories should confirm compatibility of collection devices during assay validation. # pediatrics: swab insertion distance will differ for pediatric patients. swabs with stoppers make estimating distance easier for mt self-collection. two-sided mt sampling not always performed. we identified nine studies that performed both an upper respiratory tract (urt) swab and lower respiratory tract (lrt) sample collection consecutively on the same patient (supplement e). two reported on viral load and did not report on sensitivity [47, 48] . seven studies reported on sensitivity, of which three had a case control design [35, 49, 50] and one reported results per sample and not per patient [51] . the three cohort studies [43, 52, 53] were used to inform the panel's decision-making process. the sample type varied by study and included throat and nasal swabs for urt sampling and sputum and bronchoalveolar lavage (bal) fluid specimens for lrt sampling. summary statistics for urt versus lrt sampling in 3 cohort studies are shown in table 6 . the timing of specimen collection with regards to clinical course was not reported for all these studies and different diagnostic reference standards were a c c e p t e d m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t 0 (0 to 9) 0 (0 to 9) 0 (0 to 6) 0 (0 to 6) explanations: this table is based on applying the sensitivity and specificity estimates to calculate true and false positives and negatives in a hypothetical population of 1000 individuals a. studies reported test accuracy results but did not report on patient-important and population-important outcomes based on the results. b. considering the lower vs upper limit of the sensitivity confidence interval may lead to different clinical decision, and the low number of patients lead to very serious imprecision c. typically seen in symptomatic outpatients who have not reached a hospital facility d. typically seen in patients meeting clinical definition for covid-19 who were hospitalized e. certainty of evidence (coe) m a n u s c r i p t missing data in the studies included timing of specimen collection in relationship to onset of clinical symptoms and specimen type used for testing. additionally, the performance and accuracy of different rapid tests was very inconsistent. given all these issues, the overall certainty of the effect of using rapid tests on patients was very low. the major benefit of a rapid result is the ability to make clinical decisions while the patient is present in a timely manner and implement interventions to protect others. a possible harm of rapid tests is the potential for increased numbers of false negative results, which could lead to missed diagnoses and patients not being isolated when they are indeed infected, if sensitivity is lower than non-rapid tests. diagnostic accuracy should be stratified by duration of symptoms and severity of disease. furthermore, the diagnostic reference standard must be clearly defined. performance characteristics of eua rapid tests, especially those that are clia-waived, should be collected in the field and performed by the non-laboratory staff running the test (which is how they are used in real life). ideally, studies should assess the impact of rapid results on clinical outcomes, such as time to appropriate treatment or therapeutic intervention. individuals who are either known or suspected to have been exposed to covid-19 (conditional recommendation, very low certainty of evidence).  known exposure was defined as direct contact with a laboratory confirmed case of covid suspected exposure was defined as working or residing in a congregate setting (e.g., longterm care, correctional facility, cruise ship, factory, among others) experiencing a covid-19 a c c e p t e d m a n u s c r i p t  the risk of contracting sars-cov-2 may vary under different exposure conditions.  this recommendation assumes the exposed individual was not wearing appropriate ppe.  the decision to test asymptomatic patients will be dependent on the availability of testing resources. summary of the evidence: we did not identify any studies that directly assessed a strategy of testing versus no testing of asymptomatic individuals exposed to sars-cov-2. therefore, the effect of testing on the pre-specified outcomes could not be directly assessed. we also did not identify test accuracy studies directly assessing the performance of sars-cov-2 naats in asymptomatic individuals. however, based on evidence that asymptomatic or pre-symptomatic patients may have similar viral loads and shedding compared to those who are symptomatic [15, 62, 63] , the panel agreed that it is reasonable to apply test accuracy data based on symptomatic patients to the asymptomatic populations. hence, it was essential to determine the pre-test probability or prevalence of covid-19 in the asymptomatic groups. we assessed studies that reported the prevalence of covid-19 among asymptomatic individuals in household clusters [15, 17, 19] , a nursing home outbreak [14] , active surveillance of passengers quarantined on a cruise ship or passengers of repatriation flights [18] , hospital employees with close contact to covid-19 positive patients [13] , and customers and employees of a restaurant that had a covid-19 outbreak [16] . overall, prevalence ranged from 10 to 50% in settings where substantial transmission was suspected prior to testing. summary statistics for single versus repeated testing are shown in table 8 and supplement h. we acknowledge that information on individual exposure was limited in the evidence base. all these limitations led to very low certainty in the evidence overall. testing asymptomatic individuals who have been exposed, or suspected to have been exposed, allows for isolation for those who are positive. whether in an institutional cluster or a wider community outbreak, isolation will help reduce further transmission. there is potential harm in a false negative naat result collected from an exposed individual who is a c c e p t e d m a n u s c r i p t actually infected; these individuals may incorrectly consider themselves non-infected, and unknowingly expose others to sars-cov-2 as a result. given the lack of evidence, a negative test post-exposure does not mean quarantine can be discontinued. some individuals may still be in the incubation phase, subsequently develop active viral shedding, and incorrectly consider themselves non-infected. as a result, a negative post-exposure test cannot necessarily be used to avoid quarantine. a positive result, however, would reinforce the importance of isolation as well as inform contact tracing, cohorting, or other mitigation strategies. additional considerations: diagnostic test performance in asymptomatic individuals has not been established. assuming an overall test sensitivity between 75%-95% [35, 36, [38] [39] [40] 44] , false negative test results are expected. there is also cost to testing asymptomatic exposed individuals; since quarantine may still be indicated regardless of test results, such testing may add cost without changing practice. data are limited to define definitions of close contact. risk stratification of a given exposure can be made in consultation with public health authorities. in addition, the cdc has published guidance on defining healthcare exposures and categorizing exposure risks [64] .the ideal time to test an asymptomatic contact of a known or suspected covid-19 case is also unknown. timing also becomes complicated for household contacts with ongoing exposure. the average incubation period for sars-cov2 has been determined to be five days [65] . thus, 5-7 days following exposure may be a reasonable time frame to consider post-exposure testing. in addition, data to inform the definition of a significant exposure or close contact are limited. considerations when assessing the risk of a known contact include the duration of exposure and the clinical symptoms (e.g., cough) of the person with covid-19. with known or suspected exposures should be coordinated with local public health officials. this indication for testing is especially important in situations where knowledge of asymptomatic or pre-symptomatic infection is essential for determining medical follow-up, defining risks for other vulnerable individuals in the household, congregate setting or hospital. special consideration should also be given to healthcare personnel exposed without a c c e p t e d m a n u s c r i p t appropriate ppe in healthcare settings. definitions of appropriate ppe can be found on the cdc website [66] . comparative studies (preferably randomized controlled trials) along with cost-effectiveness analyses of testing strategies in asymptomatic populations are needed. studies on the ideal time and collection method to test asymptomatic individuals who have been exposed to covid-19 should be performed. in addition, what constitutes an exposure that would justify testing requires further research. whether early diagnosis of covid-19 might provide an opportunity to intervene therapeutically and change the ultimate course of infection (i.e., prevent severe pneumonia) is unknown. if this is shown to be the case, the opportunity for therapeutic intervention might justify screening exposed individuals. this table is based on applying the sensitivity and specificity estimates to calculate true and false positives and negatives in a hypothetical population of 1000 individuals a. reference standard considered to be nasopharyngeal specimen rt-pcr. b. studies report test accuracy results but do not report on patient-important outcomes based on these results. c. a small number of patients included. d. we assessed studies that reported the prevalence of covid-19 among asymptomatic individuals who were exposed to covid-19 and determined that the prevalence may range from 10% to 50% based on household clusters, nursing home outbreak, active surveillance of passengers quarantined on a cruise ship or passengers of repatriation flights, hospital employees with close contact with covid-19 positive patients and customers and employees of a restaurant that had a covid-19 outbreak. e. certainty of evidence (coe)  asymptomatic individuals are defined as those with no symptoms or signs of covid-19.  a high prevalence of covid-19 in the community was considered communities with a prevalence of 10%. a c c e p t e d m a n u s c r i p t  the decision to test asymptomatic patients (including when the prevalence is between 2 and 9%) will be dependent on the availability of testing resources. we did not identify any studies that directly assessed a strategy of nucleic acid testing for sars-cov-2 versus no testing before hospitalization for non-covid-19 related reasons. we also did not identify test accuracy studies directly assessing the performance of sars-cov-2 viral rna tests in asymptomatic individuals. however, based on existing evidence suggesting that asymptomatic or pre-symptomatic patients may have similar virus loads and shedding as those who are symptomatic [62, 63] , the panel agreed to infer test accuracy for asymptomatic populations before being hospitalized. it was also essential to determine the pre-test probability or prevalence of the disease in asymptomatic patients admitted to the hospital. we assessed studies that reported prevalence  this recommendation defines immunosuppressive procedures as cytotoxic chemotherapy, solid organ or stem cell transplantation, long acting biologic therapy, cellular immunotherapy, or high-dose corticosteroids.  testing should ideally be performed as close to the planned treatment/procedure as possible (e.g. within 48-72 hours).  many of these patients require frequent, repeated or prolonged visits to receive treatment.  this recommendation does not address risks or strategies to deal with sars-cov-2 transmission in outpatient settings such as infusion centers. we did not identify any studies that directly assessed a strategy of testing for sars-cov-2 versus no testing of asymptomatic individuals before receiving chemotherapy or transplantation. in addition, we were unable to evaluate the risks of delaying necessary treatments or transplants if testing was not available and quarantine/delay of treatment was then required. we also did not identify any test accuracy studies directly assessing the performance of naat in asymptomatic individuals. based on existing evidence supporting that asymptomatic or pre-symptomatic patients may have similar virus loads and a c c e p t e d m a n u s c r i p t shedding as those who are symptomatic [62, 63] , the panel agreed that test accuracy data from symptomatic patients would apply to asymptomatic populations being hospitalized. it was essential to determine the pre-test probability or prevalence of covid-19 in asymptomatic patients who will be receiving chemotherapy. we assessed studies that evaluated prevalence of covid-19 among asymptomatic individuals and patients with cancer to estimate prevalence a between <1 to 10%. we identified three studies reporting data on the prevalence of cancer among covid-19 patients and the percentage of complications (e.g., icu admission, death) among these patients. liang et al [67] showed that the prevalence of cancer among covid-19 patients to be 1%, which was higher compared to their general population (0.2%). yu et al [21] showed the prevalence of covid-19 among patients admitted to the radiation and medical oncology floor to be 0.8%. lastly, a systematic review conducted by desai et al. (2020) [68] showed the pooled prevalence of cancer among covid-19 cases to be 2-3%. the overall certainty of the evidence about testing effects in immunocompromised individuals was very low due to extremely limited data in this population. the panel determined that a maximum threshold of <2-5 missed cases per 1000 would be acceptable. not testing individuals regardless of low versus high prevalence areas would lead to higher numbers of missed cases which the panel considered to exceed the acceptable threshold. the threshold was set very low due to concern about catastrophic outcomes in this population. although data is limited, there are reports documenting outbreaks of respiratory viruses in hospitalized immunocompromised hosts [69] . in addition, increased risks of severe adverse respiratory virus-related outcomes in this population are documented [70] . for false negative test results, so caution should be exercised by those who will be in close contact with/exposed to the upper respiratory tract (e.g., anesthesia personnel, ent procedures). the decision to test asymptomatic patients will be dependent on the availability of testing resources.  the panel defined time-sensitive procedures as medically necessary procedures that need to be done within three months.  procedures considered to be aerosol generating are listed in table 9 .  decisions about ppe will be dependent on test results because of limited availability of ppe. however, there is a risk for false negative test results, so caution should be exercised for those who will be in close contact with/exposed to the patient's airways.  procedures considered to be aerosol generating are listed in table 9 .  the decision to test asymptomatic patients will be dependent on the availability of testing resources.  this recommendation does not address the need for repeat testing if patients are required to undergo multiple procedures over time. the panel did not identify any studies that directly assessed a strategy of testing for sars-cov-2 versus no testing of asymptomatic individuals before undergoing major surgery or aerosol generating procedures (agps). the panel also did not identify test accuracy studies directly assessing the performance of sars-cov-2 naats in asymptomatic individuals. however, based on existing evidence supporting that asymptomatic or presymptomatic patients may have similar viral loads and shedding as those who are symptomatic, the panel agreed that test accuracy data from symptomatic patients could be applied to asymptomatic populations before surgery. it was essential to determine the pre-test probability or prevalence of disease in the asymptomatic patients who will undergo surgery. we assessed studies that evaluated the a c c e p t e d m a n u s c r i p t prevalence of covid-19 among asymptomatic individuals and determined that the range of prevalence would be between <1 to 10% based on assessing rates of infection in asymptomatic individuals in the general population in low prevalence and in "hotspot" areas [8, 20, 21] . the panel recommendation was based on emphasizing the importance of preventing infection in healthcare providers during major time-sensitive surgeries and agps. in addition, the limited data showing poor outcomes in covid-19 positive patients undergoing a major surgical procedure requiring intubation informed decisions to reduce this risk for asymptomatic patients [71] . there are no data that assess the outcome of agps in sars-cov-2 positive patients. the benefit of suggesting testing for sars-cov-2 in asymptomatic patients undergoing major time-sensitive surgery is that it allows for the identification of infected patients before the procedure; thus allowing surgery to delayed based on the limited data suggesting that patients testing positive may have poor outcomes [71] . this approach also has the potential to inform healthcare workers in terms of ppe use, particularly in areas where ppe is limited. of note, there is very low certainty evidence from retrospective case series suggesting poor outcomes of time-sensitive surgeries for those with covid-19. the surgeries included were variable in complexity and it was not clear if the poor outcomes came mostly from major or minor surgeries. however, it is plausible that poor outcomes were driven by the major surgeries. a potential harm of testing of immunocompetent, asymptomatic patients before a major surgery or agp is depletion of testing supplies and the diversion of all associated resources away from symptomatic patients. an additional harm of testing is related to the sensitivity of the naats for sars-cov-2, which will not detect all asymptomatic patients with covid-19 infection. therefore, some patients may be missed and healthcare workers at high risk could be exposed. thus, the panel suggests that healthcare workers at the highest risk during surgical procedures (e.g., those performing intubation or ent procedures) consider wearing ppe at all times, regardless of test results. this would be especially important in high prevalence areas a c c e p t e d m a n u s c r i p t (i.e., "hotspots"). an additional harm is that false positive tests for sars-cov-2 may unnecessarily delay a major time-sensitive surgery. there is no standard definition of what constitutes a major surgery. in general, the panel in consultation with surgical colleagues, agreed that major surgeries would be defined as more complicated and/or prolonged surgeries that require general anesthesia and intubation (which is an agp). additionally, time-sensitive surgeries/procedures were defined as those for which a delay greater than 3 months would negatively affect outcomes. in addition to the clinical questions addressed above, there is significant interest in the use of serologic sars-cov-2 tests both for diagnosis and public health surveillance. at the time of our literature review, however, additional data were needed to formulate recommendations. important areas that need to be addressed include assessment of the sensitivity and specificity of commercial antibody tests, determinations of protective immunity and measures of antibody responses over time. whether seroconversion can inform return to work or hospital staffing policies needs to be assessed. in the absence of evidence to guide the use of sars-cov-2 serologic testing, the idsa diagnostics committee published a serology primer for clinicians which highlights potential benefits, limitations and unmet research needs [73] . antigen detection tests may be on the horizon. how these will compare with naat needs to be defined. in addition, current naats detect viral rna but cannot distinguish infectious from noninfectious virus. this determination requires viral culture, which is not routinely performed in clinical laboratories for biosafety reasons and is likely less sensitive than naat. it will be important to define whether individuals who remain nucleic acid positive after symptom resolution, and potentially seroconversion, are infectious to others because this will have important ramifications for quarantine and reducing restrictions around social distancing. some "test of cure" algorithms require two sequential negative naats, yet some studies describe a c c e p t e d m a n u s c r i p t prolonged rna positivity. future studies are required to determine the significance of nucleic acid or antigen shedding after clinical recovery. molecular tests designed to detect sars-cov-2 nucleic acids are essential both for confirming covid-19 diagnosis and for public health responses aimed at curbing the pandemic. several countries have deployed naat on a massive scale as the cornerstone of a successful containment strategy. although the u.s. was hampered by limited test availability early in the outbreak, there are now more than 25 different commercially available sars-cov-2 assays and multiple clinical laboratories have developed their own laboratory-developed tests. aggressive efforts are underway to assure access to testing, but regional differences in availability persist. individual medical centers and clinics are likely to have different testing capacity as well. furthermore, which test a laboratory or facility chooses to perform will vary based on the resources of a given setting (e.g., near-patient versus high complexity laboratory) and turnaround-time to result requirements (i.e., rapid versus standard). the primary recommendations set forth in this guideline assume that sars-cov-2 testing is available to healthcare providers on the front lines. however, the panel also recognized that resources may vary, and contingency recommendations were developed for situations where naat supplies or ppe are limited. individual institutions will need to prioritize testing based on available resources and unique patient populations. testing for symptomatic patients should be prioritized. when testing capacity for symptomatic individuals is considered sufficiently robust, testing for asymptomatic individuals should be considered. there will undoubtedly be challenges prioritizing and implementing testing strategies for asymptomatic groups. the strongest recommendation for testing in asymptomatic individuals in this guideline pertains to immunocompromised patients being admitted to the hospital or in advance of immunosuppressive procedures. a c c e p t e d m a n u s c r i p t molecular tests have been central to our understanding of sars-cov-2. however, much about the biology of sars-cov-2 remains unknown. early experience suggests that sars-cov-2 is detectable in the upper respiratory tract, with peak levels typically measurable during the first week of symptoms [48, 62, 74] . rna detection rates, however, appear to vary from patient to patient and change over time. some patients with pneumonia, for example, have negative upper respiratory tract samples but positive lower airway samples [35, 75] . much less it known about the frequency of viral detection in asymptomatic individuals, although the concentration of detectable virus in some people with infection may be quite high [62, 63] . a better understanding of the spectrum of viral load kinetics over time at different anatomic sites is needed to inform decisions about the optimal testing strategies, including when and how to repeat if the first test is negative. like other respiratory viruses, shedding of viral rna in respiratory secretions may persist beyond resolution of symptoms and seroconversion [76] . whether such patients remain infectious to others is uncertain and this is an important area for future study. the clinical performance of commercially available sars-cov-2 molecular diagnostic tests has not yet been defined and will depend in large part on the biology of the virus. typically, when tests for the detection of viral respiratory pathogens are submitted to the fda, both analytical and clinical performance data are provided. under eua, however, only analytical data are required. diagnostic developers may test contrived specimens, by spiking viral rna or inactivated virus into the desired matrix, rather than using real clinical specimens collected from patients with covid-19. thirty contrived positive and 30 negative specimens tested, with 95% sensitivity and 100% specificity required for eua. therefore, while we have information regarding the limit of detection of the test and evidence (both in vitro and in silico studies) that the primer design is specific for sars-cov-2, there is no information on how each test performs clinically at the time the eua is issued. clinical laboratories using commercial eua tests must verify analytic test performance at some level in their own hands, including evaluation of different specimen types and collection methods (e.g., swab types and transport media). a c c e p t e d m a n u s c r i p t clinical performance metrics include sensitivity, which is the ability of the test to correctly identify those with infection, and specificity, the ability of the test to correctly identify those without the disease. in practice, the positive and negative predictive values of the test are also essential for interpreting test results. estimations of community prevalence and patient pre-test probability combined with knowledge of test sensitivity and specificity are essential for determining the likelihood that an individual has covid-19. in practice, however, the true prevalence of covid-19 in the community may not be well-defined and may be underestimated when test availability is limited. in addition, while sars-cov-2 rna tests are highly specific, their respective sensitivities are likely to vary. recognizing these complexities, estimates of prevalence/pre-test probability and assay sensitivity were varied in our analyses based on the available literature in an attempt to mirror what may be encountered in clinical practice. going forward, robust prevalence studies are needed. clinical test performance should also ideally be determined in prospective multicenter studies using a well-defined reference standard as the benchmark for test comparisons. table 2 outlines the type of clinical studies needed to address the most pressing covid-19 diagnostic knowledge gaps. one of the most important problems with current covid-19 diagnostic literature is the lack of a standard definition to define covid-19. the studies included in the systematic reviews that informed this guideline used variable case definitions and many classified disease based in part on the results of the index test under investigation. incorporation of the investigational index test into the diagnostic "gold" standard falsely inflates sensitivity and specificity estimates (i.e. incorporation bias). table 10 outlines options for defining a confirmed covid-19 case in diagnostic trials. it is recognized that not all individuals with covid-19 will have detectable sars-cov-2 nucleic acid. therefore, a "probable" case definition is also proposed. false negative naat results may be due to a variety of factors, including assay limit of detection, anatomic location and adequacy of specimen collection, timing of sampling relative to symptom onset, and underlying biology of disease. to fully understand sars-cov-2 viral dynamics, studies need to be designed to obtain specimens from multiple sites, ideally from the same a c c e p t e d m a n u s c r i p t patient at the same time. in addition, information on the duration of symptoms (if present), assessment of potential exposures and longitudinal follow-up of outcomes will be essential to define optimal diagnostic test strategies across a variety of patient populations. nucleic acid sequencing matches sars-cov-2 reference sequences positive results from at least 2 different naats (one of the two may be the index test) option 3 dual positive results from a single naat targeting 2 different genes (cannot be the index test) compatible clinical signs and symptoms in a setting with known community transmission, negative reference naat and documented sars-cov-2 seroconversion. compatible clinical signs and symptoms in a setting with known community transmission, negative reference naat and positive index test from two different anatomic sites. compatible clinical signs and symptoms in a setting with known community transmission, negative reference naat and positive sars-cov-2-specific serology. the guideline panel used a methodologically rigorous process to critically appraise the available diagnostic literature and formulate sars-cov-2 testing recommendations. the quality of existing evidence, however, was limited and not all of the data used to inform these recommendations had undergone peer-review. based on low certainty evidence, the idsa panel recommends nucleic acid testing for all symptomatic individuals suspected of having covid-19. in addition, testing selected asymptomatic individuals is suggested when the results will have significant impact on isolation/quarantine/ppe usage, dictate eligibility for surgery, or inform use of immunosuppressive therapy. ultimately, institutional resources will dictate test this project was funded by idsa. the following list displays what has been reported to the idsa. to provide thorough transparency, the idsa requires full disclosure of all relationships, regardless of relevancy to the guideline topic. evaluation of such relationships as potential conflicts of interest is determined by a review process which includes assessment by the board of directors liaison to the standards and practice guideline committee and, if necessary, the conflicts of interest (coi) and ethics committee. the assessment of disclosed relationships for possible coi is based on the relative weight of the financial relationship (i.e., monetary amount) and the relevance of the relationship (i.e., the degree to which an association might reasonably be interpreted by an independent observer as related to the topic or recommendation of consideration). the reader of these guidelines should be mindful of this when the list of disclosures is reviewed. k.h. serves as an advisor for biofire and quideland and receives centers for disease control and prevention. health care infection prevention and control faqs for covid-19 prevention strategies for seasonal influenza in health care settings advice on the use of masks in the context of covid-19 epidemic-and pandemic-prone acute respiratory diseases -infection prevention and control in health care day zero, visby, and chroma code; receives research funding from arcbio and hologic; and has served as an advisor for luminex. c.a. receives royalties from uptodate and receives research funding from merck entasis pharmaceuticals and the national institute of allergy and infectious diseases (niaid)/nih. j.e. serves as a consultant for sanofi pasteur; an advisor/consultant for meissa vaccines; and receives research funding from the centers for serves as an advisor for sanofi, seqirus, and medicago; has served as an advisor for pfizer, sunovion, and md brief; and receives research funding from the canadian institutes of health research and the medical research council (united kingdom). r.p. receives grants from shionogi the infectious diseases society of america (idsa), the national board of medical examiners, uptodate, and the infectious disease board review course; y.f.y. receives honoraria for evidence reviews and teaching from the evidence foundation, honoraria for evidence reviews for the american gastroenterological association, and serves as a director for the evidence foundation and for the u.s a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the species severe acute respiratory syndromerelated coronavirus: classifying 2019-ncov and naming it sars-cov-2 novel coronavirus-2019 events-as-they happen adminsitration fad development of rapid guidelines: 3. gin-mcmaster guideline development checklist extension for rapid recommendations grade guidelines: 2. framing the question and deciding on important outcomes spread of sars-cov-2 in the icelandic population preliminary results of initial testing for coronavirus (covid-19) in the emergency department. the western journal of emergency medicine optimizing diagnostic strategy for novel coronavirus pneumonia, a multi-center study in eastern china positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in sars-cov-2 infection in health care workers asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility -king county clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing covid-19 outbreak associated with air conditioning in restaurant epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts estimated effectiveness of symptom and risk screening to prevent the spread of covid-19 presymptomatic transmission of sars-cov-2 -singapore universal screening for sars-cov-2 in women admitted for delivery sars-cov-2 transmission in cancer patients of a tertiary hospital in wuhan quadas-2: a revised tool for the quality assessment of diagnostic accuracy studies grade guidelines: 21 part 1. study design, risk of bias, and indirectness in rating the certainty across a body of evidence for test accuracy grade guidelines: 21 part 2. test accuracy: inconsistency, imprecision, publication bias, and other domains for rating the certainty of evidence and presenting it in evidence profiles and summary of findings tables available from gradepro.org world health organization recommendations are often strong based on low confidence in effect estimates guideline panels should not grade good practice statements differential diagnosis of illness in patients under investigation for the novel coronavirus (sars-cov-2) featuring covid-19 cases via screening symptomatic patients with epidemiologic link during flu season in a medical center of central taiwan screening and managing of suspected or confirmed novel coronavirus (covid-19) patients: experiences from a tertiary hospital outside hubei province centers for disease control and prevention. priorities for testing patients with suspected covid-19 infection comparison of throat swabs and sputum specimens for viral nucleic acid detection in 52 cases of novel coronavirus (sars-cov-2) infected pneumonia detecting sars-cov-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (lamp) to detection of sars-cov-2 in different types of clinical specimens evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections evaluation the auxiliary diagnosis value of antibodies assays for detection of novel coronavirus (sars-cov-2) causing an outbreak of pneumonia nasal swab sampling for sars-cov-2: a convenient alternative in time of nasopharyngeal swab shortage patient-collected tongue, nasal, and mid-turbinate swabs for sars-cov-2 yield equivalent sensitivity to health care worker collected nasopharyngeal swabs quantitative detection and viral load analysis of sars-cov-2 in infected patients saliva as a non-invasive specimen for detection of sars-cov-2 sars-cov-2 viral load in upper respiratory specimens of infected patients self-collected oral fluid and nasal swabs demonstrate comparable sensitivity to clinician collected nasopharyngeal swabs for covid-19 detection self-collection: an appropriate alternative during the sars-cov-2 pandemic viral kinetics and antibody responses in patients with self-collected compared with professional-collected swabbing in the diagnosis of influenza in symptomatic individuals: a meta-analysis and assessment of validity viral load kinetics of sars-cov-2 infection in first two patients in korea virological assessment of hospitalized patients with covid-2019 evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections evaluation the auxiliary diagnosis value of antibodies assays for detection of novel coronavirus (sars-cov-2) causing an outbreak of pneumonia (covid-19) quantitative detection and viral load analysis of sars-cov-2 in infected patients viral kinetics and antibody responses in patients with comparison of throat swabs and sputum specimens for viral nucleic acid detection in 52 cases of novel coronavirus (sars-cov-2) infected pneumonia analysis of factors associated early diagnosis in coronavirus disease clinical features and outcomes of 197 adult discharged patients with covid-19 in yichang rapid colorimetric detection of covid-19 coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification (rt-lamp) diagnostic plat-form rapid detection of sars-cov-2 using reverse transcription rt-lamp method rapid molecular detection of sars-cov-2 (covid-19) virus rna using colorimetric lamp reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of comparison of abbott id now and abbott m2000 methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from symptomatic patients comparison of abbott id now, diasorin simplexa, and cdc fda eua methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from individuals diagnosed with sars-cov-2 viral load in upper respiratory specimens of infected patients cluster of coronavirus disease 2019 (covid-19) in the french alps guidance for risk assessment and public health management of healthcare personnel with potential exposure in a healthcare setting to patients with coronavirus disease the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application centers for disease control and prevention. infection control guidance for healthcare professionals about coronavirus (covid-19). available at cancer patients in sars-cov-2 infection: a nationwide analysis in china covid-19 and cancer: lessons from a pooled meta-analysis respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles respiratory virus infections in hematopoietic cell transplant recipients clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection idsa covid-19 antibosy testing primer viral load of sars-cov-2 in clinical samples negative nasopharyngeal and oropharyngeal swabs do not rule out covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study a c c e p t e d m a n u s c r i p t key: cord-312905-8xlt92pl authors: li, guanjian; li, weiran; he, xiaojin; cao, yunxia title: asymptomatic and presymptomatic infectors: hidden sources of covid-19 disease date: 2020-04-09 journal: clin infect dis doi: 10.1093/cid/ciaa418 sha: doc_id: 312905 cord_uid: 8xlt92pl nan a c c e p t e d m a n u s c r i p t dear editor, with interest, we read the study by sarah et al. who reported the first mildly ill, nonhospitalized case of covid-19 in the united states [1] . the authors conducted rigorous public health surveys, contact tracking and serial sarscov-2 testing. in their research, despite specimens testing positive 18 days after diagnosis, no sars-cov-2 transmission occurred among 10 high-risk contacts of this patient, including 1 intimate contact. this study provided important insights for the spread of covid-19. however, they promoted the possibility that mild cases can be released from isolation based on symptom resolution. some of the limitations of this study have been well summarized by the authors. in fact, the key problem should be that the use of a single covid-19 case to demonstrate the infectivity of all mild cases is insufficient. at the time of writing this paper, the authors claimed that "evidence regarding transmission from individuals with asymptomatic infection or mild illness is limited", but that the current evidence may need an update. recently, two epidemiological studies on covid-19 have reported that typically asymptomatic carriers transmit the virus to close contacts, resulting in aggregated infection of symptomatic covid-19 diseases [2, 3] . on march 28, the chinese journal of epidemiology published a prospective study from zhejiang province [4] . this study used a prospective design to follow up the viral load and clinical manifestations of 2147 close contacts of symptomatic and asymptomatic covid-19 cases. their conclusion is that the virus infection rates of close contacts were 6.30% (contact with symptomatic patients) and 4.11% (contact with asymptomatic patients), respectively. the authors suggested that the transmission ability of asymptomatic individuals should not be ignored. a c c e p t e d m a n u s c r i p t in fact, even if patients with mild symptoms with a low ability to transmit the virus, the degradation of control measures still needs careful consideration. there are individual differences in the symptoms of the infectious disease, and the symptoms are often subjective and constantly changing, so it may be difficult to accurately define the severity of the symptoms in clinical practice. it is also difficult to distinguish patients with prodromal symptoms (with subsequent progression) from mild symptoms patients. in a recent article published in mmwr on april 1, a team from the national centre for communicable diseases in singapore reported seven clusters of cases in which presymptomatic transmission is the most likely explanation for the occurrence of secondary cases [5] . similar cluster infections caused by presymptomatic infectors have been reported in other areas [6] . in addition, the proportion of asymptomatic patients among all patients may be higher than previously expected, with recent reports suggested a range of 30%-60% [7] .the huge number of asymptomatic infected individuals makes their role in the spread of sarscov-2 cannot be ignored. in short, asymptomatic or mild cases of infection should be paid more attention, as china and some regions of italy have done [8] . inactive diagnosis and isolation may result in accumulation of infectious sources in the community, making it more difficult to control the "pandemic" of covid-19. all authors have no potential conflicts of interest. first mildly ill, non-hospitalized case of coronavirus disease 2019 (covid-19) without viral transmission in the united states presumed asymptomatic carrier transmission of covid-19 clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing the epidemiological characteristics of infection in close contacts of covid-19 in ningbo city presymptomatic transmission of sars-cov-2 -singapore a covid-19 transmission within a family cluster by presymptomatic infectors in china covert coronavirus infections could be seeding new outbreaks covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village a c c e p t e d m a n u s c r i p t key: cord-297612-swc2pitd authors: nosyk, bohdan; armstrong, wendy s; del rio, carlos title: contact tracing for covid-19: an opportunity to reduce health disparities and end the hiv/aids epidemic in the us date: 2020-04-27 journal: clin infect dis doi: 10.1093/cid/ciaa501 sha: doc_id: 297612 cord_uid: swc2pitd sars-cov2 testing and contact tracing have been proposed as critical components of a safe and effective covid-19 public health strategy. we argue that covid-19 contact tracing may provide a unique opportunity to also conduct widespread hiv testing, among other health promotion activities. m a n u s c r i p t massive sars-cov2 testing and contact tracing at a scale and speed never before seen have been proposed as critical components of a covid-19 public health strategy that could, in theory, safely allow us to relax social distancing measures and begin to bring back the world we left behind before a cure or effective vaccine is delivered. one of a number of challenges this strategy faces is that contact tracing is extremely labor intensive. deployed in sexuallytransmitted infections such as hiv, a recent study from new york reported costs of over $500 per contact interviewed with 85% of state-level costs attributable to personnel [1] . for coronavirus these efforts are simpler but the volume of contacts-an average of 36 per positive case [2] pose substantial logistic challenges. digital applications and other technological supports may help, though it appears inevitable that a large labor force will be needed. already, massachusetts has begun hiring to fill their state's projected need for 1,000 contact tracing workers [3] . health canada has an open call for a volunteer workforce to do contact tracing and other related tasks [4] . south korea employed a massive workforce of public health workers at the peak of the epidemic [5] . the value of a such a public health workforce extends beyond contact tracing for covid-19 and could lead to progress fighting many other health conditions. programs in african communities have combined hiv testing with screening for infectious diseases like tuberculosis and malaria as well as non-communicable diseases like hypertension and diabetes [6] . using this model, we can take this opportunity to scale-up testing for infectious diseases as well as noncommunicable diseases and by doing so improve community health. as sars-cov-2 testing is evolving with, not only serological but saliva testing, similar approaches could be taken for optout hiv, hcv and hemoglobin a1c testing for which finger stick methods are already available. this will allow us to begin to address the unacceptable health disparities that have existed for many of these conditions and, not surprisingly, also occur in covid-19 [7, 8] . aside from the potentially profound health benefits of a combination implementation approach, pairing covid-19 contact tracing with testing for hiv may serve to offset the immense costs of such an approach. it is well-established that hiv testing provides outstanding value and can even be cost-saving in the long-term in high-prevalence populations and settings [9] . simply learning of a new hiv infection is known to change behavior, with meta-analyses estimating an 80% reduction in sexual activity with partners of negative or unknown hiv status [10] , and starting arvs with subsequent viral suppression stops hiv transmission [11] . testing focused on the geographic regions with the highest rates of new diagnoses and further targeted using phylogenetics to identify the largest and fastest-growing clusters of infection, has a c c e p t e d m a n u s c r i p t been positioned as a key pillar of the united states' 'ending the hiv epidemic' strategy [12] . these efforts will be familiar to public health leaders in the us -anthony fauci was an architect of the strategy [13] , which was motivated by the profound successes in the african continent, engineered in part by the president's emergency plan for aids relief (pepfar) and headed by deborah birx [14] . yet testing remains one weak link in our efforts to end the hiv epidemic, in part because of persistent stigma and because the communities at greatest risk often have limited access to healthcare and/or are young and without established healthcare. we [bn, cdr] have recently written that for six of the largest us cities comprising nearly 1 in 4 people living with hiv/aids in the us, implementing a wide range of interventions to diagnose, treat and protect against infection at even near-ideal levels would fall short of the ehe targets [15] . we excluded contact tracing because of the relatively limited experimental evidence supporting its effectiveness in hiv and the uncertainty regarding the potential scale this type of effort could actually reach. contact tracing for hiv is difficult to implement and may be profoundly threatening to already marginalized individuals. covid-19 contact tracing may provide a unique opportunity to also conduct widespread hiv testing with modified contact tracing that could be acceptable and important for ending the hiv epidemic. though the task is indeed monumental, the necessary temporary labor force is readily available. were implemented [16, 17] . some project the unprecedented stimulus packages and protections for workers may not return the hardest-hit industries to pre-covid levels, and high unemployment may linger. job loss is a major life stressor and is associated with declines in psychological and physical well-being along with a host of other negative social effects [18] . creating jobs, even if only for a limited term, is a necessary government intervention to not only kickstart the economy but also to mitigate further growth in the concurrent epidemic of loss and despair [19, 20] . mobilizing this labor force in the covid-19 response will allow communities to take meaningful action to protect themselves, an act which in itself may have transformative benefits over the long-term. hiv is known to disproportionately impact urban african american and hispanic/latinx communities [21, 22] as do other chronic health conditions such as diabetes and hypertension. now covid-19 shows the all too familiar progression into vulnerable populations. early cases were seen primarily in international travellers, but currently african american and a c c e p t e d m a n u s c r i p t latinx populations are disproportionately impacted. a public health workforce with point of care screening for these conditions as well could make meaningful strides to reduce the widespread disparities inherent in health. furthermore testing to promote health allows for stigma-free messaging and broad acceptance. testing and contact tracing for covid-19 will provide an entry into social networks in communities where risk factors for conditions like hiv or noncommunicable diseases may be shared. it is imperative however that contact tracing does not increase stigma and discrimination of minority populations and thus the development of a workforce dedicated to culturally competent contact tracing, point of care testing and overall health promotion [23] has to be a priority in order to seize this opportunity. the speed with which covid-19 contact tracing must be conducted is on a different scale, though more intensive efforts can be triaged to specialized staff. the greatest effort howeverand valueis in testing. of course, these potential benefits hinge on the rapid development of antibody testing technology, not to mention sufficient availability of personal protective equipment, and the public health system's ability to rapidly train and deploy the massive influx of temporary workers that would be needed. aside from the devastating death toll, the potential long-term effects of covid-19 on the global economy are severe and may ultimately prove to be greater than any other period in living memory. difficult decisions regarding how and when to restart the economy lie ahead, but the balance sheet can include the long-term benefits of widespread hiv testing, screening for chronic diseases and potentially narrowing health disparities. though it may feel like a distant priority, now more than ever, as the official language of the plan goes, we have unprecedented opportunity to end the hiv epidemic in america. accomplishing this and expanding the impact to other health conditions is an opportunity we must not let go to waste. notes: this study was funded by the national institutes of health/national institute on drug abuse grant no. r01-da-041747 to emory center for aids research and emory vteu. the authors have no conflicts of interest to declare. cost analysis and performance assessment of partner services for human immunodeficiency virus and sexually transmitted diseases the efficacy of contact tracing for the containment of the 2019 novel coronavirus (covid19) coronavirus disease-19: summary of 2,370 contact investigations of the first 30 cases in the republic of korea. osong public health res perspect hiv testing and treatment with the use of a community health approach in rural africa covid-19 and african americans failing another national stress test on health disparities localized economic modeling study group. the impact of localized implementation: determining the cost-effectiveness of hiv prevention and care interventions across six united states cities meta-analysis of high-risk sexual behavior in persons aware and unaware they are infected with hiv in the united states-implications for hiv prevention programs hptn 052 study team. prevention of hiv-1 infection with early antiretroviral therapy ending the hiv epidemic: a plan for the united states at 15 years. lancet localized hiv modeling study group. ending the hiv the usa: an economic modelling study in six cities mortality and morbidity in the 21 st century the epidemic of despair among white americans: trends in the leading causes of premature death trends in racial/ethnic disparities of new aids diagnoses in the united states what lessons it might teach us? community engagement in hiv research addressing hiv criminalization: science confronts ignorance and bias a c c e p t e d m a n u s c r i p t key: cord-310390-7pkbd3kg authors: han, xiaoyu; cao, yukun; jiang, nanchuan; chen, yan; alwalid, osamah; zhang, xin; gu, jin; dai, meng; liu, jie; zhu, wanyue; zheng, chuansheng; shi, heshui title: novel coronavirus pneumonia (covid-19) progression course in 17 discharged patients: comparison of clinical and thin-section ct features during recovery date: 2020-03-30 journal: clin infect dis doi: 10.1093/cid/ciaa271 sha: doc_id: 310390 cord_uid: 7pkbd3kg background: to retrospectively analyze the evolution of clinical features and thin-section ct imaging of novel coronavirus pneumonia (covid-19) in 17 discharged patients. methods: serial thin-section ct scans of 17 discharged patients with covid-19 were obtained during recovery. longitudinal changes of clinical parameters and ct pattern were documented in all patients during 4 weeks since admission. ct score was used to evaluate the extent of the disease. results: there was a marked improvement of fever, lymphocytes count, c-reactive protein and erythrocyte sedimentation rate within the first two weeks since admission. however, the mean ct score rapidly increased from the 1(st) to 3(rd) week, with a top score of 8.2 obtained in the 2(nd) week. during the 1(st) week, the main ct pattern was ground-glass opacities (ggo,76.5%). the frequency of ggo (52.9%) decreased in the 2(nd) week. consolidation and mixed patterns (47.0%) were noted in the 2(nd) week. thereafter, consolidations generally dissipated into ggo and the frequency of ggo increased in the 3(rd) week (76.5%) and 4(th) week (71.4%). opacities were mainly located in the peripheral (76.5%), subpleural (47.1%) zones of the lungs, and presented as focal (35.3%) or multifocal (29.4%) in the 1(st) week and became more diffuse in the 2(nd) (47.1%) and 3(rd )week (58.8%), then showed reduced extent in 4(th )week (50%). conclusions: the progression course of ct pattern was later than the clinical parameters within the first two weeks since admission; however, there was a synchronized improvement in both clinical and radiologic features in the 4(th) week. in late december 2019, a new pneumonia of unknown cause broke out in wuhan city, china [1, 2] , and the infection has spread rapidly in china and worldwide [3] [4] [5] [6] . on 7 th of january 2020, a novel coronavirus (sars-cov-2) was identified as the cause via deep sequencing analysis of the respiratory tract samples [2, 4] . sars-cov-2 possess a strong ability to infect humans, with the capability of human-to-human transmission [7, 8] . the number of patients is increasing rapidly. by march 7, 2020, more than 8,0000 cases of pneumonia were reported in china, including 3073 cases of death (the mortality rate is round 3.8 %). the epidemiological and clinical characteristics of the initial novel coronavirus pneumonia population in wuhan have been recently published [9] [10] [11] [12] . the common presentation of these patients was as a rapidly progressing lower respiratory tract illness with fever and cough. the diagnosis of covid-19 mainly depends on the results of viral nucleic acid, which possesses a high specificity but a poor sensitivity. according to a recent publication, half of the patients were diagnosed as covid-19 without fever in the early stage [12] , and even with negative nucleic acid tests at first few times [13] . therefore, combining imaging features with clinical and laboratory findings can help make an early diagnosis of covid-19. at present, a limited number of reports have focused on chest imaging findings of covid-19 [13] [14] [15] [16] . the common ct findings of patients with covid-19 in those reports include bilateral ground-glass opacities or areas of consolidation of the peripheral lung, which bear some resemblance to sars-cov [17] [18] [19] and mers-cov [20, 21] . in a recent report of 81 patients, ct findings across different timepoints throughout the disease course has been described [16] .however, the longitudinal progression of thin-section ct imaging changes in 5 patients with covid-19 and its correlation with changes in clinical parameters remains unclear. hence, this study aimed to analyze the serial thin-section ct changes of 17 discharged patients with covid-19, and to compare the progression trend of imaging pattern and clinical parameters. this retrospective study was approved by the institutional review board. the requirement for informed patient consent was waived by the ethics committee for the emerging infectious disease. between december 20 th , 2019 and february 2 nd , 2020, 17 patients with confirmed covid-19 who underwent at least three serial chest ct scans were retrospectively included. throat swab samples were collected for confirmation of covid-19 by the rt-pcr as previously described [9] [10] . all the patients showed improvement both clinically and on thin-section ct imaging on discharge. the standard for survive and discharge of patients was according to the guideline of diagnosis and treatment of pneumonia caused by sars-cov-2 (trial sixth version) published by the china ministry of health [22] , which include: temperature returning to normal for more than 3 days, both the clinical and chest imaging showing significant improvement, and two consecutive respiratory pathogen nucleic acid tests turning negative (the interval at least 24 hours). three readers (x.h.,y.c., m.d.) recorded the clinical parameters of the patients during treatment. ct was performed using the following ct scanners: somatom definition as+, siemens healthineers, germany. ct scan parameters were as described in our previous study [16] . all follow-up scans were obtained by using the same scanner used to obtain the initial scans. of 17 6 cases, four scans were available in 14 cases, and three scans in 3 cases. the initial images and follow-up chest ct scans obtained from 17 patients were reviewed by three experienced radiologists (h.s., n.j., y.c.). all digital imaging and communications in medicine (dicom) images were analyzed from the ct studies without access to clinical findings of the patients. after separate evaluations, disagreements wherever found were solved by discussion and consensus. on each ct scan, lung segment involved, the location of lesion categorized as central, peripheral or both; and the distribution of opacities classified as being predominantly subpleural, peribronchovascular or random were recorded. the extent of lesion involvement was categorized as focal, multifocal, diffuse. the predominant pattern was categorized as ground glass opacities, consolidation, reticular and mixed pattern. in addition, the margin definition, interlobular/septal thickening , crazy paving (thickened interlobular septa and intralobular lines superimposed on a background of ground-glass opacity) [23] , air bronchogram, bronchiolectasis, cavitation, calcifications, thickening of the adjacent pleura , evidence of pulmonary fibrosis , tree-in-bud, pleural effusion and lymphadenopathy were also documented. the extent of pulmonary abnormalities on thin-slice ct was also evaluated. a semi-quantitative ct scoring system was used to quantitatively estimate the pulmonary involvement of all these abnormalities on the basis of the area involved, as previously reported by ooi et al [24] . after evaluation, the scans were classified according to the time from the admission to 1, 2, 3, and 4 weeks. the analyses were performed using sas (sas, version 9.4, sas institute, cary, nc, usa). distribution normality was assessed using the kolmogorov-smirnov test. normally, non-normally distributed data and categorical variables were expressed as the mean ± standard deviation, median (interquartile range) and frequency (percentage), respectively. differences between weeks were analyzed by generalized linear mixed models (for categorical data) or linear mixed models (for continuous data without/with square root transformation). a p value <0.05 (two-tailed) was considered to be statistically significant. clinical characteristics and laboratory findings in all patients on admission are summarized in the mean time of treatment was 22 days (range, 18-31 days). patients were treated by anti-viral drug (ganciclovir, abidol hydrochloride, oseltamivir, or interferon) and empirical antibacterial drug (moxifloxacin hydrochloride, amoxicillin, meropenem or linezolid). abidol hydrochloride is approved in china and russia as an antivirals drug for influenza treatment. the hexadecadrol (5mg) was given to three patients (p6, p14 and p17), and the methylprednisolone(40mg) and auxiliary ventilation were used in patient 1 and 2. there was a marked resolution of fever within the first two week since admission (figure1a, p＜0.0001) and improvement in oxygen saturation and heart rate ( figure 1b ) within the first two weeks ( table 2) . there was also a trend of improvement in lymphocyte (figure 1d ), crp and esr (figure1e) within the first week (table 2 ). there was an initial increase of alt level in 3 rd week followed by improvement, and a slow continuous increase in ast level from 2 nd week ( figure 1f , table 2 ). as table 3 and 4 illustrate, the initial chest ct in first week was abnormal in all patients (100%). the mean ct score was 4.3 per/ patient (ranged from 1 to 11). the predominant ct feature was poor-defined (88.2%) ground-glass opacities (ggo, 76.5%) with enlarged pulmonary vessels as figure 3 showed, there was a marked increase of mean ct score of all patients from 1 st week to 3 rd week (4.3, 8.2 and 7.2 per/ patients in 1 st , 2 nd and 3 rd week, respectively). after that, a decrease to a mean ct score of 4.2 occurred in the 4 th week(p=0.0190). when analyzing the evolution of ct findings, the present study had identified three patterns of radiographic progression ( figure 3 ). type 1 evolution pattern was the most common observed in 12 patients (70.6%), with initial radiographic deteriorations in the 2 nd week followed by radiographic improvement in the 3 rd and 4 th week ( figure 4 ). four patients (23.5%) had type 3 evolution pattern (static radiographic change, figure 5 ). one patient (patient 15, 5.9%) had type 2 pattern (progressive radiographic improvement, figure 6 ). the predominant abnormality of ct changes through 4 weeks are shown in table 4 and figure 7 . crazy paving appearance had the highest frequency in 2 nd week (76.5%), but disappeared in the 4 th week(p＜0.0001). pleural effusion appeared from 2 nd week (11.8%) and peaked at 3 rd week(23.5%). in the 4 th week, bronchiectasis was identified in 2 patients (15.4%) and evidence of pulmonary fibrosis was seen in 3 patients (23.7%, p=0.0012). presence of subpleural nodule was 10 found in one scan(p7) at 3 rd week, which was dissipated on follow-up ct scans. the other findings such as air bronchogram, enlarged pulmonary vessels, thickening of the adjacent pleura and interlobe fissure displacement were observed over 4 weeks. in our study, the age of patients was 40±6 years old, younger than previously reported [9] [10] [11] [12] . besides, five patients (29.4%) had underlying diseases, similar to previous studies [9, 10] . there was a female gender predilection (male: female, 6:11). on the contrary, covid-19 patients were mostly men (73%) in a previous study [9] . such a discrepancy may be due to the different study [19, 20] . there was a marked resolution of fever within the first week, and improvement in oxygen saturation and heart rate within the first two weeks. there was also a trend towards improvement in lymphocyte, crp and esr within the first week. however, there was an initial increase of alt level in the 3 rd week followed by improvement, and a slow continuous increase of ast level from 2 nd weeks in those patients. the preliminary results indicate that patients with covid-19 may develop liver damage during hospitalization. this may due to the viral infection of liver cells [25] , therapeutic medications [25, 26] or immune-mediated inflammation [25] causing damage to liver tissue. initial chest cts were abnormal in all patients (100% infections, which was linked to variable histopathologic changes, such as diffuse alveolar damage or interstitial (intrapulmonary or airway) inflammatory cell infiltration [27, 28] . moreover, the enlarged pulmonary vessels also indicated an inflammatory cell infiltration of the vessels. the peripheral, subpleural lung involvement bare some resemblance to sars-cov [17] [18] [19] and mers-cov [20, 21] . however, in contrast, sars-cov infection usually presents with unifocal opacification [24] . the focal or multifocal of lesion involvement were common ct findings in our case series of covid-19 in early stage, consistent with the report by chuang et al. [15] . other common ct findings included thickening of the adjacent pleura (41.2%), crazy paving (35.3%), air bronchogram (29.4%) and interlobar fissural displacement (23.5%). a marked increase of mean ct score was noted during the 1 st to 2 nd week (from 4.3 to 8.2). then, there was a slow decline in ct score (7.2) at 3 rd week in 58.8% (10 of 17) of patients. thereafter, the extent of changes rapidly decreased to a mean ct score of 4.2 in 4 th week indicating resolution. when analyzing the evolution of ct finding of all patients by time, the 12 present study found that most patients recovering from covid-19 showed an initial radiographic deterioration to a peak (mean time, 2 nd week) followed by radiographic improvement in the 3 rd and 4 th week. nevertheless, there was remarkable resolution of fever and improvement in oxygen saturation and heart rate, lymphocyte, crp and esr within the first two weeks. accordingly, we may conclude that the progression course of ct pattern was later than the clinical parameters within the first two weeks. this may result from inflammatory reaction occurring earlier than the morphological changes of lung. thereafter, the radiologic findings and clinical parameter showed a synchronization of improvement at 4 th week. although, the four patients had a static radiographic course, the ct score remained low (＜4) in those cases. therefore, evolution course type 1, 2 or 3 with a consistent low ct score may be associated with a favorable outcome. present study. the crazy paving reached the highest frequency in the 2 nd week (76.5%), but was not seen in the 4 th week. this phenomenon suggests that the presence of crazy paving may be associated with an early period of disease. pleural effusion appeared from the 2 nd week (11.8%) and peaked at the 3 rd week (23.5%). this feature is different from sars-cov [17] [18] [19] and other recent publications on covid-19 [13] [14] [15] , which barely present with pleural effusion. previous studies showed that the presence of pleural effusion in patients infected with mers-cov or avian flu (h5n1) was a poor prognostic factor [20, 29] . nevertheless, in three patients, pleural effusion was totally absorbed on follow-up ct scans. in the 4 th week, bronchiectasis in 2 patients (15.4%) and evidence of pulmonary fibrosis in 3 patients (23.7%) were identified. as antonio et al [30] described, pulmonary fibrosis included parenchymal bands, traction bronchiectasis, and irregular interfaces. they found 15 (62%) of sars patients to have evidence of fibrosis on ct after discharge. since the natural history of covid-19 has not been fully studied, it may be premature to define these pulmonary changes as irreversible and longer-term follow-up is needed. lymphadenopathy, tree-in-bud, masses, cavitation and calcifications were not observed in our case series, which is in line with the previous studies on covid-19 [15] and sars-cov [17] [18] [19] . our study had several limitations. first, this study was performed at a single center with a small sample size. second, the subjects included in our study were all discharged patients, cases of death or other outcomes were not included. hence, natural selection bias may have occurred thirdly, only patients with serial chest ct thin-section ct images available were included, which make the findings non-generalizable to people with milder disease who did not undergo serial ct or those with asymptomatic disease. finally, due to the short time of follow up, the ct findings 14 after discharge were not documented. in conclusion, most patients with covid-19 showed an initial radiographic deterioration to a peak at the 2 nd week since admission followed by radiographic improvement in the 3 rd and 4 th week. the progression course of ct pattern was later than the clinical parameters within the first two weeks, but showed synchronization of improvement in both clinical and radiologic features in the 4 th week. longer-term follow-up is required to determine whether the findings (bronchiectasis and pulmonary fibrosis) in the 4 th week represent irreversible fibrosis. we would like to thank all colleagues for helping us during the current study. we highly appreciate drs. xiaoming yang, md, phd, fsir (radiology, university of washington) and hanping potential conflicts of interest. the authors declare no competing non-financial/financial interest. t, temperature; crp, c-reactive protein, esr, erythrocyte sedimentation rate; ast, aspartate aminotransferase. alt, alanine aminotransferase, copd, chronic obstructive pulmonary disease. data are presented as the n (n/n %), where n is the total number of ct scans with available data. # 14 ct scans were available at 4 th week. *p<0.05 between over 4 weeks since admission determined from serial chest ct scans. type 1 stands for an initial radiographic deterioration to a peak level followed by radiographic improvement, with maximum difference in overall mean lung involvement greater than 25%; type 2 is progressive radiographic improvement. type 3 represents a static radiographic change with no discernible radiographic peak or change in overall mean lung involvement of less than 25% for more than 2 weeks. world health organization. who/novel coronavirus -china notes from the field: a novel coronavirus genome identified in a cluster of pneumonia cases-wuhan evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission novel coronavirus -thailand (ex-china) us centers for disease control and prevention. first travel related case of 2019 novel coronavirus detected in united states genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding early transmission dynamics in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of 2019 novel coronavirus infection in china. medrxiv. preprint first posted online feb chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia. radiology ct imaging features of 2019 novel coronavirus (2019-ncov). radiology online radiological fndings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study severe acute respiratory syndrome: radiographic and ct findings thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease clinical and imaging findings in patients with severe acute respiratory syndrome ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings notice on the issuance of a program for the diagnosis and treatment of novel coronavirus (2019-ncov) infected pneumonia fleischner society: glossary of terms for thoracic imaging severe acute respiratory syndrome: temporal lung changes at thin-section ct in 30 patients liver injury in covid-19: management and challenges drug-induced liver injury -types and phenotypes viral pneumonia after hematopoietic stem cell transplantation: high resolution ct findings high-resolution computed tomography of diffuse lung disease the radiologic manifestations of h5n1 avian influenza thin-section ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience key: cord-317940-yg91bsmm authors: chalumeau, martin; bidet, philippe; lina, gérard; mokhtari, mostapha; andré, marie-claude; gendrel, dominique; bingen, edouard; raymond, josette title: transmission of panton-valentine leukocidin–producing staphylococcus aureus to a physician during resuscitation of a child date: 2005-08-01 journal: clin infect dis doi: 10.1086/431762 sha: doc_id: 317940 cord_uid: yg91bsmm we report the first case of transmission of panton-valentine leukocidin–producing staphylococcus aureus to a physician during the resuscitation of an infant with fatal pneumonia. the physician exhibited numerous furuncles. this case highlights the necessity for health care workers to protect themselves against transmission of infectious diseases from patient to care giver. we report the first case of transmission of panton-valentine leukocidin-producing staphylococcus aureus to a physician during the resuscitation of an infant with fatal pneumonia. the physician exhibited numerous furuncles. this case highlights the necessity for health care workers to protect themselves against transmission of infectious diseases from patient to care giver. severe respiratory diseases, in immunocompetent infants, and chronic furunculosis have been associated with staphylococcus aureus strains producing panton-valentine leukocidin (pvl), a cytotoxin that causes leukocyte destruction and tissue necrosis [1] . pvl-producing s. aureus is known to cause rapidly extensive and necrotizing pneumonia in children and young adults. we report the first case, to our knowledge, of transmission of pvl-producing s. aureus to a physician during the resuscitation of an infant with fatal pneumonia. case report. a 4-month-old boy was admitted to the pediatric ward of saint-vincent-de-paul hospital (paris) with a history of 3 days of cough and 1 day of moderate fever. physical examination revealed only mild respiratory distress. findings on a chest radiogram were normal, as were the wbc count and the c-reactive protein and procalcitonin levels. bronchiolitis was diagnosed, and the patient did not receive antibiotics. progressive respiratory failure developed during the 12 h after admission, and the patient required nasal oxygenation. while a physical examination was being performed by a physician, the patient experienced cardiac arrest. resuscitation measures, including oral intubation, oxygenation, bag-valve-mask ventilation, chest compression, and intravenous administration of adrenaline, were immediately performed and were continued for 1 h, without success. it was notable that blood flow from the trachea was observed during intubation. repeated oral and tracheal suction was performed. because the resuscitation took place in the general pediatric ward, health care workers participating in the resuscitation did not wear any protection, such as a facial mask and gloves. a necropsy was performed, and the findings revealed right lobar pneumonia with a necrotizing hemorrhage of the entire right lung and one-half of the left lung. a culture specimen from tracheal aspirates grew methicillin-susceptible s. aureus. identification of s. aureus was based on colony morphology, coagulation of citrated rabbit plasma (biomérieux), and production of clumping factor (determined with the staphyslide test; biomérieux). the methicillin resistance was determined by the kirby-bauer disk-diffusion method, with the use of a 5-mg oxacillin disk, in accordance with the nccls recommendations [2] , after 24-48 h of incubation at 35њc. the s. aureus isolate was only resistant to penicillin and amoxicillin and was susceptible to methicillin, aminosides, macrolides, quinolones, and cotrimoxazole. on day 5, the physician who had performed intubation presented with numerous furuncles of the fingers and face. samples were obtained from the skin lesions. at the same time, nasal samples were obtained from the 15 health care workers who had participated in the resuscitation. s. aureus strains susceptible to methicillin were isolated from samples from the physician and from 5 other health care workers. the isolate from the infant (s1, isolated from a tracheal sample) and 7 isolates from the 6 health care workers (s2, isolated from skin lesions of 1 physician; s4 and s5, from nasal and anal samples from another physician; and s3, s6, s7, and s8, from nasal samples from 4 nurses) were genotyped and analyzed for the presence of the pvl gene. pfge, used for genotyping, was performed on dna fragments obtained by digestion with the restriction enzyme smai by use of a chef-mapper apparatus. the pfge banding patterns were compared visually. strains were considered genetically distinguishable if their restriction patterns differed by у3 bands [3] . the isolates from the infant (s1) and from 1 physician (s2) were genetically related and were unrelated to the other isolates (s3-s8) and the control strain ( figure 1) . the presence of the pvl gene was determined by use of a pcr method described elsewhere [4] . as expected, a fragment of 433 bp was obtained from the s. aureus isolates from the infant (s1) and the physician (s2) but not from the isolates from the other health care workers. discussion. this report describes the transmission of a pvl-producing s. aureus strain from a patient to a health care worker during resuscitation. previous studies have reported transmission of different pathogens during mouth-to-mouth resuscitation: salmonella [5] , herpes simplex [6] , helicobacter pylori [7] , and more recently, severe acute respiratory syndrome coronavirus [8] . in the present case, it is likely that transmission was related to the events leading up to intubation, and contact, droplet, or airborne transmission might have occurred. the transmission might have been promoted by the particular pathogenicity of pvl-producing strain. the transmission of the s. aureus strain to the physician was revealed by furunculosis. as previously reported, most cases of furunculosis (93%) are due to pvl-producing s. aureus [1] . unfortunately, no nasal sample was obtained from this physician. this study highlights the necessity for health care workers to protect themselves against transmission of pathogens from patient to care giver, even in situations where benign infection is suspected. association between staphylococcus aureus strains carrying gene for panton-valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically interpreting chromosomal dna restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing involvement of panton-valentine leukocidin-producing staphylococcus aureus in primary skin infections and pneumonia transmission of salmonella via mouth-to-mouth resuscitation primary herpes simplex infection following mouth-to-mouth resuscitation mouth-to-mouth resuscitation and helicobacter pylori infection possible sars coronavirus transmission during cardiopulmonary resuscitation potential conflicts of interest. all authors: no conflicts. key: cord-299499-66qh3r75 authors: guilamo-ramos, vincent; benzekri, adam; thimm-kaiser, marco; hidalgo, andrew; perlman, david c title: reconsidering assumptions of adolescent and young adult sars-cov-2 transmission dynamics date: 2020-09-07 journal: clin infect dis doi: 10.1093/cid/ciaa1348 sha: doc_id: 299499 cord_uid: 66qh3r75 evidence regarding the important role of adolescents and young adults (aya) in accelerating and sustaining coronavirus disease 2019 (covid-19) outbreaks is growing. furthermore, data suggest two known factors that contribute to high severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmissibility—presymptomatic transmission and asymptomatic case presentations—may be amplified in aya. however, aya have not been prioritized as a key population in the public health response to the covid-19 pandemic. policy decisions that limit public health attention on aya and are driven by the assumption of insignificant forward transmission from aya pose a risk to inadvertently reinvigorate local transmission dynamics. in this viewpoint, we highlight evidence regarding the increased potential of aya to transmit sars-cov-2 that, to date, has received little attention, discuss adolescent and young adult specific considerations for future covid-19 control measures, and provide applied programmatic suggestions. adolescents and young adults (aya), who are between the ages of 10 and 24 years, account for approximately 20% of the total population in the united states (us), but the extent to which aya contribute to forward transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is not fully understood. while, the published evidence remains inconclusive and contains methodological limitations, such as small sample sizes [1, 2] , missing information regarding the timing of test administration relative to exposure [3] , and potential selection and collider biases [4, 5] , the body of evidence supporting the importance of aya in accelerating and sustaining covid-19 outbreaks is growing [6] [7] [8] [9] . policy decisions that are driven by still prevalent assumption of insignificant forward transmission from aya pose a risk to inadvertently reinvigorate local transmission dynamics, particularly as jurisdictions relax social distancing measures, explore ways to reopen schools and universities, and as economies rebound and aya enter the workforce. in this viewpoint, we highlight evidence regarding the increased potential of aya to transmit sars-cov-2 that, to date, has received little attention, discuss adolescent and young adultspecific considerations for future covid-19 control measures, and provide applied programmatic suggestions. examination of two known factors that contribute to high sars-cov-2 transmissibility-presymptomatic transmission and asymptomatic case presentations [10] provides insights into the transmission potential of specific populations. emerging evidence a c c e p t e d m a n u s c r i p t 4 suggests both of these transmissibility factors can be amplified in aya, indicating that aya may represent an under addressed population for sars-cov-2 transmission control. presymptomatic transmission represents a key facilitator of covid-19 outbreaks. the median serial interval of infectious diseases-the time between symptom onset for an index case and the date of symptom onset for secondary cases-can provide information about the likelihood of presymptomatic transmission. for covid-19, a median serial interval that is shorter than the incubation period indicates a high probability of presymptomatic transmission [10] . importantly, emerging evidence suggests the median serial interval for aya may be significantly shorter (1-2 days) [6, 7] than in the general population (4-6 days) [11, 12] . given an estimated median covid-19 incubation period of 5 days [14] , these data suggest presymptomatic transmission may be more common among aya than among older age cohorts. variability in serial intervals has been linked to differential incubation periods, profiles of infectiousness, and contact patterns [14] , the role of which needs to be better understood in aya transmission dynamics. in addition, asymptomatic infections complicate case finding and isolation. therefore, the large proportion of asymptomatic covid-19 cases-estimated to be approximately 16% in a recent meta-analysis, where included estimates ranged from 6% to 41%-facilitates transmission [15] . while low-risk-of-bias estimates of the asymptomatic fraction by age are scarce [15] , a growing body of evidence suggests that disease severity is increasing with age and aya are more likely to experience no or only mild symptoms compared to older age cohorts [12, 16, 17] . while a growing body of evidence suggests comparable viral loads of symptomatic and asymptomatic individuals infected with sars-cov-2 [18] , accurate estimates of forward transmission from aya are not possible in the absence of conclusive data regarding the disease severity-dependent infectiousness of sars-cov-2. furthermore, a c c e p t e d m a n u s c r i p t 5 recognition of the full range of covid-19 clinical manifestations in aya continues to evolve. most recently, aya have presented with atypical symptoms, such as multi-system inflammatory syndrome in children, presumed to be associated with covid-19 [19] . given slowly expanding testing capacity, clinically defined testing criteria, and prioritization of severely symptomatic cases for covid-19 testing, timely identification and isolation of aya infected with sars-cov-2 but with no, mild, or atypical symptoms is less likely, thereby increasing the risk of forward transmission. despite recent expansions, significant gaps and regional differences in testing capacities for sars-cov-2 in the us persist. limited testing capacities complicate surveillance efforts and likely result in a substantial undercount of true infections. testing practices, which prioritize severe cases and patients who present with specific symptoms, render available covid-19 case data for aya particularly unreliable. march 29-june 27 data from commercial laboratories-which process approximately 80% of all covid-19 tests nationwide-show that individuals 5-17 years accounted for only 3.5% of all covid-19 tests conducted in the us and a corresponding 4.4% of confirmed covid-19 cases [20] , despite accounting for 17% of the population. however, testing data also show that across the department of health and human services geographic operating regions, the proportion of tests conducted among those aged 5-17 years is approximately proportional to the share of confirmed covid-19 cases accounted for by the age group ( figure 1 ). in other words, low testing rates in the 5-17 year age cohort could significantly contribute to the low covid-19 case counts, particularly given that the proportion of positive tests among individuals aged 5-17 years (12.3%) is higher than for any other age group (0-4 years: 8.4%, 18-49 years: 10.8%, 50-64 years: 9.0%, ≥ 65 years: 7.5%) [20] . a c c e p t e d m a n u s c r i p t 6 while available surveillance statistics fail to disaggregate young adults, the data presented here for children and adolescents aged 5-17 years are compelling in illustrating the impact of low testing rates on the disproportionately small number of young people in national case statistics, serving as further evidence for the increased risk of undetected infection in young people. in fact, serologic testing data from new york state [21] identified a similar seroprevalence of sars-cov-2 antibodies among young adults 18-24 years of age (10.9%) and among older adults aged 55-64 (11.9%), 65-74 (10.4%), as well as among those aged ≥ 75 (9.5%). similarly, a population-based study in geneva, switzerland, found the seroprevalence of sars-cov-2 antibodies to be similar in the age cohorts 10-19 years (9.6%), 20-49 years (9.9%), and 50-64 years (7.4%), but found lower seroprevalence among those younger than 10 years (0.8%) or older than 64 years (4.1%) [22] . importantly, available serologic data may reflect the impact of social distancing measures (e.g., school closures, shelter-in-place orders) that have been implemented promptly during the epidemic and have been shown to disproportionately reduce the contact rates of aya [23] , leaving uncertainty regarding the validity of inferences about covid-19 prevalence distributions across age groups in the absence of these control measures. it is important to note that much of the available evidence that suggests infrequent forward transmission of sars-cov-2 from young people relies on data specific to young children (<10 years), fails to disaggregate young children from adolescents (10-19 years), or excludes young adults (20-24 years) [1, 2, 24] . however, physiological and behavioral factors indicate aya may have greater susceptibility and transmission potential as compared to younger children. for example, ace-2 expression, which has been linked to covid-19 susceptibility, has been shown to increase with age, with ace-2 expression among young a c c e p t e d m a n u s c r i p t 7 adults aged 20-24 and adolescents aged 10-19 years falling within 2% and 10% of the expression among older adults (≥25 years), respectively, while young children show significantly lower expression (>20% difference) [25] . interestingly, a recent mathematical modeling analysis found that a decreased sars-cov-2 susceptibility in the age groups 0-9 years and 10-19 years by approximately 50% relative to individuals aged 20 or older is consistent with observed case data from various settings globally [17] . in contrast, susceptibility of individuals aged 20-29 was estimated to be approximately as high as that in older age cohorts. given that disease susceptibility is determined by biological factors and that adolescent physiological development is gradual and takes place over the course of adolescence rather than instantaneously, the estimated stepwise increase in susceptibility, doubling from those younger than 20 years to those aged 20 or older, is unlikely to be an accurate representation of the underlying physiological changes. a biologically more plausible scenario is that sars-cov-2 susceptibility in aya mirrors a developmental trajectory analogous to the one shown for ace-2 expression, with susceptibility among older adolescents and young adults approaching that of older adults and with susceptibility among younger adolescents increasing gradually throughout pubertal development. importantly, data from both surveillance and research studies that disregard important developmental stages in early, mid, and late adolescence as well as young adulthood and may obscure gradual but meaningful changes in sars-cov-2 susceptibility, resulting in the underestimation of true susceptibility among aya. adolescent and young adult-specific data furthermore, behavioral factors unique to aya may increase the risk of forward transmission of sars-cov-2 relative to both younger children and older adults. for example, data suggest aya tend to have a higher frequency of contacts effective in the a c c e p t e d m a n u s c r i p t 8 transmission of respiratory infections (i.e., close contact with individuals in enclosed settings for longer than 5 minutes) than do younger children and older adults [23] . in addition, motivating aya to adhere to social distancing and other covid-19 control strategies (e.g., hand washing, using hand disinfectant, etc.) appears to be particularly difficult [26] . for these reasons, we argue that aya represent a distinct population warranting specific considerations in covid-19 prevention and containment efforts. taken as a whole, the evidence regarding the prevalence of covid-19 among aya and their role in the forward transmission of sars-cov-2 in the us is not conclusive, but data suggest current estimates may inadequately reflect the contribution of aya to accelerating and sustaining covid-19 outbreaks. these insights have important implications for improving the effectiveness and durability of the covid-19 response. as an evidence base for the design, communication, and implementation of aya-centered control measures, detailed age-disaggregation of surveillance and research data as well as research on aya exposures, household data, prevention behavior, and symptom profiles is needed. the sars-cov-2 transmission potential of young people, including aya, has received increasing attention in the context of scientific, political, and public discourse about the utility of school closures for population-level covid-19 control. a growing corpus of research, including mathematical modeling studies, suggests moderate effects of school closures on population level incidence of covid-19 [17, 27] . this notwithstanding, caution is warranted when implementing gradual school and university reopening, given that the quality of available surveillance data remains questionable, the published evidence contains methodological limitations, and forward transmission of sars-cov-2 from aya is not fully understood. accordingly, there is growing consensus in the scientific community that a c c e p t e d m a n u s c r i p t 9 approaches that combine the return to in-person instruction with other effective control measures, such as reduced student density in classroom indoor settings, maintenance of adequate social distance, and use of face masks, hand disinfectant, etc. [28] , represent the most promising strategies for balancing the benefits and potential risks of reopening school and university systems in the us and globally. important to note is, however, that current public health measures do not prioritize aya-specific considerations in domains of the covid-19 response other than school closures, which represents a missed opportunity for increasing the long-term effectiveness of covid-19 control measures. we argue aya-specific considerations for testing, averting household transmission, leveraging family influence, aya brain development, and aya social context and provide a set of applied programmatic suggestions to strengthen the design, communication, and implementation of future covid-19 control measures that warrant consideration by policy makers and public health professionals (table 1) . as part of the efforts to scale-up national, regional, and local covid-19 testing capacities, targeted strategies for increasing the proportion of tests administered to aya are warranted. to date, settings frequented by youth-including schools, households, and community settings-have not been prioritized as sentinel surveillance sites involved in the testing and contact tracing of asymptomatic or presymptomatic cases, despite lower healthseeking behavior among aya. of particular importance are public health plans that enable robust testing of students, their household contacts and school staff after reopening. in addition, tailored strategies to identify and isolate priority contacts of infected students in school and university contexts are needed. furthermore, schools and universities will have to a c c e p t e d m a n u s c r i p t 10 ensure that online learning opportunities are maintained for quarantined students, in addition to regular in-person instruction. evidence suggests sars-cov-2 is frequently transmitted among cohabiting household members and at family gatherings. the household secondary attack rate of sars-cov-2 is estimated to be 10.6%, as compared to less than 1% in hospital and community settings in the us [29] . while additional data are needed to delineate the directionality of documented household transmissions (aya to non-aya vs. non-aya to aya), it is important to note that aya are more likely than older individuals to live with family members and will be more likely to have a disproportionately higher number of contacts with whom they have had prolonged, physically close indoor contact, potentially facilitating aya to non-aya sars-cov-2 transmission [30] . furthermore, the risk of sars-cov-2 transmission from aya to older individuals, who are vulnerable to severe covid-19, may be increased in multigenerational households, particularly given that aya are frequently employed in occupational setting that increase risk of exposure to sars-cov-2. aya ages 16-24 make up 22% of the service industry workforce in the us. notably, aya are markedly overrepresented in occupations with significant face-to-face human contact. for example, aya represent approximately half of all cashiers and waiters in the us [31] . despite the importance of family and household contexts in facilitating the spread of sars-cov-2, limited guidance is provided for the prevention of asymptomatic or presymptomatic transmission in such settings. further, the ability of aya to maintain 6-foot distancing from sick or infectious household members as recommended may be limited, as aya are less likely to have control of family mitigation strategies. particular attention should a c c e p t e d m a n u s c r i p t 11 be given to ethnic/racial minority youth, who are more likely to live in multigenerational households and to work in the service sector. research indicates families-parents, in particular-matter in relation to aya health behavior [32] . however, current public health messaging regarding covid-19 only minimally addresses the parental role in influencing aya behavior. specific guidance for parents regarding communication and monitoring strategies that positively influence aya health behavior in the context of covid-19 is sorely needed. limited attention has been paid to the impact of covid-19 and the associated immune system response on the developing aya brain. individuals infected with sars-cov-2 have presented with acute neuropsychiatric symptoms, including encephalopathy, stroke, and muscle weakness [33] . several neuropsychological symptoms as early signs of covid-19 have been identified, including the temporary loss of olfactory and gustatory perception, which may be common in aya [34] . furthermore, possible mechanisms for long-term neuroinvasive consequences of covid-19 in youth have been described in the literature [35] . increased attention should be given to the study of potential immediate and long-term effects of covid-19 on the developing nervous system. future research should also seek to understand whether sars-cov-2 infection can result in impairment to aya decision-making and reduce adequate preventive behaviors. a c c e p t e d m a n u s c r i p t 12 the socio-developmental context of aya and its potential influence on sars-cov-2 transmission dynamics has received limited attention. aya's decisions regarding health behaviors are more likely to be shaped by immediate social and emotional benefits rather than by consideration of more distal health consequences [36] , increasing the likelihood of non-adherence to social distancing [26] . this is of particular concern, given that the gradual loosening of shelter-in-place measures will increase opportunities for aya to engage in group interactions with peers in community settings that may facilitate transmission of sars-cov-2. elevated contact rates [23] and increased likelihood of contacts not known by name represent potential challenges for effective contact tracing among aya. however, novel mobile applications designed to facilitate covid-19 contact tracing may represent an effective and aya-friendly strategy. while evidence shows adolescents and young adults (aya) acquire and transmit sars-cov-2, aya have not been prioritized as a key population in the covid-19 response. consideration of factors unique to aya in the design, communication, and implementation of covid-19 control measures is warranted. adolescence and young adulthood is a distinct period of neurobiological development in which changing connectivity between brain regions results in the prioritization of short-term social and emotional reward (e.g., group interactions, normative life events), rather than the longterm health consequences of potential covid-19 infection [s1] a . m a n u s c r i p t 22 evidence-based best practices for a safe return to in-person instruction are needed. evaluation and optimization of recommended mitigation measures in schools/universities is needed (i.e., situate students at least 6 feet apart, reduce class sizes, minimize mixing between student class groups, assign permanent seating, use of outdoor spaces for classroomrelated activities) [s5, s6]. mobility patterns such as cross-country travel of out-of-state university students may reinvigorate sars-cov-2 transmission in communities with controlled local epidemics. robust quarantine strategies for traveling students represent an essential piece of university covid-19 mitigation plans. attendance of in-state universities may reduce travel-related spread of covid-19. adolescence and young adulthood is a period of optimal health, in which aya infected with sars-cov-2 may be more likely to experience mild, no, or atypical symptoms-making timely identification and isolation of aya with covid-19 a challenge. high-volume, high-frequency, and symptomunspecific testing in at-risk aya populations (e.g. schools, universities) can reduce reproductive rates and account for the asymptomatic transmission potential among aya [s7, s8]. frequent and widespread testing of asymptomatic aya, for example in school or university settings, may be costprohibitive and takes up laboratory capacity. batch testing is scalable, cost-effective, and should be considered for school/university testing initiatives [s9, s10]. school and university testing may exclude vulnerable aya who are often disconnected from educational institutions (e.g. criminal justice-involved youth, pregnant or parenting youth, sexual/gender minority youth, youth with mental health issues). once available, point-of-care covid-19 testing should be offered routinely in youthserving health and social service organizations, particularly in vulnerable communities most heavily affected by covid-19 [s11, s12]. family/ household transmission aya are more likely than older individuals to co-reside with others (e.g., family members, roommates), increasing the risk of household transmission [s13]. evidence-based and specific guidance to minimize transmission within households and among families, and particularly from aya to parents and other family members, is needed. there is limited guidance that addresses the needs of aya residing in households within low socioeconomic status communities. community-involved initiatives to develop culturally appropriate, feasible, effective, and replicable covid-19 mitigation strategies within socially and economically vulnerable households is warranted [s14-s16]. aya are a key population for efforts to promote uptake of covid-19 prevention methods that are currently under development, including vaccines, particularly given that vaccination rates among aya remain low and have decreased during the covid-19 pandemic [s17, s18]. aya participation in ongoing and future covid-19 trials for vaccines, hyperimmune globulin and convalescent plasma therapy, preexposure and post-exposure prophylaxis is important. behavioral interventions to promote the uptake of a novel covid-19 vaccine as well as available influenza vaccines need to account for the unique needs of aya. m a n u s c r i p t practical strategies to avoid adolescent disengagement in remote schooling, address technology/language barriers to online schooling, and ensure adequate learning environments at home are warranted. in the current recessionary economic climate, opportunities for aya to enter the workforce are limited. in the contemplated rollout of workforceintensive covid-19 mitigation initiatives such as contact tracing, community-based testing, or public health messaging, aya training and professional development programs for key positions such as community health workers, contact tracers, etc. should be considered. covid-19 prevention represents only one of many aya health priorities shaping long-term adult health and wellbeing (e.g. sexual and reproductive health, mental health, etc.) universal access to youth-friendly covid-19, primary and specialty healthcare, and auxiliary services is imperative for reducing long standing health disparities in the us. existing non-covid-19 specialty services for aya represent an opportunity for integration of covid-19 mitigation strategies. notes: a no evidence of secondary transmission of covid-19 from children attending school in ireland covid-19 in schools -the experience in nsw variation in falsenegative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure susceptibility to and transmission of covid-19 amongst children and adolescents compared with adults: a systematic review and meta-analysis collider bias undermines our understanding of covid-19 disease risk and severity rapid asymptomatic transmission of covid-19 during the incubation period demonstrating strong infectivity in a cluster of youngsters aged 16-23 years outside wuhan and characteristics of young patients with covid-19: a prospective contact-tracing study epidemiological and clinical characteristics of covid-19 in adolescents and young adults presumed asymptomatic carrier transmission of covid-19 covid-19 outbreak among college students after a spring break the future of the covid-19 pandemic: lessons learned from pandemic influenza serial interval of covid-19 among publicly reported confirmed cases epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application serial interval of sars-cov-2 was shortened over time by nonpharmaceutical interventions estimating the extent of true asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis coronavirus disease 2019 in children -united states age-dependent effects in the transmission and control of covid-19 epidemics asymptomatic transmission during the covid-19 pandemic and implications for public health strategies hyperinflammatory shock in children during covid-19 pandemic covidview: a weekly surveillance summary of u.s. covid-19 activity amid ongoing covid-19 pandemic, governor cuomo announces results of completed antibody testing study of 15,000 people showing 12.3 percent of population has covid-19 antibodies seroprevalence of anti-sars-cov-2 igg antibodies in geneva, switzerland (serocov-pop): a population-based study changes in contact patterns shape the dynamics of the covid-19 outbreak in china children are not covid-19 super spreaders: time to go back to school nasal gene expression of angiotensin-converting enzyme 2 in children and adults public attitudes, behaviors, and beliefs related to covid-19, stay-at-home orders, nonessential business closures, and public health guidance -united states school closure and management practices during coronavirus outbreaks including covid-19: a rapid systematic review re-opening schools safely: the case for collaboration, constructive disruption of pre-covid expectations, and creative solutions active monitoring of persons exposed to patients with confirmed covid-19-united states contact tracing during coronavirus disease outbreak employed persons by detailed occupation and age parental monitoring and its associations with adolescent sexual risk behavior: a metaanalysis are we facing a crashing wave of neuropsychiatric sequelae of covid-19? neuropsychiatric symptoms and potential immunologic mechanisms anosmia and ageusia: not an uncommon presentation of covid-19 infection in children and adolescents severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the central nervous system adolescent brain response to reward is associated with a bias toward immediate reward clinical characteristics and results of semen tests among men with coronavirus disease 2019 a c c e p t e d m a n u s c r i p t 18 key: cord-326138-16kpn9db authors: weinstein, robert a.; singh, kamaljit title: laboratory-acquired infections date: 2009-07-01 journal: clin infect dis doi: 10.1086/599104 sha: doc_id: 326138 cord_uid: 16kpn9db laboratory-acquired infections due to a wide variety of bacteria, viruses, fungi, and parasites have been described. although the precise risk of infection after an exposure remains poorly defined, surveys of laboratory-acquired infections suggest that brucella species, shigella species, salmonella species, mycobacterium tuberculosis, and neisseria meningitidis are the most common causes. infections due to the bloodborne pathogens (hepatitis b virus, hepatitis c virus, and human immunodeficiency virus) remain the most common reported viral infections, whereas the dimorphic fungi are responsible for the greatest number of fungal infections. because of the increasing attention on the role of the laboratory in bioterrorism preparation, i discuss the risk of laboratory-acquired infection with uncommon agents, such as francisella tularensis and bacillus anthracis. physicians who care for a sick laboratory worker need to consider the likelihood of an occupationally acquired infection while advising exposed laboratory workers about postexposure prophylaxis. in addition, physicians should be aware of the importance of alerting the laboratory if infection with a high-risk agent is suspected. formed with a sputum specimen and returned a positive result for sars coronavirus. additional epidemiologic investigation revealed that the laboratory where he worked was also involved in research on sars coronavirus and that one of the cell cultures of west nile virus was contaminated with the same infecting strain of sars coronavirus. although this case represents an exceptional event, it serves to highlight the inherent risk posed to laboratory workers by virtue of their occupation. infectious diseases specialists may be asked to evaluate an ill laboratory worker. this article provides a framework for assessment of such patients by reviewing the published literature on infections acquired in the clinical diagnostic laboratory. laboratory infections due to a wide variety of bacteria, viruses, rickettsiae, fungi, and parasites have been described in the literature. the largest survey of infections was reported in 1976 by pike [3] , who found that 4079 laboratory-acquired infections were due to 159 agents, although 10 agents accounted for 150% of the cases (table 1) [3, 4] . at least 173 deaths have resulted from laboratory-acquired infection [5, 6] . however, care should be taken in the interpretation of historical surveys, because some infections (e.g., q fever, venezuelan equine encephalitis, and dermatomycoses) occurred predominantly in research and animal laboratories, and many of these infections (e.g., psittacosis and typhoid) were reported before 1955 [1, 3] . surveys of diagnostic laboratory workers in the united kingdom conducted since 1971 have reported that tuberculosis and enteric infections (especially shigellosis) were the most common laboratory-acquired infections [7, 8] . a follow-up survey of uk laboratories from 1994-1995 reported that gastrointestinal infections predominated, particularly shigellosis [9] . similar results were obtained from a survey of clinical microbiology laboratories in utah from the period 1978-1992, with shigellosis reported to be the most common laboratory-acquired infection [10] . these results suggest a shift in the pattern of laboratoryacquired infections, with enteric infections predominating. however, no denominator data have been provided that would help determine the actual risk or incidence of infection for laboratory workers. in a 2002-2004 survey of clinical laboratory directors who participate in clinmicronet, an online forum sponsored by the american society of microbiology, 33% of laboratories reported the occurrence of at least 1 laboratory-associated infection (table 2) [11] . the 3 most common laboratory-acquired infections were shigellosis, brucellosis, and salmonellosis. in contrast, the highest incidences of infection were associated with brucella species (641 cases per 100,000 laboratory technologists, compared with 0.08 cases per 100,000 persons in the general population) and neisseria meningitidis (25.3 cases per 100,000 laboratory technologists, compared with 0.62 cases per 100,000 persons in the general population). of note, the annual number of laboratory-acquired infections has steadily decreased since 1965 [3, 5] . for example, survey results from the united kingdom from the period 1988-1989 found an infection incidence of 82.7 cases per 100,000 person-years, compared with an incidence of 16.2 cases per 100,000 person-years during the period 1994-1995 [8, 9] . this finding undoubtedly reflects an improved awareness of the hazards of working with infectious agents and placement of a greater emphasis on laboratory safety, such as through the use of personal protective equipment. in addition, there have been improvements in laboratory design, such as the use of laminarflow biological safety cabinets (bscs), which provide unidirectional airflow that entraps any aerosolized particles in the airstream and subsequently into air filters [12] . bacteria account for the largest proportion of infections (43%) in diagnostic laboratories, with over 37 different species reported [3] . below, i highlight common causes of infection that are currently of most concern. brucella species. brucellosis continues to be the most frequently reported laboratory-associated bacterial infection [13] [14] [15] [16] [17] [18] [19] . in the united states, brucella infection is one of the most common laboratory-acquired infections, accounting for 24% of laboratory-acquired bacterial infections and 11% of deaths due to laboratory infection [20] . aerosolization is the major source of transmission, but the bacterium can also be transmitted via direct contact. however, in many reported cases, it has not been possible to accurately determine the mechanism for transmission. the disease has also affected janitors and persons who have made brief visits to the laboratory [21] . person-to-person transmission is rare, although a case of brucella infection transmitted from a laboratory worker to a spouse has been documented, presumably through sexual intercourse [22] . although no controlled studies have been performed to assess the benefit of postexposure prophylaxis (pep), it should be considered for laboratory workers who have high-risk exposure to brucella species (e.g., because of direct manipulation of brucella cultures outside of laminar-flow bscs). doxycycline (or trimethoprim-sulfamethoxazole for pregnant women) and rifampin have been frequently used for pep [13, 23] . in a report from canada, 26 laboratory workers were exposed to brucella melitensis, which had been isolated from a patient from india with a draining chest sinus. six laboratory workers were offered pep with doxycycline (100 mg twice daily) and rifampin (600 mg daily) for 3 weeks. only 1 person declined pep; 10 weeks after exposure, the technologist developed fever (temperature, р40њc), and 2 sets of blood cultures confirmed brucellosis. none of the other laboratory workers developed infection or evidence of seroconversion. follow-up serologic tests should be performed for all exposed individuals, probably every fortnight for the first 3 months, then every month for an additional 6-9 months [13, 18] . n. meningitidis. sejvar et al. [24] examined the risk of laboratory-acquired n. meningitidis infection using postings on listservs, to obtain reports of laboratory-acquired meningococcal disease occurring worldwide during the period 1985-2001. sixteen cases of probable laboratory-acquired meningococcal disease were identified, including 6 cases in the united states. nine cases (56%) were due to serogroup b, and 7 (44%) were due to serogroup c. overall, 8 cases (50%) were fatal. all cases occurred among clinical microbiologists and were likely due to exposure to aerosols containing n. meningitidis. the calculated attack rate was 13 cases per 100,000 microbiologists, compared with an attack rate of 0.3 cases per 100,000 persons among the general population. the results of this analysis suggest that laboratory-acquired meningococcal disease represents a significant occupational hazard to clinical microbiologists. although primary prevention of laboratory-acquired meningococcal disease should focus on appropriate handling and manipulation of cultures in a laminar-flow bsc, all laboratory microbiologists should be offered the tetravalent vaccine [25] . it will decrease-but not eliminate-the risk of infection, because it is less than 100% effective and does not provide protection against serogroup b [26] . microbiologists who inadvertently manipulate invasive n. meningitidis isolates on an open bench-top in a manner that could result in aerosolization should consider pep with either a single 500-mg dose of ciprofloxacin or 600 mg of rifampin given twice daily for 2 days [24] . mycobacterium tuberculosis. early surveys of laboratoryacquired tuberculosis found an incidence of tuberculosis among laboratory personnel 3-9 times greater than that in the general population [7, 27] . however, unless there is some accident to which the infection can be traced, it is difficult to state with certainty that tuberculosis was laboratory acquired, because of the potential for exposure outside of the workplace and the long incubation period before symptomatic disease develops. m. tuberculosis can be isolated from a variety of clinical spec-imens, and manipulation of specimens or cultures that generate aerosols is the most important risk factor for acquiring tuberculosis in the laboratory. the high infectivity of m. tuberculosis is related to the low infective dose for humans (50% infective dose, !10 bacilli) [28] . the use of laminar-flow bscs for aerosol-generating manipulations with biosafety level у2 practices and fit-tested respirators with n-95 rating should be routinely used [12, 29] . laboratory personnel should undergo an annual mantoux purified protein derivative skin test or an interferong release assay to demonstrate conversion. persons with positive test results should be evaluated for active tuberculosis by chest radiography. in the event of accidental exposure, laboratory workers should be tested 3 and 6 months after the accident, and persons with new conversion should be offered prophylaxis [29] . francisella tularensis. f. tularensis is a fastidious, gramnegative coccobacillus that is infrequently encountered in the clinical microbiology laboratory, but it has gained increased importance because of its possible use as a bioterrorism agent [30] . the greatest hazard to laboratory workers is from exposure to infectious aerosols from manipulation of cultures. clinicians should be aware that, although patients with tularemia, brucellosis, or endemic mycoses do not pose a communicable disease risk to health care workers, specimens obtained from these patients pose a significant threat to laboratory workers. shapiro et al. [31] described 12 laboratory workers who were exposed to f. tularensis after clinicians failed to notify the laboratory about a suspected case of pneumonic tularemia in a 43-year-old man who eventually died. blood cultures, sputum cultures, and autopsy pleural fluid were all positive for gramnegative coccobacilli, which failed to grow on sheep blood agar and macconkey agar and were initially misidentified as haemophilus species. eleven laboratory workers and 2 autopsy personnel with high-risk exposure to f. tularensis received pep with doxycycline (100 mg twice daily for 14 days), with no resulting infections. to minimize the risk of exposure of laboratory workers, any suspicion about infection with a high-risk pathogen should be immediately communicated to the laboratory. this practice not only protects the staff but also benefits the patient, because a faster and more directed laboratory evaluation can be initiated. bacillus anthracis. before the 2001 outbreak of bioterrorism-related anthrax in the united states, anthrax was an uncommon illness in the united states [32] . in march 2002, the centers for disease control and prevention were alerted about a laboratory worker who had received a diagnosis of cutaneous anthrax [33] . one day after he had cut himself over the right jaw while shaving, the patient assisted a coworker in moving vials of b. anthracis from the laminar-flow bsc in the main laboratory to a freezer. the patient had handled the vials without gloves but washed his hands with soap and water. over the next 3 days, the cut over the laboratory worker's jaw increased in size, and he developed low-grade fever, cervical lymphadenopathy, and cellulitis around the scab. cultures of specimens from beneath the scab were positive for b. anthracis, and the patient was treated with intravenous ciprofloxacin and doxycycline and discharged while receiving ciprofloxacin. epidemiologic investigation of this case revealed that the tops of the vials tested positive for b. anthracis. although all specimen processing surfaces were decontaminated with 10% bleach solution, storage vials were sprayed with 70% isopropyl, because bleach caused labels to become dislodged. this case brings the number of cases of bioterrorism-related anthrax identified since 3 october 2001 to 23 and is the first laboratory-acquired case of bioterrorism-related anthrax. enteric pathogens. salmonellosis is one of the most common reported infections in published surveys [3, 5, 6, 8, 11] . blaser et al. [34] reported 32 cases of laboratory-acquired typhoid fever in the united states over a 42-month period from 1977 to 1980, representing 11.2% of the sporadic cases of typhoid fever reported in the united states. of particular concern was that a number of cases occurred in individuals who had not directly worked in the microbiology laboratory, including cases in 2 family members of a microbiologist who worked with salmonella culture, 1 of which proved to be fatal. in fact, ty-phoid fever has accounted for more reported fatalities than any other laboratory-acquired infection [5] . of note, many earlier reported cases of typhoid fever were associated with mouth pipetting and handling of proficiency test strains-practices which are now avoided [1, 35, 36] . in recent surveys, shigella species was the most frequently identified agent of laboratory-acquired infection [9] [10] [11] . one explanation for the large number of reported cases of laboratory-acquired shigellosis is that shigella species are more virulent and require a much lower inoculum to cause illness. however, it is also probably true that microbiology laboratory staff who develop diarrhea are more likely to attempt to establish a cause for their illness, compared with the general population. a number of other enteric pathogens have also been identified as less common causes of laboratory-acquired infection, including clostridium difficile and escherichia coli o157:h7 [11, 37] . viral agents transmitted through blood and bodily fluids cause most of the laboratory-acquired infections in diagnostic laboratories and among health care workers [1] . although the viral hemorrhagic fevers incite the most fear and dominate the imagination of the media and public, the viruses responsible are rare causes of laboratory infection [3, 4] . however, there is always the possibility that an agent not previously seen may be encountered. this occurred in 1967, when 31 workers were infected while handling tissue specimens from african green monkeys, with 7 deaths resulting [38] . the causative agent was named marburg virus, after the town in germany where most cases occurred. of the common blood-associated viruses, hepatitis b virus (hbv) is the most common cause of laboratory-acquired infection [1] . the incidence of hbv infection among all health care workers in the united states is estimated to be 3.5-4.6 infections per 1000 workers, which is 2-4 times than the level for the general population [39] . it is encouraging that, in the 2 most recent surveys of laboratory-acquired infections in the united kingdom, there were no reported cases of hbv infection among laboratory workers [8, 9] . this finding is probably related to the use of universal precautions when handling blood specimens, improvements in needleless devices, and the availability of immunization. because hepatitis b is a vaccine-preventable disease, all laboratory workers should be offered the hepatitis b vaccine without charge. nonimmunized laboratory workers who have percutaneous, ocular, or mucous membrane exposure to contaminated blood should receive pep with hepatitis b immunoglobulin and vaccine [39] . during 2005-2006, there were 802 confirmed cases of acute hepatitis c reported to the centers for disease control and prevention, with 5 occupational exposures (1.5%) to blood [40] . however, there are few data on the incidence of hepatitis c among laboratory workers, and only single case reports in surveys have been performed in the united states and the united kingdom [8] [9] [10] . human immunodeficiency virus (hiv) infection associated with exposure to contaminated blood or body fluids probably causes the greatest concern. the risk of hiv transmission after a percutaneous exposure to hiv-infected blood has been estimated to be ∼0.3%, and the risk has been estimated to be ∼0.09% after exposure to a mucous membrane [41, 42] . data on occupational transmission of hiv from the period 1981-1992 revealed a total of 32 health care workers in the united states with occupationally acquired hiv infection; 25% of these health care workers were laboratory workers [43] . in the event of laboratory-based exposure from a source person with hiv infection, or if information suggests the likelihood that the source person is hiv infected, immediate referral to employee health for hiv pep should be sought [44] . the dimorphic fungi blastomyces dermatitidis, coccidioides immitis, and histoplasma caspsulatum are responsible for the majority of laboratory-acquired fungal infections in the united states (table 1) [1, 3, 4] . although cutaneous infections due to accidental inoculation are documented, most laboratory-acquired infections are caused by inhalation of infectious conidia from the mold form, resulting in pulmonary infection. the mere lifting of a culture plate lid often suffices to cause the release of large numbers of conidia, and should a sporulating culture be dropped, millions of conidia would be dispersed. the risk of infection in the mycology laboratory probably is low, because handling of specimens is done in laminar-flow bscs, and culture plates are secured with shrink seal to prevent accidental opening. however, a greater risk of infection is likely on the aerobic culture bench, because colonies of b. dermatitidis and c. immitis can grow on routine media and may be visible within 2-3 days. it cannot be overemphasized that clinicians who suspect a dimorphic fungal infection should immediately alert the microbiology laboratory. laboratory-acquired parasitic infections are uncommon in the diagnostic microbiology laboratory [1, 3, 6] . approximately 313 cases of laboratory-acquired infection, with a variety of blood and intestinal protozoa, have been reported (table 3) [3, 45] . most of these cases occurred in research and reference laboratories. readers are referred to the review by herwaldt [45] . fifty-two cases of malaria among laboratory workers and health care workers have been reported, with 34 cases reviewed by herwaldt [45] ; 10 cases were due to plasmodium cynomolgi, 9 cases were due to plasmodium vivax, and 15 cases were due to plasmodium falciparum [3, 45] . most of the cases of p. vivax and p. falciparum infection occurred among health care workers and laboratory workers rather than among researchers and resulted from needlestick injuries that occurred while obtaining blood or preparing blood smears from patients [45] . infection due to intestinal protozoa are uncommon in clinical diagnostic laboratories [45] . one case of giardiasis was reported in a clinical laboratory technologist who processed specimens, many of which were in leaky containers. one case of isospora belli infection occurred in a technologist who examined numerous stool specimens from a patient infected with i. belli. laboratory-acquired infection represents an occupational hazard unique to laboratory workers, especially those in the microbiology laboratory. exposures may occur inadvertently, may not even be recalled, or may result from lapses in technique leading to accidental inoculation. however, not every exposure results in infection. a risk assessment for infection based on the host's immune system, mechanism of the exposure, infectious dose of the exposure, virulence of the agent, use of personal protective equipment, and immunization status needs to be performed. the accurate quantification of such risk is unfortunately difficult, because there is no systematic reporting system that monitors the number of laboratory-related exposures and infections. the centers for disease control and prevention has recently convened a committee to address these issues that will, i hope, provide evidence-based guidelines on exposure risk and use of pep. laboratory associated infections and biosafety laboratory-acquired severe acute respiratory syndrome laboratory-associated infections: summary and analysis of 3921 cases past and present hazards of working with infectious agents laboratory-associated infections: incidence, fatalities, causes and prevention continuing importance of laboratoryacquired infections incidence of tuberculosis, hepatitis, brucellosis and shigellosis in british medical laboratory workers infections in british clinical laboratories, 1988-1989 a survey of infections in united kingdom laboratories, 1994-1995 infections acquired in clinical laboratories in utah bacterial and fungal infections among diagnostic laboratory workers: evaluating the risks evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures prevention of laboratoryacquired brucellosis analysis of risk factors for laboratory-acquired brucella infections the spanish co-operative group for the study of laboratory-acquired brucellosis. laboratory-acquired brucellosis: a spanish national survey laboratory-acquired brucellosis brucella abortus infection acquired in microbiology laboratories exposure of hospital personnel to brucella melitensis and occurrence of laboratory-acquired disease in an endemic area epidemiology of laboratory-associated infections laboratory infections due to brucella person-to-person transmission of brucella melitensis brucellosis in pregnant women assessing the risk of laboratoryacquired meningococcal disease laboratory-acquired meningococcal disease-united states centers for disease control and prevention. prevention and control of meningococcal disease: recommendations of the advisory committee on immunization practices (acip) incidence of tuberculosis among workers in medical laboratories centers for disease control and prevention and national institutes of health. biosafety in microbiological and biomedical laboratories mycobacterium: general characteristics tularemia as a biological weapon: medical and public health management exposure of laboratory workers to f. tularensis despite a bioterrorism procedure the sverdlovsk anthrax outbreak of 1979 suspected cutaneous anthrax in a laboratory worker-texas fatal salmonellosis originating in a clinical microbiology laboratory acquisition of typhoid fever from proficiency testing specimens acquisition of typhoid fever from proficiency testing specimens nosocomial and laboratory-acquired infection with escherichia coli o157 zur atiologie einer unbekanten, von affen ausgegangen menschlichen infektionskrankheit the risk of hepatitis b infection among health care professionals in the united states: a review surveillance for acute viral hepatitis-united states occupational risk of human immunodeficiency virus infection in healthcare workers: an overview italian study group on occupational risk of hiv infection. the risk of occupational human immunodeficiency virus in health care workers surveillance for occupationally acquired hiv infection-united states updated us public health service guidelines for the management of occupational exposures to hiv and recommendations for postexposure prophylaxis laboratory-acquired parasitic infections form accidental exposures potential conflicts of interest. k.s. has served on the speakers' bureau for wyeth. key: cord-012511-fl5llkoj authors: meltzer, martin i.; gambhir, manoj; atkins, charisma y.; swerdlow, david l. title: standardizing scenarios to assess the need to respond to an influenza pandemic date: 2015-05-01 journal: clin infect dis doi: 10.1093/cid/civ088 sha: doc_id: 12511 cord_uid: fl5llkoj nan an outbreak of human infections with an avian influenza a(h7n9) virus was first reported in eastern china by the world health organization on 1 april 2013 [1] . this novel influenza virus was fatal in approximately onethird of the 135 confirmed cases detected in the 4 months following its initial identification [2] , and limited humanto-human h7n9 virus transmission could not be excluded in some chinese clusters of cases [3, 4] . there was, and still is, the possibility that the virus would mutate to the point where there would be sustained human-to-human transmission. given that most of the human population has no prior immunity (either due to natural challenge or vaccine induced), such a strain presents the danger of starting an influenza pandemic. in response to such a threat, the joint modeling unit at the centers for disease control and prevention (cdc) was asked to conduct a rapid assessment of both the potential burden of unmitigated disease and the possible impacts of different mitigation measures. we were tasked to evaluate the 6 following interventions: invasive mechanical ventilators, influenza antiviral drugs for treatment (but not large-scale prophylaxis), influenza vaccines, respiratory protective devices for healthcare workers and surgical face masks for patients, school closings to reduce transmission, and airport-based screening to identify those ill with novel influenza virus entering the united states. this supplement presents reports on the methods and estimates for the first 5 listed interventions, and in this introduction we outline the general approach and standardized epidemiological assumptions used in all the articles. given that there had not yet been (and subsequently has not been to date) a pandemic caused by the h7n9 virus, there are no relevant large-population data concerning transmission and clinical impacts of h7n9. we therefore had to consider the potential impacts of disease and interventions for a not fully defined pandemic (ie, a pandemic caused by a generic influenza strain hxny). thus, any model that we built had to allow for a wide range in virus transmissibility and resulting clinical impact. the models had to also fully consider a range of effectiveness of interventions-for example, influenza antiviral drugs could be less effective against the next influenza strain causing a pandemic. given these uncertainties, and the need for a rapid assessment of a large number of factors, the models produced had to meet a number of specifications: had to be produced in a manner that would allow the models to be easily transferred to other units in government and to public health officials, and subsequently used by people who did not build them; had to provide easy identification of all input variables, their values, and ability to rapidly change those values; can be easily stored and resurrected for future use and reference at some unspecified time in the future; and, the results from each model can be readily compared to each other. in response to these specifications, we decided to require that each model be built in a spreadsheet format, and that we would essentially have 1 model for each intervention considered. meeting these specifications had the added value of producing models that readily fit into the existing cdc emergency operations response structure. in this structure, groups called task forces are formed to focus on particular aspects of a response to a public health emergency. for example, for an influenza pandemic response, there are usually task forces that focus on vaccines (eg, recommendations regarding prioritization of vaccine supplies, issues related to distribution), medical countermeasures (eg, recommendations regarding use of drugs for treatment and prophylaxis, use of personal protective equipment such as face masks), and nonpharmaceutical interventions (eg, recommendations regarding school closures, border security, and screening). to allow easy comparison between results (a specification), we standardized a risk space defined by using ranges of transmission and clinical severity from a previously published influenza severity assessment framework ( figure 1 ) [5] . the framework can be used to plot, and compare to historical data, the relative severity of an influenza pandemic (or nonpandemic influenza season). the framework uses 2 scales: a scale of clinical severity, and a scale of transmissibility. the severity scale has a number of components in it, including case-fatality ratio and caseto-hospitalization ratio (table 1 ) [5] . the transmissibility scale is assessed by considering factors such as the clinical (symptomatic) attack rate in various locales, such as school, community, and workplace (table 1 ) [5] . we defined and chose a risk space that has a transmission scale that runs from approximately a scale of 3 (eg, comparable to a community attack rate of 11%-15%) to a scale of 5 (community attack rate of >25%) ( figure 1 , table 1 ). our defined risk space has a low-end clinical severity scale of 3, with a case-fatality ratio of 0.05%-0.1% and a death-to-hospitalization ratio of 7%-9% ( table 1 ). the upper range of severity in our risk space was defined as a scale of 5, with a case-fatality rate of 0.25%-0.5%, and a death-to-hospitalization ratio of 13%-15% ( table 1 ). note that the defined risk space encloses the 1968 and 1957 pandemics ( figure 1 ). it is essential to note that this chosen risk space is illustrative, not definitive. until there are data defining the epidemiological elements of the next pandemic, such as rate of transmission, and case-fatality rate, other risk spaces could be chosen for planning purposes. the models presented in this collection, built to the specifications listed here, allow for rapid alterations in input values. the size and shape of the epidemic curve could impact the effectiveness of interventions. for example, the impact of influenza vaccines depends upon the start of deliveries of large amounts of vaccine compared to the timing of the pandemic peak. thus, we included in the standardized epidemiological scenario 4 epidemic curves, produced using a simple simulation model (see below). we configured the model using 2 clinical attack rates of approximately 20% and 30%. these clinical attack rates represent the aggregated attack rate across the entire us population. within the population, subpopulations will typically experience different attack rates (eg, children will experience a higher attack rate than adults 20-64 years old-see description later in paper). furthermore, for each attack rate, we assumed 2 starting (seeding) scenarios. we used one scenario in which the pandemic started with the arrival of 10 infectious cases and the other when the pandemic started with 100 infectious cases ( figure 2) . to model the 4 epidemic curves, we built a simple, nonprobabilistic (ie, deterministic) model that simulates the spread of influenza through a population by moving the population into groups of susceptible, exposed, infectious, and recovered or death ( table 2 provides values used). we divided the population into 4 age groups (0-10, 11-20, 21-60, or ≥61 years of age). we table 1 . see main text for additional details. note that the 1977-1978, 2006-2007, and 2007-2008 seasons were nonpandemic seasons. they are included to provide reference points regarding the impact of nonpandemic seasons. adapted from reed et al [5] . modeled the probabilities of daily contact (and thus risk of disease transmission) by constructing a contact matrix using data from the united kingdom (see table a1 in technical appendix a). we thus produced 4 notably different epidemic curves (figure 2 ). for example, the two 30% attack rate scenarios peak in weeks 12 and 14, whereas the 20% attack rate scenarios peak in weeks 13 and 22 ( figure 2 ). the clinical attack rates by age group are presented in table 3 . obviously, the largest numbers of cases occur in the largest age group of 21-to 60-year-olds; however, children in both the 0-10 and 11-20 age groups have the highest attack rates, indicating a potentially greater degree of vulnerability (table 3) . perhaps one of the greatest strengths of the simple models presented in this collection of articles is that they highlight what is for case-fatality ratio and case-hospitalized ratio, scale 3 shows low severity, and scale 5 shows high severity (in bold). source: adapted from reed et al [5] . a these estimates related to the framework for assessing the impact of influenza pandemics, shown in figure 1 . table 3 ). seeding refers to the number of infectious cases, either 10 or 100, that arrives nearsimultaneously in the united states to start the pandemic. and is not known about the burden of disease and the potential impact of a planned intervention. to find the weaknesses of what is currently known, a reader need only consult table 1 in each article. these tables list inputs, their assumed values, and data sources. an example of an important unknown is as follows: when estimating the number of respiratory protection devices (eg, face and surgical masks) needed by first responders ( police officers, firefighters, emergency medical technicians), one could assume that first responders will need 1 mask per person whom they encounter with influenza-like illness. the problem is that there are no readily available data that report on the measurement of such [6] . similarly, when considering the potential use and impact of influenza antiviral drugs, o'hagan et al had to assume that existing influenza antiviral drugs would have the same level of effectiveness against the strain causing the next influenza pandemic as they do with existing influenza strains [7] . despite these limitations, these simple models make it fairly straightforward to rapidly assess the relative importance of each of the input variables. one assumption that may not be readily appreciated is the impact of the shape of the standardized epidemiological curves used in all the models (figure 2 ). previous influenza pandemics have produced different shapes of deaths over time (figure 3 ). such differences in deaths over time can greatly influence the success of some of the interventions. for example, when considering the number of mechanical ventilators needed at the peak of the pandemic, meltzer et al initially assumed that the peak demand for ventilators would equal approximately 13% of all patients needing mechanical ventilation [8] . however, in the 30% attack rate epidemiological curve (figure 2 ), the number of cases that figure 2 ). note that the data for 1957 were recorded once every 2 weeks, whereas all other plots used weekly data. see technical appendix b for further details. occur in the peak 10 days is approximately 30% of all cases. thus, the authors of the ventilator study conducted a sensitivity analysis by changing from 13% to 30% the assumed number of mechanically ventilated patients that occurs at the peak of a pandemic. the articles in this supplement also incorporate other important implicit assumptions. one of the more important is that each article essentially assumes that the healthcare system can absorb and/ or successfully execute any of the interventions so modeled. for example, biggerstaff et al provide some estimates of the impact of influenza vaccination in which it was assumed that 30 million persons could be vaccinated each week [9] . the us private and public health systems, collectively or separately, have never previously achieved such a rate (though the authors clearly demonstrate that achieving such a rate would have very positive public health outcomes). furthermore, the successful deployment and ultimate impact of each intervention is likely to have a wide variation. schools can close for different lengths of time, antiviral drug prescription and distribution may not be equally efficient in all areas, and healthcare workers and patients may have different levels of compliance in wearing protective gear. finally, readers will note that there are no reports in this collection that consider the simultaneous deployment of ≥2 interventions. it is realistic to assume that, during the next influenza pandemic, public health officials, healthcare providers, and other policy makers are likely to enact several interventions at once (eg, close schools, start dispensing antiviral medications, recommend use of protective personal gear). the problem arises in that such multi-intervention models become very scenario specific. for example, different locales are likely to face different unmitigated epidemic curves ( figure 3) . thus, researchers who estimate the potential impact of combining several interventions at once have to make a very large increase in the number of assumptions. this makes it more difficult to both generalize the results and to rapidly understand what assumptions are relatively more important. despite these limitations, we believe that the benefits of using these models outweigh the limitations. this assessment is based on our experience of using the models and results produced to help public health leadership reassess us influenza pandemic planning and preparedness. in the 2013 response to the h7n9 threat, the most important outcome from policy makers seeing the results from these models was the intense debate concerning the inputs and assumptions. we thus believe that the methodology used here to develop and guide the building of the models in this collection, and the subsequent interpretations and use of the results, can be a useful part of future public health responses. supplement sponsorship. this article appears as part of the supplement titled "cdc modeling efforts in response to a potential public health emergency: influenza a(h7n9) as an example," sponsored by the cdc. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. standardized epidemiological curves-contact matrix: to model the 4 epidemic curves (figure 2 ), we built a simple, nonprobabilistic (ie, deterministic) model in which we divided the population into 4 age groups (0-10, 11-20, 21-60, ≥61 years). to measure the risk of contact and possible onward spread between and within each age group, we used the contact matrix shown in table a1 . for the contact matrix, we used, in the absence of relevant data from the united states, data from the united kingdom [10] , collected as part of a study called the polymod study that collected contact data from approximately 8000 persons living in 8 european countries [10] . because the uk data are split into 5-year age groups, we had to aggregate the data into the 4 age groups used in our model. during this aggregation, we ensured that the total number of contacts between any 2 age groups is "equal in any direction" (eg, the number of contacts between 0-9 years and 10-18 years is the same as those between 10-18 years and 0-9 years). we used, for this aggregation process, the age distribution of the us population (www. censusscope.org). as noted above, the uk contact data [10] are split into 5-year age groups, which we had to aggregate into the 4 age groups used in our models. furthermore, the matrix that we constructed had to meet the condition of being symmetrical. that is, the number of contacts from age group a to age group b should equal the number of contacts in the reverse direction. we begin the explanation of how we built our contact matrix by introducing some notation: the mixing matrix elements of the published matrix [10] are denoted by u ij , i, j = 1, . . . , m, where i, j refers to rows and columns, respectively, and m is the number of age groups in the mixing matrix. as the mixing matrix required has fewer age groups than that of the published polymod matrix [10] , indexed by f, g = 1, . . . , n, then we let age group u contain narrower age groups i ¼ lð f þ to uð f þ. 1. the contact rate between someone in group i and another in group g is given by we then proceed according to the following steps: 2. if the us population distribution is such that the population in age group i is n i , we can calculate the population-weighted means of each of the elements d, to obtain contact rates between groups f and g. for f = g, this calculation is simple: 3. for the elements that are off the diagonal, the calculation becomes more complicated because we need to sum up the correct number of contacts made between each age group. the total reported rates of contact from f to g and g to f are: 4. theoretically, these values should be equal to one another; however, they differ from one another when calculated from actual reported contact rates (from self-administered surveys such as those conducted by mossong et al [10] ), and so, to ensure that they are equal, we can average them before calculating the final mixing-matrix elements e fg and e gf : here then, e fg is the rate at which an individual in age group f makes contacts with anyone in age group g, per unit time, as reported in the original data (ie, per day for the original mossong et al [10] data). an example of this procedure is given below, following the steps above, outlined theoretically: 1. we begin with the "all contacts" (ie, both conversational and physical) matrix for great britain from the polymod study. the elements of this matrix denote the daily number of contacts between an individual in one 5-year age group with those in another 5-year age group. element (1, 2) , for example, is the daily number of contacts a person aged 0-4 years has with someone aged 5-9 years. the fill matrix is as follows: summing the columns of the 5-year group matrix according to the desired group widths (eg, the first 2 columns are summed to give a 10-year age group column) gives the following intermediate 15-group by 4-group matrix: 2. next, we obtain a vector whose elements are the numbers of individuals in each of the age groups of the original matrix (here 5-year width groups, taken from the 2011 great britain census; the age distribution should correspond closely with the distribution that held at the time when the contact survey was performed), and we perform a sum of the total number of contacts to produce an aggregated age group (ie, two 5year age groups are aggregated into one 10-year age group). elements of the total contact matrix. note that the numbers in the above matrix in these 2 positions differ slightly from those in the y21, y22 calculations outlined above; this is because daily polymod contacts were rounded for the calculations illustrated. 4. once we have completed this procedure for the whole aggregated total contact matrix, we need to divide our total contact numbers by the correct number of individuals in each age group, to ensure we end up with a matrix that gives the number of contacts per person per day in the relevant age group. for example, the (1,1) element of the final matrix is the (1,1) element of the matrix produced by step (2) divided by the total number of individuals in the first age group (ie, the sum of the individuals in the first two 5-year age groups = 3 914 000 + 3 517 000 = 7 431 000); and the (1,2) element is divided by the same number, whereas the (2,1) element is divided by the number in the second age group = 3 669 000 + 3 997 000 = 7 666 000. dividing through gives the final matrix below (which is similar to table a1 , accounting for rounding in the illustrative calculation). to model the curves shown in figure 3 , we used the estimated number of deaths from previous pandemic seasons (1918, 1957, and 1968 ). we compared those to the estimated clinical cases from the epidemiological model built for this exercise, using attack rates of both 20% and 30% (ie, the curves shown in figure 2 , main text). all deaths were based on the clinical data reported during the specific pandemic season. however, in an effort to obtain current death estimates, we extrapolated the seasonal case values (either clinical data or number of deaths) into currentyear 2014 us cases at a total population of 310 million. • 1918 influenza pandemic: we obtained from the source [11] the weekly number of deaths in 1918 ( per 100 000 people) for the reported 3 different us geographic locations (west, east, and midwest/south). we then adjusted those number of deaths, per 100 000, to the approximate current us population of 310 million persons (ie, multiplied each data point by 3100). this gave us the equivalent number of deaths for the 2014 us population. • 1957 influenza pandemic: we obtained the total, all ages biweekly (ie, reported every 2 weeks) number of respiratory illnesses per 100 000 from figure 5 in the report of the cdc (then known as the communicable disease center) [12] . we then adjusted those number of cases to the approximate current us population of 310 million persons (ie, multiplied each data point by 3100). this gave us the equivalent number of cases for the 2014 us population. to obtain estimates of deaths in equivalent 2014 us population, we multiplied the estimates of cases by a case-fatality ratio of 0.001 (ie, 0.1% of all cases result in death). this case-fatality estimate was taken from table 1 in the main text [5] . • 1968 influenza pandemic: we obtained the weekly reported number of pneumonia-influenza deaths in 122 us cities from figure 3 in sharrar et al [13] . however, the total number of deaths recorded by sharrar et al was only 19 450, which is notably lower than what may be expected. we therefore used a multiplier of 7.89 to adjust upward their estimates. we constructed this multiplier by noting that meltzer et al's figure 1 [14] showed approximately 155 000 deaths for a 1968-type influenza pandemic occurring in the us population (ie, 155 000/ 19 540 = 7.89). • attack rates: we took the 2 curves plotting the 20% and 30% clinical case attack rates shown in figure2 of the main text (the 2 plots assuming 100 infectious persons start, or "seed," the pandemic in the united states). we then used a case-fatality rate of 0.001 (ie, 0.1% of all cases result in death), taken from table 1 in the main text [5] . for simplicity, we assumed a low severity (scale of 3) of 0.1% for both attack rates to generate the number of deaths. human infection with influenza a(h7n9) virus in china human infection with avian influenza a (h7n9) virus-update epidemiology of human infections with avian influenza a(h7n9) virus in china probable person to person transmission of novel avian influenza a (h7n9) virus in eastern china, 2013: epidemiological investigation novel framework for assessing epidemiologic effects of influenza epidemics and pandemics potential demand for respirators and surgical masks during a hypothetical influenza pandemic in the united states estimating the united states demand for influenza antivirals and the effect on severe influenza disease during a potential pandemic estimates of the demand for mechanical ventilation in the united states during an influenza pandemic estimating the potential effects of a vaccine program against an emerging influenza pandemic-united states social contacts and mixing patterns relevant to the spread of infectious diseases nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic the epidemiology of asian influenza. 1957-1960. a descriptive brochure national influenza experience in the the economic impact of pandemic influenza in the united states: priorities for intervention for example, to construct the matrix element pertaining to the total number of contacts between the 0-9-year age group and the 0-9-year age group (ie, itself ), we perform the following sum:3 914 000 ã 2:6 þ 3 517 000 ã 7:6 ¼ 36 905 600: this is the first diagonal element of the "total contacts" matrix and, again, it represents the total number of contacts made per day between those in the 0-9-year age group.because diagonal elements are of course the same as their off-diagonal counterparts, there is no problem.3. however, corresponding pairs of off-diagonal totals should be the same; that is, the total number of contacts between those in the 0-9-year and 10-19-year groups should be the same as the total number between those in the 10-19-year and 0-9-year age groups. y21 = n3*d31 + n4*d41 = 3 669 000*1.8 + 3 997 000*0.7 = 9 402 100 y12 = n1*d12 + n2*d22 = 3 914 000*0.7 + 3 517 000*1. these 2 total contact numbers are not the same and so we take the average of them and they become the (1,2) and (2, 1) key: cord-304487-ycvu5l5f authors: wertheim, joel o title: a glimpse into the origins of genetic diversity in sars-cov-2 date: 2020-03-04 journal: clin infect dis doi: 10.1093/cid/ciaa213 sha: doc_id: 304487 cord_uid: ycvu5l5f nan a deadly pathogen has found a new host. near the end of 2019, the novel coronavirus sars-cov-2 began transmitting among humans. in the first two months following its discovery, over 75,000 cases of covid-19 causing over 2,000 deaths have been confirmed. researchers rapidly characterized the viral genome [1] and have already identified a closely related virus in bats [2] , pointing to these animals as the likely natural reservoir of this virus. the search continues for an intermediate host animal that may have facilitated the zoonosis, like camels do for mers-cov and palm civets did for sars-cov. from a clinical and epidemiological perspective, there is obvious interest in whether sars-cov-2 will adapt to become more transmissible and/or virulent in humans. before we can address this question, it is importantly to realize that most evolutionary options available to this virus have already been explored in bats, where there is an immense array of genetically distinct coronaviruses. viruses in the betacoronavirus genus, to which sars-cov-2 belongs, have likely been infecting bats for tens of millions of years, possibly since the origin of bats themselves [3] . within this natural reservoir, viruses exist that would cause high and low virulence in humans, as well as those viruses that are easily transmissible from human to human. evolution tinkers with these viruses in bats, and the epidemiological consequences are seen both in pathogenic zoonotic diseases (e.g., sars, mers, and covid-19) and in the less-virulent circulating coronaviruses causing common colds. occasionally, however, some evolutionary avenues are not available to a zoonotic pathogen until after arrival in humans [4] . evolution requires genetic diversity, and genetic diversity arises from two distinct mechanisms: mutation and recombination. mutation is the replacement of one nucleotide base with another, or the addition/removal of nucleotide(s). mutations are the only true source of genetic novelty. recombination, on the other hand, can increase genetic diversity by placing existing mutations into new genetic backgrounds. two articles in the most recent issue of clinical infectious diseases explore these sources of genetic variation in sars-cov-2. li and colleagues describe the genetic variability within 7 covid-19 patients from china. yi explores potential instances of recombination among 84 sars-cov-2 genomes from multiple countries. molecular epidemiology can use the genetic variation of sars-cov-2 to trace its history and better understand clusters of transmission. when we examine viruses infecting two different people, the amount of genetic similarity between these viruses is a proxy for the directness of their epidemiological relationship. direct transmission partners are more likely to have identical or nearly identical sequences. bear in mind that the genetic differences separating these viruses did not arise between these individuals; rather, all mutations arise during intra-host replication. in this issue, li and colleagues reveal the intra-host genetic variation of sars-cov-2. when these researchers examined genomes from a probable transmission pair who resided in the same house, they found the consensus sequences to be identical. however, deep-sequencing of viral genomes from 7 patients (including this probable transmission pair), the authors detect substantial variation in the number and frequency of minority variants within different individuals. remarkably, despite the limited number of individuals included, they found many of the same minority variants in different, epidemiologically unrelated individuals. this pattern suggests that sars-cov-2 is predisposed towards particular mutations, which may continue to re-appear as the virus continues to spread. recombination is an ongoing process in positive-strand rna viruses [5] , and coronaviruses are no exception. the evolutionary history of mers-cov in its intermediate host, camels, is littered with recombination events [6] . the bat coronaviruses from which sars-cov-2 descends also betray a history of recombination [1] , although there does not appear to have been recombination distinguishing sars-cov-2 from its closest described relative in bats [7] . in contrast, the phylogeny for the first sars-cov, which emerged in 2002, does not display evidence for recombination. it is unlikely that this virus lacked the capacity for recombination, only the opportunity. sars-cov-2 is presumably constantly recombining with itself within every infected individual. however, a homogenous population of viruses recombining with nearly identical relatives will not produce any novel genetic combinations. genetic recombination has biological and evolutionary consequences only when the two recombining viruses are sufficiently genetically distinct. in yi's article, the prospect of recombination between genetically distinct sars-cov-2 strains is investigated. robust phylogenetic testing for recombination cannot be performed on such closely related viruses, so yi relies on detecting cycles in haplotype maps. these cycles represent potentially non-linear evolutionary histories of viral evolution that is consistent with recombination. however, among such closely related strains, the signal for recombination cannot be reliably disentangled from convergent evolution. a scenario for convergent evolution is plausible based the observation of recurrent minority variants reported by li et al. nonetheless, as more sars-cov-2 genomes are sequenced and analyzed, a clearer picture of the role recombination plays in sars-cov-2 will likely emerge. the impact of this increasing genetic diversity on a hypothetical vaccine design is likely minimal. vaccine efficacy endures against circulating human rna viruses that have accumulated decades of evolutionary diversity (e.g., measles and polio virus). the obvious exceptions to this endurance are seasonal influenza viruses, where protective immunity extends over only a matter of years of viral evolution. whether a successful vaccine is developed or sars-cov-2 continues to spread remains to be seen. at the beginning of a zoonotic event, viral adaptation is difficult. the viral effective population size (an amalgam of the number of productive infections and the genetic diversity) is low; thus, natural selection will be inefficient, and recombination will not result in particularly novel combinations. however, as the number of infections increases and the circulating viruses become more genetically distinct, natural selection will become more efficient, making viral adaptation more of a possibility. nonetheless, when sars-cov-2 arrived in humans, it was already well suited for person-to-person transmission, with pathogenic consequences. furthermore, it is worth remembering that the evolutionary landscape that sars-cov-2 will explore in humans is dwarfed by its previous exploration in bats. for now, sars-cov-2 genetic variation is likely evolutionarily inconsequential and will be more important for facilitating molecular epidemiology in tracing the origins of novel clusters of viral infection. dr. wertheim reports grants from national institutes of health (nih), and grants from centers for disease control and prevention (cdc), outside the submitted work; he has no conflicts of interest to disclose. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin a case for the ancient origin of coronaviruses identifying genetic markers of adaptation for surveillance of viral host jumps evolutionary aspects of recombination in rna viruses mers-cov spillover at the camel-human interface full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event key: cord-326494-qhxlh43t authors: beckerman, karen palmore title: pregnancy and pandemic disease date: 2020-06-10 journal: clin infect dis doi: 10.1093/cid/ciaa741 sha: doc_id: 326494 cord_uid: qhxlh43t nan m a n u s c r i p t 2 what could antisepsis, 1,2 immunosuppressive therapy, 3 prevention of transfusion reaction, 4 elimination of pediatric syphilis and aids, 5 prevention of diabetes sequellae, 6 and prevention of birth defects 7 all possibly have in common? we need not dig too deeply into the history of each to find the role that maternal health care and investigation played in heralding each of these triumphs of modern medicine. in contrast to such achievements, we have only been able to chip away at tuberculosis (tb), despite the availability of effective treatment for almost 80 years. one hardly needs to be reminded of the scope of the tb pandemic: two billion, or one-quarter of the world's population infected; 10 million new infections per year, every year; and the distinction of being the leading cause of mortality from a single infectious disease, with 1.5 million deaths in 2018 alone. once infected, those living with latent tb have a 5-15% lifetime risk of developing active disease, which, if untreated, will end in death for about half. 8 pillar 1 of the who end tb strategy calls for diagnosis, contact and high risk screening, treatment and preventioneach of which presents a formidable challenge. 9 in most countries with high disease burden, incidence and population prevalence have never been measured directly. at best, incidence estimates rely on historic tuberculin surveys in children combined with estimates of disease duration. 8 even less reliably, approximations of incidence are derived from case notification data (i.e. infection diagnosed by sputum smear microscopy of symptomatic individuals presenting for care) combined with expert opinion on case-detection gaps from regional workshops or country missions. 8 many assumptions underlying such estimates may not hold true. 9 case notification data can be confounded by a c c e p t e d m a n u s c r i p t 3 access to care as well as care-seeking patterns. for example, the accepted notion of higher tb prevalence among men than women could be because men are more likely to seek out care than women. 10 some with active tb may be smear negative, but culture positive. so, what of pregnancy and tb? globally, estimates suggest it is a disease of men (57%), however women (32%) and children < age 15 (11%) together make up close to half of those living with tb. although overall global prevalence has remained constant in recent years, best estimates suggest that new infections and deaths are falling by about 2.5% yearly. 8 in endemic regions, tb is a major cause of death in women of reproductive age, and has been reported to be the major non-obstetric cause of maternal death, especially in the setting of hiv coinfection. 11 extrapulmonary manifestations of the infection may be more common with pregnancy, and latent tb reactivation risk is increased in the postpartum period. 12 two-thirds or more of pregnant women are asymptomatic at presentation, and signs and symptoms of active infection such as fatigue may be attributed to normal pregnancy. with active disease, increased risk of adverse outcomes such as preterm delivery, low birth weight and perinatal death has recently been confirmed in a comprehensive meta-analysis. 13 peripartum lymphatic and hematogenous vertical transmission have been described, but the greatest risk of newborn infection is not congenital, but post-partum, via newborn inhalation of infected droplets. 12 in this issue of clinical infectious diseases, walles and colleagues from adama regional laboratory in ethiopia and lund university, sweden confirm the truism that pregnancy may be one of the few opportunities to assess a woman's health, particularly in resource-limited regions. 10 they report an unexpectedly high proportion of women (37%) living with tb, the strong implication of these pregnancy data, that exposure and transmission in the population studied is both continuous and ongoing, is striking. the authors also found that none of the 85 women with past or currently active tb had known exposure to active infection, and that most women (61%) with past or current tb were hiv-uninfected. taken together, ample evidence is provided that successful tb control will likely require vigorous case finding beyond traditional target populations such as individuals living with hiv and those exposed to known active tb. we might go even further and suggest that walle et al. have provided proof of concept data that routine testing during pregnancy could be an ideal method of assessing the extent of community spread of both recognized endemic infection as well as emerging infectious diseases. other proof-of-concept data for such a proposal exist. first, pregnancy not only provided us with our earliest evidence that antiretroviral prophylaxis (or even better, treatment 14 ) prevents transmission. 15 the universally accepted and lauded goal of prevention of pediatric aids also created the political/social will and mandate to introduce hiv testing, prevention and treatment to much of the world. today, we continue to rely on universal hiv testing of pregnant women to estimate community hiv prevalence and to prevent vertical transmission, both in resource-rich and resource-limited regions. a c c e p t e d m a n u s c r i p t 5 second, our earliest evidence-based insights into community prevalence in the months-old covid-19 pandemic in the united states came from the study of pregnant women. nine days after the first diagnosis of a pregnant woman with covid-19 their hospital system, despite severe testing shortages, obstetricians at columbia university were able to begin universal testing of all women admitted for delivery. their report 16 a c c e p t e d m a n u s c r i p t m a n u s c r i p t the contagiousness of puerperal fever die aetiologie, der begriff und die prophylaxis des kindbettfiebers (the etiology, concept, and prophylaxis of childbed fever the potential reversibility of rheumatoid arthritis the prevention of rh immunization elimination of mother-to-child transmission of hiv and syphilis (emtct): process, progress, and program integration effect of euglycemia on the outcome of pregnancy in insulin-dependent diabetic women as compared with normal control subjects impact of folic acid fortification of the us food supply on the occurrence of neural tube defects world health organization global epidemiology of tuberculosis. cold spring harbor perspectives in medicine tuberculosis infection in women of reproductive age -a cross-sectional study at antenatal care clinics in an ethiopian city the neglected global burden of tuberculosis in pregnancy maternal and perinatal mortality and morbidity associated with tuberculosis during pregnancy and the postpartum period: a systematic review and meta-analysis control of maternal hiv-1 disease during pregnancy reduction of maternal-infant transmission of hiv-1 with zidovudine treatment. paediatric aids clinical trials group protocol 076 study group universal screening for sars-cov-2 in women admitted for delivery a c c e p t e d m a n u s c r i p t 7 key: cord-279167-fj36mzm5 authors: gwaltney, jack m. title: acute community-acquired sinusitis date: 1996-12-17 journal: clin infect dis doi: 10.1093/clinids/23.6.1209 sha: doc_id: 279167 cord_uid: fj36mzm5 nan managing acute community-acquired sinusitis (acas) has been a continuing challenge for physicians, who frequently have trouble making the diagnosis with accuracy and evaluating the effectiveness of treatment with precision. these problems would not exist if the paranasal sinuses were accessible to direct examination and to noninvasive sampling for microbial culture. because they are not, for diagnosis physicians have had to rely on clinical evaluations that are either insensitive or nonspecific [1] and on imagining studies, which are also insensitive or nonspecific. the problem can be explained in large part by the recent finding that sinus disease is an inherent part of the common cold syndrome itself, in that 87% of patients with colds have sinus cavity disease [2] . thus, the common cold is in reality a viral rhinosinusitis (vrs), not simply a rhinitis as traditionally held. not appreciating this difference has led to confusion, since historically the term acute sinusitis has been used to identify what is considered a bacterial infection of the sinus. undoubtedly this term has often been misapplied to cases of viral etiology or of a combined viral-bacterial etiology. without the benefit of sinus puncture, these distinctions cannot be made with accuracy. in cases of combined viral-bacterial infection, the clinical features of the illness will reflect its dual nature. the viral component will contribute to the overall severity and duration of the illness and thus complicate the diagnosis and confound the assessment of antimicrobial effectiveness. likewise, cases of pure vrs that are misdiagnosed as acute bacterial sinusitis will be a cause of confusion in case management, and these cases will contaminate patient samples in clinical trials of antimicrobial therapy. the perspective provided by the new information on vrs leads to a better understanding of the pathogenesis of acas and permits the development of improved approaches to diagnosis and management, which will be discussed in this review. the maxillary antrum was described by vesalius (1514-1564) and later by highmore (antrum highmori), who died in 1664 [3] . in 1707, william cowper developed a method of treating sinusitis by draining the antrum through the alveolus after removal of a tooth. following this, john hunter (1728-1793) recommended opening the antrum by way of the middle meatus, and gooch, who died in 1780, and later, mikulicz (1896) introduced antral puncture through the inferior meatus [4] . in this same period, caldwell (1893) and luc (1897) described an approach to the maxillary antrum via the canine fossa. these procedures were used for diagnosis and treatment of acute and chronic maxillary sinus infection. drainage and irrigation were a mainstay of treatment for acute and subacute cases, while creation of a permanent drainage site was employed for cases that had become chronic. scandinavian investigators began using sinus aspiration to obtain specimens for bacterial culture in the 1940s [5] [6] [7] [8] [9] . this work established that the maxillary sinus cavity is sterile under normal conditions and disclosed the bacterial species that are the most important causes of bacterial acas (acabs). when antibiotics became available, pretherapy and posttherapy sinusaspirate cultures were used to evaluate (and thus demonstrate the effectiveness of) antimicrobial treatment for acabs [10] . more recently, the nature ofthe bacterial etiology of acabs has been confirmed by investigators in the united states [11, 12] , who also recovered respiratory viruses from sinus aspirates of patients with acas [11, 13] . in addition, the recent development of computed tomography (ct) has provided a powerful tool for studying the pathogenesis of sinus infections and for em 1996; 23 (december) improved management of cases, especially those of chronic sinus disease. as discussed above, sinus ct scanning has demonstrated the importance of common cold viruses in causing acute disease of the sinuses [2] . paranasal pneumaticity originated in animals that lived somewhat earlier than the common ancestor of the dinosaurs [14] . in the dinosaurs, an air sac is believed to have filled a large paranasal space, the antorbital cavity. the derivative of this structure, the antorbital or cavionchal sinus, is present in birds and crocodilians, respectively, but not in lizards, snakes, and turtles. a similar structure, the maxillary sinus, arose in the evolutionary line leading to humans and is present in placental mammals but not in marsupials and egg-laying mammals. the ethmoid, frontal, and sphenoid sinuses are of more recent origin and have been described as characteristic of conventional mammals [15] . despite this long history, the function of the paranasal sinuses remains somewhat in doubt. it has been proposed that these structures reduce the bony mass and weight of the skull, participate in warming and humidification of inspired air, and add resonance to the voice. the maxillary sinus is a bony cavity located within the maxilla (figure i) [16, 17] . the chamber has a pyramidal shape, and the base of the pyramid is formed by the lateral wall of the nasal cavity, with the apex extending toward the zygomatic process. in the adult, the maxillary sinus cavity has a volume of 15 -30 ml. the sinus is lined with ciliated pseudostratified epithelium and is covered with a mucus blanket. the epithelium is well supplied with goblet cells (table 2) [18, 19] . in contrast, seromucous gland densities in the sinus cavity are low in comparison with those in the nasal passages [20] . the maxillary ostium is located on the highest part of the medial wall of the sinus cavity (figure 1) [21] . it is connected to the nasal cavity by a small tubular passage, the infundibulum, which is encased in bone and lies directly under the lamina papyracea of the orbit. the infundibulum leads to the hiatus semilunaris of the middle meatus, which is posterolateral to the uncinate process in a shielded location. the anterior ethmoid and frontal sinuses also empty into the middle meatus. this area and the region of the anterior ethmoid are described together as the ostiomeatal complex. the infundibulum is ~6 mm in length and has an average diameter of 3 mm, which is of adequate size to drain 30 ml (the maxillary sinus volume) of water by gravity in ~ 11 seconds. mucus and other fluids produced in the maxillary sinus figure 2 . ct scan views of the maxillary sinus of a patient: a, the natural ostium opening into the infundibulum; h, an accessory ostium located posteriorly and superiorly on the medial wall of the sinus and emptying into the middle meatus; and c, a second accessory ostium located further posteriorly at the level of the middle turbinate and emptying into the nasal passage. cavity are transported by ciliary action in a spiral direction up to and through the infundibulum and delivered into the hiatus semilunaris of the middle meatus [22, 23] . mucociliary transit times of 4.6-12.3 mm/min have been measured in the nose [24] , and transit times are presumably similar in the sinus. the mucous blanket changes 2-3 times each hour [25] , and normally mucus does not accumulate in the sinus cavity. in 10%-30% of adults, the maxillary sinus cavity is connected to the nasal passage by one or more accessory ostia that are located inferiorly to the infundibulum in the area of the anterior and posterior nasal fontanelles (figure 2). it is not known if these accessory ostia are congenital in origin or are acquired as a result of rupture during sinus infection in early life. rupture of an anterior nasal fontanelle has been observed by endoscopy to occur during the course of an acute sinus infection (d. kennedy, personal communication). the fontanelles are areas of very thin bone or membrane located in the lateral nasal wall at the level of the middle meatus and lying anteriorly and posteriorly to the uncinate process. if accessory ostia are present, fluid may enter or leave the maxillary sinus cavity through these openings, although ciliary transport remains directed to the natural ostium. the paranasal sinuses, although directly connected to the nasal passages, which are colonized with bacteria [26] , are themselves sterile under nonnal conditions [6, 11, 27, 28] . sterility is maintained in the sinus by mechanisms that are not fully understood but are believed to include the mucociliary clearance system and, possibly, antibacterial concentrations of nitric oxide gas in the sinus cavity [29] . components of the hu11loral and cellular immune systems also probably have a role in maintaining sterility in the sinus. the composition of nasal secretions has received considerable study, but secretions from the sinus cavity itself have not received comparable attention because of the difficulty of obtaining them, especially from healthy persons. constituents of human nasal secretions include a variety of mucous and serous products, plasma proteins, and inflammatory mediators [30] . the epidemiology ofvrs is the well-characterized epidemiology of common colds and related acute viral respiratory illnesses. in the united states, the incidence of acute respiratory illness in children is 6-8 illnesses per person per year, and in adults, 2 -3 [31] . in temperate areas, acute respiratory illness rates follow a well-established seasonal pattern, with annual epidemics in the fall, winter, and spring and periods of relative inactivity during the summer months. the different virus families tend to have characteristic periods of high prevalence, which for rhinovirus are early fall and late spring and for coronavirus, respiratory syncytial virus, and influenza virus are winter and early spring. if the published findings are representative [2] , then ~90% of patients with colds would be expected to have viral sinusitis as part of the basic illness. a small propoliion of colds are, in turn, complicated by an acute bacterial infection of the sinus. this has been reported to occur in 0.5%-2% of cases (table 3) [32, 33] . it should be pointed out that in these studies sinus aspirate cultures were not performed and, thus, the incidence of secondary bacterial sinusitis complicating vrs cannot be precisely determined from this work. the seasonal trends in the incidence of presumed bacterial sinusitis have been shown to correlate with those of common colds [34] . in addition to vrs-related cases, acabs occurs cm 1996;23 (december) * data are from [32] . t data are from [33] ; ent = ear, nose, and throat. throughout the year and is associated with allergy, swimming, and nasal obstruction due to polyps, foreign bodies, and tumors. other, less common risk factors are immune deficiencies such as agammaglobulinemia and aids; abnormalities of wbc function, as found in chronic granulomatous disease; structural defects, especially cleft palate; and disorders of mucociliary clearance, including cilial dysfunction and cystic fibrosis. on the basis ofthe epidemiological findings described above, ~ 1 billion cases of vrs can be expected to occur annually in the united states (260 million people [adults and children] x4 acute respiratory illnesses = ? 1 billion cases ofvrs annually) and, in turn, to be complicated by 20 million cases of acabs, assuming a 2% complication rate. data from the national ambulatory medical care survey for 1991 indicated the occurrence of 11,570,000 physician-patient contacts per year for acute upper respiratory illness and 2 million patient visits per year for presumed acute bacterial sinusitis [35] . thus, in the united states, an estimated 1 in 100 patients with vrs and 1 in 10 patients with acabs seek the care of a physician for their illness. in the survey [35] , upper respiratory tract infection (url) was the second most frequent diagnosis after essential hypertension. however, physician-patient contact for normal pregnancy, general medical examination, and child-health supervision also ranked above url on the list of most frequent reasons for visits. the total annual nonprescription cost of medications for acute url in the united states is >3 billion dollars [36] . expenditures for antibiotics, for other drugs requiring prescriptions, and for physician visits add substantially to this amount. additional large costs to society result from time lost from work and school, of which acute respiratory illness is the leading cause. most investigative work in the area of the pathogenesis of colds has been done with rhinovirus. a central feature of the pathogenicity of the rhinovirus is its ability to evade the host's protective defenses in the upper airway. the vulnerability of the nose to rhinovirus is shown by the fact that intranasal inoculation of virus in nonimmune volunteers routinely leads to a ?90% infection rate [37] . following infection, however, only three-quarters of persons have symptoms of a cold; the rest have an inapparent infection. the fact that these asymptomatic infections are apparently terminated as efficiently as those that are associated with illness suggests that, in this situation, the symptoms of a cold do not result from any beneficial activity of the immune system. key factors in initiation of infection include viral deposition in the nose, followed by presumed transport to the posterior nasopharynx [38] and attachment to rhinovirus receptor (icam-1) [39] . in biopsy specimens from uninfected volunteers, icam-1 has been located on clusters ofm (membranous) cells present in the adenoid crypts but not on adjacent ciliated epithelial cells [40, 41] . after initiation of infection, there is stimulation of several inflammatory pathways and of the parasympathetic nervous system. these events, rather than direct viral cytopathogenicity, are believed to be the major causes of symptom generation [42] . studies of rhinovirus with use of nucleic acid probes have shown only sparse and widely scattered foci of infection in biopsy specimens of nasal turbinates of experimentally infected volunteers [43] . the activation of inflammatory pathways results in engorgement of the capacitance vessels in the venous erectile tissue of the nasal turbinates, intercellular leakage of plasma into the nose and (presumably) sinuses, discharge of seromucous glands and goblet cells, and stimulation of pain nerve and sneeze and cough reflexes. sinus cavity abnormalities were seen in 87% of patients with early natural colds and included all of the different sinuses (table 4) [2] . the nature of the abnormality in the sinus cavity has not been well defined. the typical finding observed by imaging in cases of acute sinusitis has traditionally been labeled "mucosal thickening," except when a classic air-fluid level is present. however, the presence of gaseous bubbles in the material seen on ct and its irregular distribution on the walls of the sinus suggest that it is, in reality, a highly viscous substance that is adherent to the floor, the sides, and-in some casesthe ceiling of the sinus cavity (figure 3). because goblet cells but not seromucous glands are prevalent in the sinus cavity (table 2), the material probably consists of excess amounts of mucus discharged from these cells. in addithe uneven distribution of the abnonnalities is not compatible with mucosal swelling, which would be expected to be more unifonn. bilateral disease of the ethmoid sinuses is also present (large white arrows). tion, transudation of plasma into the sinus cavity may contribute to the amount and viscosity of the material. sinus ostial obstruction is often cited as a major cause of sinusitis. by ct scan, the infundibulum was occluded in 77% of patients with vrs, and the ostiomeatal complex was also frequently congested (table 4) [2] . however, this may not be the major cause of the cavity disease. observations from paired scans, performed over a span of hours, include failure of the cilia to move deposits of the material toward the ostium (figure 4) [44] . thus, a major part ofthe disease process appears to be malfunction of mucociliary clearance as a result of increased amounts of the viscous material, which occurs in addition to infundibular and ostiomeatal obstruction from mucosal swelling. another possible cause of failure to clear the material would be cilial slowing or paralysis. nasal mucus transit times were shown to be moderately delayed in subjects with experimental rhinovirus colds [45] , but the amount of slowing observed does not appear to be sufficient to explain the degree of stasis seen on the ct scans. furthermore, cilial dysfunction seems unlikely as the major problem because of the patchy nature of the cavitary abnormalities, as discussed below. with other, more destructive viral infections, such as influenza, epithelial damage is a more likely possibility. the actual mechanisms by which virus causes disease in the sinus cavity are not known. viruses have been recovered from sinus cavity aspirates taken from patients with acas [13] , but it is not clear if viral invasion of the cavity is necessary to cause the disease. the erratic distribution of the abnormalities among the different sinuses in the same patient (figure 5) seems more compatible with random viral invasion of a sinus than with a response to generalized activation of inflammatory mediators in the nasal passage. the latter might be expected to cause a more unifonn distribution of disease among the various sinuses. the nasal passages and nasopharynx are colonized with the same bacterial species that cause acabs [26] , and, undoubtedly, the bacteria in these areas serve as the reservoir for such infection (table 5). the specific factors that determine whether bacterial invasion of the sinus will occur during vrs are unknown. sneezing, coughing, and nose-blowing may create pressure differentials that cause deposition of bacteria-containing nasal secretions into the sinus. in a rabbit model, introduction of streptococcus pneumoniae or haemophilus injluenzae into an acutely obstructed maxillary sinus led to infection and disease, while obstruction of the sinus ostium without bacterial instillation did not [46] . experimental obstruction of the sinus in rabbits leads to reduced oxygen tension and increased concentrations oflactic acid [47] . once bacteria are deposited into the cavity of an obstructed sinus, growth conditions are favorable, as indicated by the high titers measured in sinus aspirates at concentrations of ~ 10 7 cfu/ml [11] . in these cases, the infection was associated with leukocytosis (~10,000 wbcs/mm 3 ) in sinus aspirates; however, granulocyte phagocytosis may be impaired by the reduced oxygen tension in an obstructed sinus. in a rabbit model, maxillary sinus infection with s. pneumoniae or h. injluenzae resulted in a modest increase in ciliarybeat frequency for 2 to 3 days, which was followed by marked destruction of ciliated epithelial cells, starting on day 2 (s. pneumoniae) and day 4 (h. injluenzae) [46] . by the fourth day, there was a 70%-80% reduction in viable ciliated cells. by the fifth day, the sinus was completely filled with extremely viscous material described as "mucopus." the authors of that study point out that reversing the disease process would involve more than just relieving ostial obstruction and that generation of new ciliated epithelium would be necessary to remove the considerable debris and bacteria present. in other studies using rabbit sinusitis models, squamous cell metaplasia and increased numbers of goblet cells were observed as part of the process [48, 49] . there is evidence that acas in humans is a process of sufficient severity to require several weeks to heal. studies using serial sinus imaging have shown slowly resolving sinuscavity abnormalities that persist after clinical complaints have resolved [11, 50, 51] . in one study of 13 previously healthy adults in which serial sinus mri was performed, mean aeration figure 4 . serial sinus ct scans of an adult with a common cold of 4 days' duration. the first scan (a) was obtained at 2:00 p.m. there are abnormalities in both maxillary sinuses (black arrows). a gaseous bubble is present on the left (white arrow), indicating that the material causing the abnormality is thick fluid. the nasal turbinates are engorged (asterisk), and the middle meatus is congested (square). immediately after the scan was obtained, the patient was given 60 mg of pseudoephedrine orally. the second scan (b) was obtained at 4:00 p.m. on the same day. the material in the maxillary sinuses (black arrows) has not moved, and the findings are essentially unchanged after treatment with pseudoephedrine. the turbinates remain engorged (asterisk) and the middle meatus congested (square). immediately after the scan was obtained, the patient was given two intranasal sprays of .05% oxymetazoline. the third scan (c), obtained 10 minutes after oxymetazoline administration, shows shrinkage of the turbinates (asterisk) and decongestion of the middle meatus (square). omnipaque dye was given intranasally immediately before the scanning while the patient was supine and is present in the middle meatus and olfactory recess (small black arrows). the location of the material in the maxillary sinus cavities (large black arrows) has not changed. of the sinus showed a steep increase up to day 10 and then a more gradual improvement up to day 56, the last day of observation [51] . however, there was still a mean aeration of only ~80% when observations were discontinued. sinus aspirate cultures were not performed in that study, but the clinical presentation with fever and facial pain and the radiological finding of an air-fluid level in some cases make it probable that at least some of the patients had bacterial infection. specimen collection has an important influence on the accuracy of sinus culture results. unless sinus-cavity specimens are collected without contamination by nasal secretions, there is always the danger that the specimens will yield bacteria that are growing in the nose instead of the sinus. for this reason, culture of sinus-cavity samples obtained by puncture and aspiration has been the "gold standard" for microbial diagnosis. sinus puncture is a relatively painless and safe procedure when performed by an experienced operator, although it is not suitable for routine clinical use (table 6) . more recently, a spring-loaded device has become commercially available for performing sinus puncture. the bacteria recovered from sinus aspirates have shown features indicating a causal relation to disease, including high titers on quantitation, correlation with simultaneous gram stains, and association with leukocytosis in the aspirate [11] . the development of modem endoscopes has raised the possibility of collecting sinus specimens endoscopically. this presents several difficulties. first, it is not possible to enter the sinus cavities by way of the natural ostia with an endoscope. with the maxillary sinus, the sheltered location of the hiatus semilunaris behind the turbinate and uncinate process [21] and the small diameter of the infundibulum and its acute angle relative to the lower nasal passage (figure 1) make it physically impossible to pass an endoscope into the cavity. it may be possible to enter the maxillary sinus cavity in some of the 10%-30% of persons with accessory ostia ( figure 2 ), but it is still very difficult to do so without contaminating the specimen with nasal secretions. therefore, most efforts at endoscopic sampling have focused on collecting secretions from the middle meatus and shielding the endoscope in an attempt to prevent specimen contamination. secretions obtained from the middle meatus have been considered to contain material that has been discharged from the sinus cavity, but it is not clear whether the middle meatus is normally sterile or is colonized or contaminated with nasopharyngeal bacteria. in one study of 47 patients with acabs, endoscopic sampling was associated with a sensitivity of 65% and a specificity of 40% in comparison with the" gold standard" of sinus aspirate culture [52] . the sensitivity and specificity increased (to 79% and 85%) when the data were analyzed for only s. pneumoniae, h injluenzae, and moraxella catarrhalis. further work is necessary to determine the ultimate utility of endoscopic sampling in sinus disease. the limited amount of information from direct comparisons of aspiration and endoscopy does not permit replacement of aspirate cultures as the' 'gold standard" for microbiological diagnosis at this time. only a limited number of sinus aspirates from patients with acute sinusitis have been tested for virus. viruses were recovered from 11 (16%) of 70 positive aspirates (table 7) [13] . most of these specimens were collccted after the first 3 days of illness, when the chance of isolating the virus declines. rhinovirus, parainfluenza virus, and influenza virus were identified, findings that support the role of viruses in the pathogenesis of sinusitis. the etiology of acabs has been well defined by puncture studies since the late 1940s and 1950s [5] [6] [7] [8] [9] [10] [11] [12] . there has been 7) . m. catarrhalis, other streptococcal spccics (including streptococcus pyogenes, streptococcus intermedius, and other 0'hemolytic streptococci), staphylococcus aureus, and anaerobic bacteria each account for a small proportion of cases. m. catarrhalis is more prevalent in children than in adults, while anaerobic infections are infrequent in children [12] . most sinusitis due to anaerobic bacteria arises from infection of the roots of the premolar teeth, thus representing a pure bacterial infection. some of the anaerobic infections have involved up to six different species of microaerophilic and anaerobic bacteria. as discussed above, most acute bacterial infections of the sinuses are a complication of vrs. viruses and bacteria have been recovered simultaneously from the same sinus aspirate, confirming the dual nature of the infection (table 8) . the relative importance of the different bacteria has not changed in the last half century, but there have been important changes in their antimicrobial susceptibilities. the appearance of penicillin resistance in s. aureus was followed by the emergence of resistance to ,6-lactam agents in strains of table 6 . method of sinus puncture and aspiration for specimen collection. (1) disinfect the anterior nares and the area below the inferior turbinate (puncture site). (2) anesthetize the puncture site with a topical anesthetic. (3) puncture the medial wall of the antrwn with a 12-gauge needle (or spring-loaded puncture device). (4) aspirate the sinus contents into a syringe; if necessary, add 1-2 ml of sterile normal saline (without preservatives) to obtain a specimen. [13] . i data are from [5] [6] [7] [8] [9] [10] [11] [12] . mean % of cases involving: lone study had a 24% rate of isolation of gram-negative bacteria, but in four other studies the recovery rate was ~ 5%. gram-negative bacteria recovered included pseudomonas aeruginosa, klebsiella pneumoniae, and escherichia coli. h. injluenzae and m. catarrhalis. the most recent and serious event has been the emergence of multiply resistant strains of s. pneumoniae. only ~60% of sinus aspirates in suspected cases of acabs yield bacteria [53] . the etiology of the culture-negative cases is not clear, but undoubtedly many are due to viruses. chlamydia pneumoniae has been identified in patients with respiratory illness that includes features of sinusitis [54] . however, until c. pneumoniae is identified in sinus aspirates, its role as a cause of sinusitis cannot be considered established. mycoplasma pneumoniae also has been suggested as a cause of acabs, but there are no reports of attempts to isolate it in cultures of sinus aspirates. atypical pneumonia and bronchitis are the characteristic syndromes associated with m pneumoniae infection [55] . the clinical features of sinusitis have not been described with this infection. on the other hand, fungi are well established as a cause of occasional cases of acas [56] . most of these fungal cases present with pressure changes such as masses, proptosis, and bony erosion. communityacquired fungal sinusitis and nosocomial sinusitis are beyond the scope of this report. because most cases of acabs are superimposed on preexisting vrs, the clinical features of the illness reflect the dual nature ofthe infection. the profiles of natural and experimental rhinovirus colds have been described in detail and include the well-known upper and lower respiratory tract symptoms of the common cold (figure 6) [57, 58] . it is not possible to separate the symptoms associated with nasal vs. sinus pathology, but sneezing, rhinorrhea, nasal obstruction, facial pressure, and headache are common sinonasal complaints. the clinical presentation of acabs has traditionally been described as including these same complaints, with the addition of purulence or color to the nasal discharge, a temperature of ~38â°c, and facial pain or erythema. cough has been reported as characteristic of acabs in children [12] and is also a common complaint in adults with acute sinusitis. hyposmia may also be noted. when the sinusitis follows dental infection, molar pain and a foul odor to the breath are additional characteristic features. patients with bacterial infection of the sphenoid sinus have presented with severe frontal, temporal, or retro-orbital headache that radiates to the occipital region and hypesthesia or hyperesthesia of the ophthalmic or maxillary dermatomes of the fifth cranial nerve [59] . lethargy and the clinical findings of cavernous sinus or cortical-vein thrombosis may also be present, as well as signs of orbital cellulitis and abscess. with severe frontal sinusitis, pus may collect under the periosteum of the frontal bone, causing swelling and edema of the forehead, which is known as pott's puffy tumor [60] . diagnosis of acas continues to present a difficult challenge to the physician, despite modem technology. an initial distinction must be made between infectious and allergic or other noninfectious syndromes. differentiation must then be made between a viral, combined viral-bacterial, or pure bacterial etiology, and finally, when possible, the specific microbial cause must be determined. an allergic etiology can usually be established by a history of paroxysmal sneezing, itching eyes, allergen exposure, and similar prior episodes. in one small study, adult patients were accurate in making the distinction between allergy and infection by self-diagnosis [61] . separating viral from viral-bacterial or bacterial infections is a more difficult problem. evaluation of symptoms and signs with use of either a positive sinus roentgenogram or the presence of purulent secretions (not cultured for bacteria) in a sinus aspirate as the comparison standards showed that no parameters were both sensitive and specific for this purpose (table 9) [1, 33] . the results might have been somewhat different if the , 'gold standard," sinus aspirate culture, had been used, but the general conclusions of the study would probably have been similar. another study examined adults with paranasal symptoms in whom pus was observed coming from the middle meatus; a purulent sinus aspirate (not cultured for bacteria) was used as the comparison standard [62] . the presence of two of three * in the setting of an illness beginning as a common cold, "flu"-like illness, or allergic rhinitis or associated with swimming or other risk factors. t or other antibiotics with a favorable profile of activity against intermediately resistant s. pneumoniae; adult dosages are given. findings-local pain with unilateral predominance, unilateral purulent rhinorrhea, and an erythrocyte sedimentation rate of > 12 rnm/h-were associated with a sensitivity of 79% and a specificity of 83%. in another study of adult patients from a general practice, in which a purulent maxillary sinus aspirate was the criterion standard, the finding of an elevated c-reactive-protein concentration (> 10 mg/l) combined with an elevated erythrocyte sedimentation rate (10 rnm/h for men and 20 rnm/h for women) was associated with a sensitivity of 82% and a specificity of 57% [63] . other standard laboratory tests such as wbc and differential cell counts are not useful because of their lack of sensitivity and specificity. ct scanning is a very sensitive method of detecting disease in the paranasal sinuses and has largely supplanted conventional roentgenography as the imaging method of choice. the cost of a ct scan limited to the sinuses is comparable to that of plain sinus roentgenography in many clinics and hospitals. however, imaging studies are not recommended for the routine diagnosis of community-acquired sinusitis because of their lack of specificity. most patients with vrs have sinus ct scan abnormalities that cannot be distinguished from those associated with acabs [2] . however, if a classic air-fluid level with a flat meniscus (indicating thin fluid in the cavity) is observed, there is a good correlation with a positive bacterial aspirate culture. in one study of adults, the finding of an air-fluid level on a conventional sinus roentgenogram had a specificity of 89% when compared with a positive aspirate-culture result, although such a level was noted in only 37.5% of 48 positive examinations [13] . without help from imaging or the laboratory, short of sinus aspirate culture, the physician must continue to depend on clinical parameters for the differential diagnosis. three diagnostic categories of acabs can be recognized (table 10). the first presentation, which is rarer than the other presentations, is that in which the sinusitis has been complicated by meningitis, brain abscess, or orbital infection. in these cases, the clinical features of the sinus infection are overshadowed by the more serious illness. the second presentation is that in which the classic and relatively specific features of acabs are present. these include fever (temperature of ~38â°c) and facial pain, marked tenderness, erythema, or swelling. also in this category are patients with molar pain or other evidence of an odontogenic cause of the infection. the third presentation, which is the most common, is that in which an illness with sinonasal symptoms of vrs has continued for 8 -1 0 days or more and the symptoms of colored nasal discharge, nasal obstruction, facial pressure, and sometimes cough are no better or are worse. uncomplicated rhinovirus colds have a median duration of 1 week (figure 7) [64, 65] . most colds, if still symptomatic, are improved by the end of 1 week, so the worsening or persistence of symptoms raises the suspicion of a complication. in addition, sinus-puncture studies have shown that for ~60% of patients who initially present with vrs and whose sinonasal symptoms do not abate after 1 week, a bacterial aspirate culture will be positive [53] . complications acabs may lead to intracranial, orbital, and respiratory complications. the intracranial complications include meningitis, brain abscess, subdural empyema, and cavernous-sinus and cortical-vein thrombosis. orbital complications are most common in young children and include orbital cellulitis, subperiosteal abscess, and orbital abscess. sinusitis also is associated with the onset or exacerbation of asthma and bronchitis. sinopulmonary disease is a well-recognized combination, especially when the condition has become chronic. whether the usual type of acabs is a cause of chronic sinus disease is unknown. certain specific microorganisms might predispose to chronic sinus disease, or chronic sinus disease may be an entirely different process related to allergy or other, as-yet-undetermined risk factors of the host. general considerations. randomized placebo-controlled trials of antimicrobial therapy for acabs with use of pretreatment and posttreatment sinus aspirate cultures have not been conducted. however, in several nonrandomized studies, such aspirate cultures have been done [la, 13, 53, 66] . the findings of these studies show that antimicrobials with appropriate antibacterial spectra and given in adequate doses are highly effective in eradicating or substantially reducing bacterial titers in the sinus cavity, while those with an inadequate spectrum or given in an inadequate dose are not (table 11) . correlations of bacterial titers and histopathologic findings in the sinus mucosa of humans have not been reported, but if epithelial damage occurs in humans with acute bacterial sinusitis as it does in rabbits [46] [47] [48] , then early eradication of a bacterial infection in the sinus cavity is an important treatment goal. table 11 . comparative bacteriologic cure rates (as determined by sinus puncture) among patients with acute community-acquired bacterial sinusitis. reference, comment regarding treatment [10] antibiotic concentration* was;;;, mic of causative bacteria antibiotic concentration* was < mic of causative bacteria [13] appropriate antimicrobial and dose given inappropriate antimicrobial given t [66] appropriate antimicrobial and dose given suboptimal dose given! [53] appropriate antimicrobial and dose given suboptimal dosage given! suboptimal dosage given ⧠over the past 20 years, a number of antimicrobials have been tested in cases of acabs with use of pretreatment and posttherapy aspirate cultures. when a lo-day course of therapy with an antimicrobial that has an appropriate [53] antibacterial spectrum has been used (at the correct dosage), a ~90% bacteriologic cure rate has been routinely obtained (table 12). in formal clinical trials of antimicrobial therapy for acabs, pretreatment bacterial aspirate culture is desirable to exclude the cases of pure vrs that will invariably contaminate the patient sample if inclusion criteria are based solely on clinical and imaging parameters. posttreatment aspirate culture is also necessary to determine effectiveness of therapy because of the recognized difficulty in accurately monitoring the clinical course of sinusitis [67] . this difficulty is due to the dual viral-bacterial etiology of the infection and to its self-limited natural history. currently the u.s. food and drug administration does not require posttreatment aspirate cultures for approval of a sinusitis-treatment indication. in addition, some reports of "bacteriologic cures" have appeared in the literature, involving cases in which posttherapy aspirate cultures were not performed; the "bacteriologic cure" assessment was based on clinical response, not findings of aspirate culture. recommendations for the treatment of acabs have changed as the antimicrobial susceptibility of the causative bacteria continues to evolve. the emergence of methicillinresistant s. aureus was not a major problem because of the relative infrequency of acabs due to this species, especially methicillin-resistant strains. the emergence of ,b-lactamaseproducing strains of h injluenzae and m catarrhalis was a more important event that reduced the usefulness of ampicillin. a number of other effective antimicrobials are still available (table 12) , although the cost of most is considerably more than that of ampicillin. the recent emergence of intermediately and highly resistant strains of s. pneumoniae is a much more serious problem that is not satisfactorily addressed with currently available antimicrobials. current recommendations. because treatment with the most effective antimicrobials against multiply resistant s. pneumoniae-vancomycin and third-generation cephalosporinsis not appropriate for acabs, the currently recommended first-line therapy is dependant on drugs with unreliable activity. susceptibility testing has shown that of the drugs proven effective in previous sinus-aspirate studies (table 12) , cefuroxime axetil and amoxicillin clavulanate are currently the most active against strains of intermediately resistant s. pneumoniae [68] [69] [70] [71] . cefprozil and cefpodoxime have shown in vitro activity similar to that of cefuroxime axetil against intermediately resistant pneumococci. in addition, some of the new quinolones under development have good activity against intermediately resistant pneumococci. a 10-day course of treatment with one of these antibiotics is recommended when the diagnosis of acabs is made (table 10). the symptoms of acas usually abate following 2 or 3 days of treatment and are generally resolved by 7-10 days [51] . however, it is important to be aware that the symptoms of patients with acute sinusitis may be substantially diminished despite the persistence in the sinus of purulent material containing high titers of bacteria [11] . in patients with evident severe infection or in whom intracranial or orbital extension of infection is suspected, intravenous therapy should be started with vancomycin and ceftriaxone or cefotaxime until the results of culture and susceptibility testing are available for directing treatment (table 10) . patients in these latter categories should have emergency evaluations by ct and/or mri and may also require diagnostic lumbar puncture and/or surgical decompression and drainage. recommendations for the treatment of the complication of acabs are beyond the scope of this review. ancillary treatment should be directed at drainage of the nasal passages and sinuses and the relief of sneezing, coughing, and systemic complaints [72] . the traditional approach to improving drainage has been the use of decongestants and drugs that are believed to aid in evacuation of mucus. this approach has a theoretical basis, but evidence of its effectiveness in cases of sinus disease in controlled clinical trials is lacking. topical and, to a lesser extent, oral decongestants are rapidly effective in shrinking the erectile vascular tissue of the turbinates and thus in helping relieve ostiomeatal and nasal obstruction (figure 4) [44] . however, sequential ct scans have shown that decongestants have little or no effect in promptly draining the sinuses. ct scans emphasize how different the anatomy of the sinus drainage passages is from that of the nose. in the maxillary sinus, for example, the drainage passage (infundibulum) is encased in bone, has a very small diameter (3 mm) compared with that of the nasal passage, and is lined with a thin nonerectile mucosa (figure 1). scans taken before and after topical or oral decongestant treatment failed to show an increase in the diameter of the infundibulum or the relief of its obstruction ( figure 4) . compounding the problem is the observation that the material in the sinus cavity of patients with vrs is too viscous to be moved by mucociliary clearance (figure 4). therefore, it is not surprising that immediate clearance of the sinus cavity was not detected by ct scans performed before and after decongestant treatment. in another study, manometric measurements in patients with acute rhinosinusitis failed to show a significant increase in the functional size of the infundibulum after administration of 100 mg of phenylpropanolamine [73] . on the other hand, after nasal decongestion, even thick secretions can be cleared from the nasal passages by sneezing and nose-blowing. oral decongestants are preferred over topical preparations. although their activity is less immediate and potent, oral decongestants avoid rebound vasodilatation and obstruction and the pharyngeal irritation that often accompanies the use of nasal decongestants. oral decongestants are safe for patients with stable hypertension who are receiving antihypertensive treatment [74] . topical and sometimes oral steroids have been used as decongestants (to reduce inflammation), but the effectiveness of this form of treatment has not been rigorously evaluated [72] . intranasal beclomethasone used alone had little, if any, beneficial effect on nasal symptoms and nasal mucus weights in volunteers with rhinosinusitis due to experimental rhinovirus infection [75] . steroids are not recommended for use against acabs unless there is evidence of an allergic component to the patient's illness. the value ofmucoevacuant drugs such as guaifenesin in sinusitis is also not established, but they are used on theoretical grounds. nonsteroidal antiinflammatory drugs are useful in treating systemic complaints such as fever and malaise and may also help in reducing cough [76, 77] . more traditional cough suppressants such as dextromethorphan and codeine may be needed for cough control. first-generation antihistamines have not been widely recommended in the past for treating vrs or acabs because of their anticholinergic activity and the possibility of their drying secretions and thus impairing drainage. while this is a reasonable theoretical consideration, testing under randomized, controlled, blinded conditions has shown a reduction of ~50% in sneezing and a 30% reduction in rhinorrhea and nasal mucus weights in volunteers with experimental rhinovirus colds [78] . in addition, there was no evidence of worsening of other symptoms or prolongation of the overall illness, indicating that drying of secretions and impairment of drainage were not problems. the theoretical case can be made that by reducing sneezing, antihistamines reduce the chance for viral and bacterial dissemination in the nasal passages and for their deposition in the sinus cavity. in addition, experimental upper-airway challenge with histamine in volunteers stimulates the release of nasal secretions with an increased sulfate concentration characteristic of mucus, suggesting that histamine stimulates goblet-cell exocytosis [79] . thus, there is the possibility that antihistamine therapy may reduce the amount of mucus that accumulates in the sinus cavity during acute sinusitis. however, controlled clinical trials of antihistamine treatment need to be done in patients with acabs before its value can be accurately assessed. preventing colds may be possible to some extent by avoiding contact with people who have colds and by hygienic measures such as handwashing when contact occurs between infected and noninfected persons. covering the mouth with disposable nasal tissues when coughing or sneezing also is desirable. vaccine is effective in preventing influenza, as is prophylactic amantadine or rimandatine during periods of epidemic influenza. there are no proven measures for preventing secondary bacterial infection of the sinuses, although suppression of sneezes and coughs can be considered of theoretical value. promotion of decongestion and drainage is possible in the lower nasal passages and ostiomeatal area, but as discussed above, its value in clearing the sinus cavity is problematic. prophylactic antimicrobial administration to prevent recurrent acabs is not recommended, and if used widely for such a common illness as vrs, it would undoubtedly hasten the emergence of bacteria with new patterns of antibiotic resistance. better treatments for colds may be available in the future, and these, when given early in the course of the illness, might modify the viral sinusitis and in tum lower the 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special reference to paranasal pneumaticity and nasal conchae definition, diagnosis, and the origin ofmammalia paranasal sinuses: anatomic terminology and nomenclature quantitative histology of the maxillary sinus quantitative histology of the normal sphenoid sinus density of goblet cel\s in the normal adult human nasal septum endoscopic view of maxillary sinus ostia the physiology of drainage of nasal mucus. iii. experimental work on the accessory sinuses uber die drainage der menschlichen nasennebenhohlen unter normalen und pathologischen bedingnngen 1. mitteilung mschr ohrenheillk lar physiology and pathology of the mucociliary system nasal physiology the nose: upper airway physiology and the atmospheric environment sinusitis in children at home and in day-nurseries bacteriology of the maxillary sinus of rhesus monkeys studies of mucociliary activity and blood flow in the upper airways, with special reference to endothelins and nitric oxide human nasal host defense and sinusitis viral infection of humans: epidemiology and control illness in the home: a study of 25,000 illnesses in a group of cleveland families occurrence of asymptomatic sinusitis in common cold and other acute ent infections chronic sinusitis: relation to upper respiratory infections and allergic rhinitis national ambulatory medical care survey: 1991 summary. series 13: data from the national health survey health industries handbook sites of rhinovirus recovery after point inoculation of the upper airway the major human rhinovirus receptor is icam-1 the human adenoid: a morphologic study the distribution of rhinovirus receptors in the upper airway clinical virology localization of human rhinovirus replication in the upper respiratory tract by in situ hyhridization virus induced rhinitis mucociliary function during experimentally induced rhinovirus infection in man early mucosal changes in experimental sinusitis experimental acute sinusitis in rabbits: energy metabolism in sinus mucosa and secretion nord ceo mucosubstance histochemistry of the maxillary sinus mucosa in experimental sinusitis: a model study on rabbits nord ceo mucosal pathology of the nose and sinuses: a study in experimental maxillary sinusitis in rabbits induced by streptococcus pneumoniae, bacteroidesfragilis, and staphylococcus aureus comparison of subjective and radiological findings during the course of acute maxillary sinusitis clinical course of acute maxillary sinusitis documented by sequential mri scanning utility of sinus endoscopy versus sinus aspiration for microbiologic documentation of acute maxillary sinusitis (ams) [abstract d42 the microbial etiology and antimicrobial therapy of adults with acute community-acquired sinusitis: a fifteen-year experience at the university of virginia and review of other selected studies association of chlamydia pneumoniae (strain tw ar) infection with wheezing, asthmatic bronchitis, and adult-onset asthma s principles and practice of infectious diseases sinusitis: pathophysiology and treatment nasal mucus weights in experimental rhinovirus infection symptom expression in natural and experimental rhinovirus colds sphenoid sinusitis: a review of 30 cases radiological evaluation of pott puffy tumor allergic rhinitis or virus cold?: nasal smear eosinophilia in differential diagnosis analysis of symptoms and clinical signs in the maxillary sinus empyema predicting acute maxillary sinusitis in a general practice population rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response the influence of signal variation, bias, noise, and effect size on statistical significance in treatment studies of the common cold treatment of sinus empyema in adults: a coordinated nordic multicenter trial of cefixime vs. cefaclor the inflammatory response of the sinus and nasal mucosa during sinusitis: implications for research and therapy management of infections caused by antibiotic-resistant streptococcus pneumoniae pharmacokinetics and pharmacodynamics of antibiotics in otitis media increasing penicillin resistance in streptococcus pneumoniae in the us: effect on susceptibility to oral cephalosporins antimicrobial resistance of streptococcus pneumoniae recovered from outpatients in the united states during the winter months of 1994 to 1995: results of a 30-center national surveillance study prospects for ancillary treatment of sinusitis in the 1990s studies of the effect of peroral fenylpropanolamin on the functional size of the human maxillary ostium does pseudoephedrine increase blood pressure in patients with controlled hypertension? a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection effects of naproxen on experimental rhinovirus colds: a randomized, double-blind, controlled trial combined antiviral and antimediatortreatment of rhino virus colds randomized controlled trial of c1emastine fumarate for treatment of experimental rhinovirus colds biochemical analysis of nasal secretions induced by methacholine, histamine, and allergen provocations this test affords you the opportunity to assess your knowledge and understanding of the material presented in the preceding clinical article, "acute community-acquired sinusitis," by jack m. gwaltney, jr., and to earn continuing medical education (cme) credit.the office of continuing medical education, ucla school of medicine, is accredited by the accreditation council for continuing medical education to sponsor continuing medical education for physicians. the office of continuing medical education, ucla school of medicine, designates this educational activity for up to 1 hour in category i credit towards the physician's recognition award of the american medical association. each physician should claim only those hours of credit that he/she actually spent in the educational activity.to earn credit, read the state-of-the-art clinical article carefully and answer the following questions. mark your answers by circling the correct responses on the answer card (usually found toward the front of the issue), and mail this card after affixing first-class postage. to earn credit, a minimum score of 80% must be obtained.certificates of cme credit will be awarded on a per-volume (biannual) basis. each answer card must be submitted within 3 months of the date of the issue.this program is made possible by an educational grant from roche laboratories. key: cord-322204-kc7dy2za authors: khalil, asma; hill, robert; wright, alison; ladhani, shamez; o’brien, pat title: sars-cov-2-specific antibody detection in healthcare workers in a uk maternity hospital: correlation with sars-cov-2 rt-pcr results date: 2020-08-08 journal: clin infect dis doi: 10.1093/cid/ciaa893 sha: doc_id: 322204 cord_uid: kc7dy2za nan m a n u s c r i p t dear editor, during the ongoing covid-19 pandemic, staff shortages due to illness, self-isolation and redeployment have been a major challenge. universal healthcare worker (hcw) testing is potentially useful in ameliorating workforce depletion and reducing asymptomatic spread of sars-cov-2. nasopharyngeal swab polymerase chain reaction (rt-pcr) can diagnose only current or recent infection; testing for antibody responses against sars-cov-2 could enhance the ability to expedite reinstatement of services, while ensuring patient and staff safety. tests are now available for immunoglobulin (ig) g against the sars-cov-2 nucleocapsid protein; the abbott sars-cov-2 igg elisa is reported to have high specificity we previously reported that 32% of hcw testing positive for sars-cov-2 on nasopharyngeal swab were asymptomatic at the time. 2 symptomatic and asymptomatic sars-cov-2 positive adults have similar viral loads and infectious virus isolation. 3 our finding that both of these groups developed sars-cov-2 igg antibodies is reassuring. of those testing positive for sars-cov-2 igg, 39% had an earlier negative nasopharyngeal swab. possible explanations are that either infection occurred at an interval before or after the swab test, or the swab rt-pcr gave a false negative result (due to poor swabbing technique, suboptimal storage conditions, delay in testing, or poor sensitivity of nasopharyngeal swabs, reported to be as low as 70%). 4 the overall prevalence of sars-cov-2 igg (22%) among hcw was higher than in the general population in london (17%) or across the uk (5%). 5 both symptomatic and asymptomatic infections were associated with sars-cov-2 igg antibodies, as were 10% of performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in covid-19 screening of health-care workers in a london maternity hospital asymptomatic transmission, the achilles' heel of current strategies to control covid-19 evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections we would like to thank ms jane whitney-smith (chief executive officer, the portlandhospital for women and children) and gregory bale (acting chief operating officer, the portland hospital for women and children) for their invaluable support. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-280571-ntgt5hy9 authors: ginocchio, christine c. title: strengths and weaknesses of fda-approved/cleared diagnostic devices for the molecular detection of respiratory pathogens date: 2011-05-01 journal: clin infect dis doi: 10.1093/cid/cir046 sha: doc_id: 280571 cord_uid: ntgt5hy9 the rapid, sensitive, and specific identification of the microbial etiological characteristics of respiratory tract infections enhances the appropriate use of both antibiotics and antiviral agents and reduces the risk of nosocomial transmission. this article reviews the current nucleic acid amplification tests approved by the u.s. food and drug administration (fda) for the detection of respiratory pathogens. in addition, emergency use authorization tests for the detection of 2009 influenza a h1n1 are discussed. the advantages and limitations of the current fda-approved/cleared tests are reviewed. the identification of the causative agent(s) of respiratory tract infections is essential to provide an accurate diagnosis, appropriately manage patient care, and reduce the risk of nosocomial transmission within health care facilities. with the steady rise of antibiotic resistance and limited or no options available for the treatment of multidrug or panresistant bacterial infections, pathogen identification is a key component in restricting antibiotic use to those circumstances in which antibiotic therapy is clearly indicated [1] . initial empiric therapeutic choices may be standardized and initiated on the basis of patient clinical status, underlying disease, and/or risk for infection with a multidrug-resistant pathogen. however, subsequent bacterial pathogen identification and accurate susceptibility data should assist in promoting switches to targeted specific therapies and reduce the use of broad-spectrum antibiotics when not indicated, thereby promoting good antibiotic stewardship [2] . viral infections probably cause between 75% and 80% of respiratory tract diseases. nonetheless, it is estimated that 22.6 million (55%) of 41 million antibiotic prescriptions for respiratory tract infections, including both lower and upper respiratory infections, were for causes unlikely to have a bacterial etiology [3] . although in many cases of otitis and sinusitis and probably 20%-40% of cases of community-acquired pneumonia a bacterial superinfection may occur, respiratory infections due to a virus(es) alone are common in the adult and pediatric outpatient populations. in addition, the vast majority of respiratory tract infections in nonimmunocompromised hospitalized children (especially those ,5 years of age) are due to 1 or more viruses, without a secondary bacterial infection. the overuse of antibiotics for the treatment of outpatients is primarily due to the fact that in adults respiratory virus testing is either not performed or generally limited to rapid antigen direct tests (radts) for influenza. testing for the elderly, for persons with chronic obstructive pulmonary disease, and for pediatric patients is limited to radts for influenza and respiratory syncytial virus (rsv). radts have highly variable sensitivities (10%-75%) and specificities (50%-100%) depending on the viral target, age of the patient, sample collection, and duration of symptoms prior to testing [4] [5] [6] . in general, radts perform better when testing pediatric samples, because children shed higher titers of virus and for longer time periods than adults [4, [7] [8] [9] . in addition to radts, there are u.s. food and drug administration (fda)-approved/cleared nonmolecular-based viral diagnostic methods with a more rapid time to result, compared with traditional viral tube culture, eg, direct fluorescent antibody (dfa) testing and rapid cell culture. both methods can readily detect 7 of the common respiratory viruses (adenovirus, influenza a, influenza b, parainfluenza 1, 2, and 3 [piv-1, piv-2, piv-3], and rsv). in addition, dfa testing can detect human metapneumovirus (hmpv) [6] . the specificity of dfa testing and rapid cell culture are high, but the sensitivities of the tests can vary from a low of 50% (rsv culture) to a high of .80% (influenza a), when compared with nucleic acid amplification tests (naats) [5, 6, 10] . dfa testing can be performed in as little as 30-60 min, and shell vial and r-mix rapid cell cultures (quidel/diagnostic hybrids) generally identify respiratory viruses in 24-48 h [6] . if these tests are performed on site, the time to virus detection can be within a time frame that could affect patient management. however, these tests are not widely available outside larger hospitals and reference laboratories. although these 8 viruses are responsible for a large number of respiratory tract infections, bocavirus, selected coronaviruses (229e, oc43, nl63, and hku-1), parainfluenza 4, and rhinovirus are also important causes of respiratory disease and are generally only detected using naats. because antiviral therapies are currently limited to the treatment of influenza a, influenza b, cytomegalovirus pneumonia, and varicella zoster virus pneumonia, it is often argued that the specific identification of other viruses is not relevant, because the information would not change patient management. from a treatment standpoint, this may currently be true; however, the respiratory viruses cause similar illnesses, and diagnosis based on clinical symptoms alone can be highly inaccurate [11] . for example, a study by poehling et al revealed that physicians who used only clinical symptoms recognized influenza in only 28% of hospitalized children and 17% of nonhospitalized children with laboratory-confirmed influenza [11] . therefore, establishing the viral etiological characteristics of the illness is often highly dependent on accurate diagnostic testing. in addition, new therapeutic agents for respiratory viruses are in development, and clinical trials for these agents will need rapid diagnostics that detect a broad range of viral pathogens. once these new drugs are approved, clinical laboratories will need the tools to identify each virus so that appropriate antiviral therapy can be rapidly initiated. in addition, during the first weeks of the 2009 influenza a h1n1 outbreak in the spring of 2009, multiple influenza viruses were cocirculating [5] . it was necessary to subtype influenza a strains to differentiate seasonal influenza a/h1, seasonal influenza a/h3, and 2009 influenza a h1n1. subtyping is necessary to provide relevant information needed for appropriate selection of antiviral therapy, in particular for acutely ill patients. antiviral resistance testing of influenza isolates from 2009 (www.cdc.gov/flu) revealed that the circulating seasonal influenza a/h1 strains were resistant to oseltamivir (99.6%), and seasonal influenza a/h3 and 2009 influenza a h1n1 were resistant to the adamantanes (100%). in addition, several patients with 2009 influenza a h1n1 infections developed oseltamivir resistance [12, 13] . other clinical factors, such as the potential for the development of more severe disease on the basis of the virus etiology, may need to be considered in the management of certain patients. for example, studies from our laboratory have revealed that children with hmpv infections have a higher incidence of admission to an intensive care unit and more often require mechanical ventilation than children with rsv infections [14, 15] . especially in the treatment of inpatients, the costs of rapid viral diagnostics can be offset by the improvement in patient care and financial outcomes [16] [17] [18] [19] . hendrickson et al showed that rapid respiratory virus diagnosis can lead to benefits in several areas, including up to a 50% reduction in hospital days, 30% reduction in antibiotic use, and 20% reduction in unnecessary diagnostic tests and procedures [16] . the burden of nosocomial influenza can be high, incurring additional costs for diagnostic tests, increased morbidity, and extended hospitalization [17, 18] . therefore, rapid diagnostic tests are needed to identify patients with influenza at admission, in order to prevent nosocomial transmission by facilitating isolation and cohorting decisions [18] . studies have documented substantial nosocomial transmission of hmpv in pediatric units [20] , as well as in chronic care facilities [21] , similar to what is seen with rsv. during the height of rsv season, many institutions must cohort rsv-positive children because of a lack of private rooms. however, dual infections with rsv and hmpv do occur. limiting diagnostics to rsv alone in a cohorting scenario could put other seriously ill children at risk for acquisition of a second viral infection with hmpv. the meaning of mixed viral infections can be defined only if testing is comprehensive. broad test panels also allow for monitoring the epidemiologic patterns of respiratory disease and for identifying new or reemerging pathogens. finally, the identification of the exact respiratory virus is essential for the accurate assessment of the efficacy of vaccines. the costs for testing using fda-approved/cleared naats are highly variable and can range from approximately $30 to .$200 per test. factors that determine test cost include test volume, the price of the test kits, the number of tests per kit, size of the testing run, the number of controls required per testing run, and the type and amount of ancillary supplies. often fda-approved/ cleared kits do not contain nucleic acid extraction reagents, so additional equipment and reagents are necessary and can add $3-$15 per test. batch testing of samples may reduce the cost per test; however, often batching is not practical if the time to result is delayed beyond a period that would affect either clinical management or infection control practices. instrumentation costs for molecular tests can be substantial, with real-time instrumentation costing on average $35,000-$85,000 per instrument, and higher volume laboratories may require multiple instruments. laboratories must also calculate the cost for technical time, which is again highly variable depending on the test complexity and run size. finally, the costs for quality control, proficiency testing, and competency assessment all affect the overall cost per test. in deciding which tests are appropriate and at what cost for the patient population at a particular health care facility, all of the above factors need to be considered in light of the clinical impact of the test result. the use of naats is an intrinsic part of infectious disease diagnostics. the ability of naats to rapidly and accurately detect a novel pathogen was best exemplified during the 2009 influenza a h1n1 pandemic [5, [22] [23] [24] . naats are especially suited for the identification of respiratory pathogens that are not routinely or easily cultured (eg, hmpv, bocavirus, parainfluenza 4, and chlamydophila pneumoniae), pathogens that are dangerous to culture (eg, severe acute respiratory syndrome coronavirus), pathogens for which the time to detection by traditional means is often too delayed to affect patient care (eg, tube cell culture for influenza), and pathogens for which serologic testing is difficult to interpret (eg, c. pneumoniae). in most cases, naats offer enhanced sensitivity over culture methods, radts, and dfa testing [5, 6, 10] . the specificity of naats varies with target and assay design but is generally very high. in addition, laboratorydeveloped naats validated in accordance with the clinical laboratory improvement amendments (clia) requirements can be used to identify new pathogens until fda-approved/ cleared in vitro diagnostic (ivd) devices become available. with clinical integration of real-time polymerase chain reaction (pcr) and fda-approved/cleared simple cartridge-based naats, laboratories of all sizes are now able to perform molecular diagnostic tests. the detection of mycobacterium tuberculosis directly in a clinical sample from a patient not yet identified as m. tuberculosis positive is always clinically relevant. detection of such bacterial pathogens as bordetella pertussis, bordetella parapertussis, legionella pneumophila, mycoplasma pneumoniae, or c. pneumoniae usually indicates active infection. in contrast, the detection of other bacteria, such as streptococcus pneumoniae, may indicate infection or simply colonization. therefore, interpretation of the molecular detection of many bacterial pathogens must be viewed in light of the clinical specimen (sterile vs nonsterile body site) and determination of the quantity of organisms present. in addition, molecular methods must include all potential pathogens, even though culture is still required for antimicrobial susceptibility testing. for these reasons, there is currently a scarcity of fda-approved/cleared assays for nonviral respiratory pathogens (table 1 ). for such targets as the atypical pneumonia pathogens, the interpretation of naat results is generally not an issue, quantitative tests are not necessarily indicated, and naats could replace traditional test methods. however, the lack of fda-approved/cleared naats for these pathogens most probably relates to the ivd device manufacturers' reluctance to perform costly clinical trials for targets with a relatively low prevalence rate and for which the potential volume of testing may be minimal. in light of the rise in m. tuberculosis drug resistance worldwide, the rapid identification of patients with pulmonary tuberculosis is essential and allows for improved patient outcomes, the appropriate use of isolation facilities, the initiation of appropriate treatment, and the identification of possible infected contacts [25] [26] [27] . in addition, studies have shown that the estimated costs associated with an inaccurate diagnosis of m. tuberculosis infection are substantial ($11,576 per patient) because of institutional isolation procedures [27] . direct sample testing can detect m. tuberculosis within 24-48 h of sample collection, compared with 1-4 weeks for liquid and traditional solid media culture methods. although acid-fast bacilli (afb) smear microscopy is inexpensive and rapid to perform, the sensitivity of the test can be poor (45%-80% with culture-confirmed pulmonary m. tuberculosis colonization or infection) and cannot differentiate m. tuberculosis from mycobacteria other than m. tuberculosis [26] . the 2009 centers for disease control and prevention (cdc) guidelines recommend that naats should be performed on at least 1 respiratory sample from patients with signs and symptoms of pulmonary m. tuberculosis colonization or infection for whom a diagnosis of m. tuberculosis colonization or infection has not been established or when the results would alter case management or infection control procedures [28] . mycobacterial culture is still required, because the m. tuberculosis isolate is needed for drug susceptibility testing. currently, there are 2 fda-approved naats available for the detection of m. tuberculosis complex from respiratory samples, the amplified mycobacterium tuberculosis direct test (amtd, gen-probe) [25, [29] [30] [31] and the amplicor mycobacterium tuberculosis test (amplicor, roche diagnostics) [32] [33] [34] [35] . recently, a new assay called the xpert mtb/rif (cepheid) was developed to detect the presence of m. tuberculosis and rifampin resistance directly from processed clinical samples [reviewed in 36] . the assay uses a heminested real-time pcr with molecular beacon detection and is performed on the genexpert instrument (cepheid). benefits of this assay include low technical complexity, allowing for potential point-of-care testing; minimal hands-on time (15-20 min); test turnaround time of ,2 h; and containment of the sample, extracted nucleic acids, and amplicons in the test cartridge. when compared with culture, the sensitivity of the assay for the detection of m. tuberculosis ranged from 98.2% (1 sample tested per patient) to 99.8% (3 samples tested per patient) for afb smear-positive samples and from 72.5% (1 sample tested per patient) to 90.2% (3 samples tested per patient) for afb smear-negative samples. the specificity of the assay ranged from 98.1% to 99.2%. the assay is currently available for diagnostic testing only in europe. amplified mycobacterium tuberculosis direct test (amtd, gen-probe) the amtd is a target-amplified nucleic acid probe test for the direct detection of m. tuberculosis complex (mycobacterium bovis, m. bovis bcg, mycobacterium africanum, mycobacterium microti, and m. tuberculosis) ribosomal rna (rrna) [25, [29] [30] [31] . amtd is approved for testing both afb smear-positive and smear-negative respiratory specimens collected from patients suspected of having m. tuberculosis colonization or infection. the amtd test is based on isothermal (42 o c) transcription-mediated amplification that uses 2 enzymes, reverse transcriptase (rt) and t7 rna polymerase, and targetspecific primers. a hybridization protection detection assay that uses a chemiluminescent-labeled, single-stranded dna probe complementary to the m. tuberculosis complex-specific sequences detects the amplified rrna using the gen-probe leader luminometer. numerous studies have evaluated amtd and have revealed an average sensitivity and specificity of .95% and .98%, respectively, for the detection of m. tuberculosis complex in smear-positive respiratory samples and a sensitivity and specificity of .60% and .72%, respectively, for smear-negative respiratory samples [29] [30] [31] . additional studies have evaluated the use of the test for several types of nonrespiratory samples, such as cerebrospinal fluid and lymph nodes [29] [30] [31] . amplicor mycobacterium tuberculosis test (amplicor, roche diagnostics) the amplicor mycobacterium tuberculosis test is fda approved for use with afb smear-positive respiratory specimens from patients suspected of having m. tuberculosis infection or colonization [32] [33] [34] [35] . the amplicor mycobacterium tuberculosis test uses traditional pcr to amplify a region of the gene encoding the 16s rrna of all mycobacteria. members of the m. tuberculosis complex are identified after hybridization with a dna target-specific probe, followed by substrate addition and colorimetric detection [32] . when testing afb smear-positive samples, the amplicor mycobacterium tuberculosis test demonstrated sensitivities and specificities for the detection of m. tuberculosis complex ranging from 92.9% to 100% and from 77.3% to 100%, respectively [32, 33] . the sensitivities and specificities of amplicor mycobacterium tuberculosis for afb smear-negative specimens ranged from 51.2% to 73.1% and 99% to 99.8%, respectively [32, 33] . the amplicor mycobacterium tuberculosis test has also been evaluated for detection of m. tuberculosis meningitis [34] and for use with nonrespiratory sample types [35] . viral respiratory pathogens are particularly suited for detection using naat, since the number of targets is relatively limited, compared with the numerous potential bacterial pathogens that can cause respiratory disease (table 1) . although there is much to learn regarding the clinical relevance of mixed viral respiratory infections, the detection of a respiratory virus is generally considered diagnostic at this time. today, most laboratories do not have the facilities for comprehensive tissue culture based viral diagnostics and therefore are limited to testing with less sensitive and less specific radts for only influenza a, influenza b, and rsv. naats offer an excellent alternative to greatly expand the test menu of clinical laboratories, thereby providing rapid, accurate comprehensive diagnostics. the first multiplex naat to receive clearance by the fda was the xtag respiratory virus panel (rvp) assay in january 2008. the fda-approved version of rvp detects adenovirus, influenza a (with subtyping of seasonal influenza a/h1 and seasonal influenza a/h3), influenza b, piv-1, piv-2, piv-3, hmpv, rhinovirus, rsv a, and rsv b [37, 38] . the us/canadian research use-only version of the assay also detects 4 coronaviruses (oc43, 229e, nl63, and hku-1), parainfluenza type 4, and enterovirus. the test is approved for use with nasopharyngeal swab samples collected from persons symptomatic for a respiratory virus infection and placed in viral transport media. the xtag rvp is a multistep test that takes approximately 8-10 h to complete, depending on the number of samples to be tested (figure 1 ). viral nucleic acid and an internal control (e. coli ms2 phage) are coextracted from clinical samples using the qiaamp minielute (qiagen), the easymag (biomã©rieux), or the minimag (biomã©rieux) extraction platforms. a multiplex reverse transcription polymerase chain reaction (rt-pcr), using primer sets specific for the test targets, amplifies the viral nucleic acid and internal control nucleic acid. pcr products are treated with exonuclease i to degrade any remaining primers and with shrimp alkaline phosphatase to degrade any remaining nucleotides. the next step consists of target-specific primer extension (tspe). when a viral target(s) is present, the target-specific primer (containing a unique tag sequence) is extended and biotin-deoxycytidine triphosphate is incorporated into the extending chain. on completion of the tspe, the detection of amplified products is performed using luminex's universal tag sorting system. the tspe reaction is added directly to microwells containing spectrally distinguishable beads with antitags, which are complementary to the sequence tags on the primers. each tagged primer will hybridize only to its unique antitag complement associated with a specific colored bead. a fluorescent reporter molecule (streptavidin-phycoerythrin) will bind to the biotin on the extended primers. the beads are then analyzed with the luminex xmap 100/200 instruments. two lasers read each bead; the first identifies the virus-specific color-coded bead, and the second determines whether an amplicon is hybridized to the bead on the basis of the detection of fluorescence (mean fluorescence intensities [mfis]) above a background threshold. the clinical performance characteristics of the xtag rvp assay were established by the manufacturer through prospectively collected nasopharyngeal swab samples (n 5 544) tested during the 2005-2006 influenza season at 4 north american clinical laboratories [101] . all specimens were tested by means of viral culture and/or dfa testing for the following targets: influenza a, influenza b, rsv, piv-1, piv-2, piv-3, and adenovirus. the comparator methods for influenza a subtyping, rsv subtyping, and hmpv and rhinovirus detection were wellcharacterized rt-pcr assays followed by bidirectional sequencing. xtag rvp sensitivity for each target was determined as follows: the benefits of the xtag rvp assay include the broad spectrum of viruses detected by a single test, with a cost per test comparable to real-time assays that only target up to 3 analytes. the subtyping of influenza a viruses as seasonal influenza a/h1 or seasonal influenza a/h3 or the identification of an ''unsubtypeable'' virus has proven to be an important aid in identifying novel influenza a strains. a limitation of the assay is the decreased sensitivity for the detection of certain adenovirus strains. in-house validation studies by our laboratory have found that by reducing the positive cutoff mfi level from 300 to 150 for adenovirus, improved sensitivity for the detection of adenovirus was obtained without loss of specificity (unpublished data). additional limitations of the assay include the time to final results, number of required steps, technical handson time, and a potential for amplicon contamination. there is a second-generation assay called rvp fast (luminex) available in europe that addresses several of these issues by reducing the number of steps and the time to results by 3-4 h, making it possible to provide comprehensive results within a single shift. gen-probe/prodesse proflu,1proflu-st, pro hmpv1, and proparaflu1 assays (gen-probe/prodesse) currently, there are 3 gen-probe/prodesse fda-cleared multiplex real-time rt-pcr assays for the qualitative detection and discrimination of respiratory viruses. the proflu1 assay targets the matrix gene for influenza a, nonstructural genes ns-1 and ns-2 for influenza b, and the polymerase gene for rsv a and rsv b. the pro hmpv1 assay targets highly conserved regions of the nucleocapsid (n) gene for hmpv and a transcript derived from escherichia coli bacteriophage ms2 a-protein gene (internal control). the proparaflu1 assay targets conserved regions of the hemagglutinin-neuraminidase gene for piv-1, piv-2, and piv-3 and a transcript derived from e. coli bacteriophage ms2 a-protein gene (internal control). all 3 assays are approved for testing nasopharyngeal swab specimens obtained from symptomatic persons. viral nucleic acids from patient samples are coextracted with an internal control that monitors assay performance and the presence of amplification inhibitors that could lead to false-negative results. nucleic acids are extracted using a magna pure lc instrument (roche diagnostics corp) and the magna pure total nucleic acid isolation kit (roche) or a nuclisens easymag system and the automated magnetic extraction reagents (bio-mã©rieux). the purified nucleic acids are amplified by means of rt-pcr using target-specific oligonucleotide primers and taqman probes complementary to highly conserved regions of the target gene. the taqman probes are labeled with a quencher dye attached to the 3'-end and a reporter dye at the 5'-end. when amplified target is present, the probes bind and the 5'-3' exonuclease activity of taq polymerase cleaves the probe, thus separating the reporter dye from the quencher. because the quencher and reporter dye are now physically separated, there is an increase in fluorescent signal upon excitation from a light source. the fluorescent signal increases with each cycle as additional reporter dye molecules are cleaved from their respective probes. during each pcr cycle, the fluorescent intensity is monitored by the realtime instrument, the smartcycler ii (time to results, including extraction, is approximately 3-3.5 hr for each test run). the performance characteristics of the assays were established by the manufacturer through prospective and retrospective clinical studies used for fda clearance of the tests. proflu1 assay results obtained by testing 891 nasopharyngeal samples were compared with results of rapid shell vial culture. proflu1 sensitivities and specificities for the detection of influenza a were 100% and 92.6%, respectively; for influenza b were 97.8% and 98.6%, respectively; and for rsv were 89.5% and 94.9%, respectively [102]. a study by liao et al compared the prodesse proflu-1 assay, a previous version of the proflu1 assay, with viral culture and radts for rsv (now rsv, binax, inverness medical) and for influenza a and b (directogen a1b, bd diagnostics) [10] . the specificities of all methods were found to be .99%. the sensitivities for detection of influenza were 59% for directogen a 1 b, 54% for viral culture, and 98% for proflu-1; the sensitivities for the detection of rsv were 82% for rsv now, 57% for viral culture, and 95% for proflu-1. in another study, legoff et al evaluated the performance of proflu-1 in 353 pediatric nasopharyngeal specimens [40] . results were compared with dfa testing and viral culture. the sensitivities and specificities of proflu-1 ranged from 97% to 100%, and proflu-1 detected viruses in 9% of the samples that had negative results by conventional methods. in response to the 2009 influenza a h1n1 outbreak, an influenza a subtyping assay (proflu-st) was developed by prodesse and received emergency use authorization (eua) from the fda in july 2009 (see eua section). proflu-st is a qualitative multiplex real-time rt-pcr assay that targets the nucleoprotein gene of 2009 influenza a h1n1, the specific hemagglutinin genes of seasonal influenza a/h1 and seasonal influenza a/h3, and an internal control (ms2 phage). the identification of 2009 influenza a h1n1 is aided by an algorithm that relies on seasonal influenza a/h1 virus and seasonal influenza a/h3 virus results in nasopharyngeal swab specimens from patients who receive a diagnosis of influenza a by a currently available fda-cleared or authorized device. this assay is intended for use in only clia high-complexity laboratories. the performance of the assay was evaluated retrospectively using nasopharyngeal swab specimens that were previously tested with either the cdc rrt-pcr flu panel (ivd device) to detect seasonal influenza a/h1 and influenza a/h3 or the cdc rrt-pcr swine flu panel (eua). the positive and negative agreements for the detection of seasonal influenza a/h1were 95.8% and 100%, respectively; for seasonal influenza a/h3 were 100% and 100%, respectively; and for 2009 influenza a h1n1 were 96.2% and 100%, respectively [103] . the pro hmpv1 assay clinical trial study for fda clearance evaluated the assay's clinical performance using 1275 nasopharyngeal swab specimens tested by 4 clinical laboratories across the united states. using the luminex rvp assay as the predicate device, the sensitivity of pro hmpv1 was 94.1% and the specificity was 99.3% [104]. the performance of the proparaflu1 assay was evaluated during the clinical trials for fda clearance. using 857 nasopharyngeal swab specimens tested by 4 clinical laboratories across the united states [105], the sensitivities and specificities of the assay for the detection of piv-1 were 88.9% and 99.9%, respectively; for the detection of piv-2 were 96.3% and 99.8%, respectively, and for the detection of piv-3 were 97.3% and 99.2%, respectively [105] . at this time, no additional independent performance data are available. the benefits of the gen-probe/prodesse assays include ease of use, with approximately 1.5 h of hands-on time for nucleic acid extraction preparation and a 1-step rt-pcr setup. the overall time to results is 4.5-5.5 h. multiple smartcycler instruments can be run simultaneously, with as many units per cycler used as needed, giving flexibility to run sizes. because tubes containing amplicons are never opened, the risk of amplicon contamination is minimal. one limitation of the assays is a maximum of 3 targets plus an internal control that can be detected in a single reaction. therefore, a comprehensive viral diagnostic panel requires multiple pcrs. multiple pcrs are costly to the laboratory in both technical time and reagent cost. however, the limited panel size could provide a mix-and-match test menu, allowing clinicians the option of selecting 1 or several panels. the first-generation verigene respiratory virus nucleic acid test (vrnat) was cleared by the fda in may 2009. this test has been replaced by the automated verigene vrnat sp , a clia moderately complex test that is intended for the identification of influenza a, influenza b, and rsv (types a and b inclusive) from nasopharyngeal swab specimens placed in viral transport media. the verigene system consists of 2 instruments (the fully automated verigene processor and the verigene reader) and single-use test cartridges (figure 2 ). the entire test process only requires 1 user pipetting step, less than 5 min of technical handson time, and a sample-to-result turnaround time of about 3.5 h. the basis of vrnatsp is nanosphere's proprietary gold nanoparticle hybridization technology [41] . the gold nanoparticles contain a high density (200) of sequence-specific oligonucleotides with a high affinity for complementary dna, which allows for very efficient hybridization kinetics. the clinical sample is pipetted into a single-use extraction tray, which is loaded into the verigene processing unit. chaotropic agents are added to the sample to lyse cells, viral particles, and an internal control (ms2 phage) that is added prior to the extraction step. the released nucleic acids are then captured on magnetic microparticles (mmps). the mmp-bound nucleic acids are washed, and the purified nucleic acids are eluted from the mmps and transferred to the amplification tray. a 1-step rt-pcr is performed using primers that target the influenza a matrix gene, the influenza b matrix gene and nonstructural gene, and the rsv l gene and f gene. the rt-pcr reaction is followed by the primary hybridization step. during primary hybridization, target dna is simultaneously hybridized to target-specific capture dna oligonucleotides arrayed in replicate on a solid substrate (a microarray) and to target-specific mediator dna oligonucleotides. after removal of uncaptured target nucleic acids and unhybridized mediator oligonucleotides, the process continues with the secondary hybridization. each microarray spot, where an appropriate target is hybridized to capture both an oligonucleotide and a mediator oligonucleotide, is saturated with silver-coated gold nanoparticle probes. after hybridization, the cartridge is removed from the processor unit and the glass slide (microarray) is separated from the cartridge and inserted into the verigene reader. a light-scattering technique, in which the slide is illuminated internally with light parallel to the slide surface, is used to analyze the results. spots where silverenhanced gold nanoparticle probes are present scatter this light, and the light scatter is detected optically and translated into a measurable signal. the performance characteristics of the first-generation vrnat assay were established during the clinical trials for ivd device clearance. vrnat was compared with viral culture/dfa testing with bidirectional sequencing to resolve discordant results. test sensitivities and specificities for the detection of influenza a were 100% and 99.8%, respectively; for influenza b were 100% and 99.1%, respectively; and for rsv were 95.7% and 98.2%, respectively [106] . comparison of vrnat with vrnatsp revealed an overall positive agreement of 97.9% and a negative agreement value of 100% [107] . the benefits of the vrnatsp system include the scalability of the system (addition of multiple processors and readers), individual sample processing with random access format, minimal hands-on time, and minimal technical expertise required to perform the test. because this test is clia moderate complexity, trained laboratory technicians could perform the testing. this test would be well suited for small-to medium-sized laboratories, in particular for laboratories with little or no molecular testing experience. limitations of the assay include the single test format that requires a dedicated processor for each sample for 3-3.5 h. multiple processor units would be needed for larger volume laboratories, and the additional instrumentation would increase costs for the lab, compared with using an instrument that can run multiple tests at a time. table 2 were rescinded as of 23 june 2010. as of july 2010, only 2 of these tests have received fda approval for use as an ivd device for the diagnosis of 2009 pandemic influenza a h1n1. the first test approved was a new optimized cdc h1n1 assay that is available in cdc-qualified laboratories. the second test is the focus diagnostics simplexa influenza a h1n1 (2009), which is performed using the 3m integrated cycler (3m). the use of fda-approved/cleared naats, in contrast to laboratory-developed tests, has substantial benefits for the laboratory. approved tests have undergone extensive analytical and clinical validations during the course of the fda evaluations. therefore, performance parameters are well characterized, and the fda monitors postapproval performance. most laboratories do not have the expertise and resources to perform extensive (table 3) , thereby saving the laboratory considerable cost and technical time. the regulatory requirements for ongoing quality assurance monitoring are also fewer for fda-approved/cleared naats than for laboratory-developed tests [42] . in addition, for regulatory reasons, some health care institutions will only allow the use of fda-approved/cleared naats. because the clinical relevance of the assays has been established, the use of fda-approved/cleared naats has a higher chance of reimbursement from both federal and private insurance payors. however, fda approval/clearance does not guarantee that these tests will be reimbursed, and, if reimbursed, the actual amount of the reimbursement can vary from state to state and by payor. finally, the simple-to-use fully automated molecular platforms, such as the genexpert and genestat, which incorporate all steps of the test process in a single test cartridge and have random-access sample-in result-out reporting, enable laboratories of all sizes to perform rapid, accurate, and sensitive molecular diagnostic testing. the most important limitation of the current fda-approved/ cleared naats is the lack of tests for the detection of nonviral targets. in particular, naats are needed for the detection of the atypical pneumonia pathogens. however, the costs of clinical trials for ivd device clearance can sometimes be quite prohibitive (.$10 million, not including the costs of developing and manufacturing the ivd device) and depend on the complexity and clinical relevance of the test and what is required for fda approval (eg, number of trial sites, number of patients enrolled, clinical indications sought, need for long-term patient follow-up, associated diagnostic procedures [such as biopsy], device evaluations, predicate device comparisons, legal and regulatory components, and so forth). manufacturers must consider the potential number of tests that will be purchased and the difficulty of the trials for low-prevalence pathogens before committing the finances and resources to bring such tests through the approval process. these factors will continue to limit the scope of fda-approved/cleared naats until the approval process can be modified to encourage the submission of naats for low-prevalence targets. the current formats of the naats require laboratories to choose between real-time, easier to perform, more rapid assays that have limited targets (generally up to 3) and more highly multiplex tests (.10) that require more hands-on time and technical steps and more time to result reporting. naats with limited targets would require laboratories to run multiple tests if a broader range of target detection is indicated. multiple tests would increase both the required technical time and the cost per patient diagnosis. conversely, the use of multiple, lower multiplex tests allows for the selection of the most appropriate panels as related to age of the patient, clinical status, underlying disease, state of immune competence, and the suspected virus(es). currently in development are modified faster versions of the highly multiplex assays that have been designed to reduce technical hands-on time and the time to results. some of the fda-approved/cleared real-time naats do not allow the user to see and evaluate the actual amplification curves. the inability to see the curves makes it difficult to troubleshoot when problems occur. although the user should not be able to alter cutoff values established during the fda trials, access to all or part of the raw data is desirable. one caveat for most qualitative naats is that they cannot distinguish between live and dead organisms and therefore, depending on the time for nucleic acid clearance, can be limited in monitoring response to therapy. in addition, with multiple viral infections, determining the relevance of each virus present in the sample is difficult, because residual virus detected may have been from a previous infection and may not be contributing to the current illness. the development of quantitative assays and evidence of declining viral load may clarify or resolve both of these issues. finally, issues relating to billing and reimbursement are substantial. the lack of specific current procedural terminology (cpt) codes for each of the individual targets requires laboratories to bill for multiple targets using the same generic amplified probe cpt code (87798). although it is appropriate to bill for each target present in a multiplex assay, the use of the same cpt code multiple times for a single test can be problematic. for example, many laboratory or hospital billing systems do not recognize multiple similar cpts for a single test and will only drop 1 cpt code charge. some insurance payors will cover only the charge of the first cpt code, and there are also limitations on how many times per day a similar cpt code can be billed. as a result, reimbursement for a highly multiplexed assay could be limited to 1 charge, thereby significantly increasing the costs to the laboratory and decreasing test profitability. under these circumstances and with pressure to reduce medical costs, many administrators will not approve the implementation of this testing if reimbursement is questionable. laboratories need analyte-specific cpt codes, and payors should reimburse for medically relevant naats. the north shore-long island jewish health system (ns-lijhs) infectious diseases molecular diagnostics laboratory opened 12 years ago. we performed 3 tests: human immunodeficiency virus type 1 viral load, amtd, and chlamydia trachomatis/ neisseria gonorrhoeae naats, with a total testing volume of 5000 tests per year. today, the laboratory performs naats for 40 different pathogens (including bacterial, mycobacterial, fungal, parasitic, and viral targets), with a volume of more than 225,000 tests per year, and in space that has tripled in size. molecular diagnostics for infectious diseases, along with molecular pathology, are 2 of the fastest growing laboratory departments in the ns-lijhs. in addition, as best exemplified by the 2001 anthrax bioterrorism event and the 2009 influenza a h1n1 pandemic, laboratories must be able to respond immediately by rapidly expanding testing capabilities, including sensitive and specific molecular diagnostics [43] . at the onset of the queens, new york, area 2009 influenza a h1n1 outbreak, the ns-lijhs laboratories' respiratory virus testing volume increased from a baseline of 225 tests per day to more than 950 tests per day, within 3 days. it was clear from the first week of the outbreak that the laboratory urgently needed to switch all influenza testing to molecular methods to obtain sufficient diagnostic sensitivity and to subtype the influenza strains. fortunately, an fda-approved test, the luminex xtag rvp test, was available and met our diagnostic requirements [5, 24, 43] . laboratories must continue to plan for future pandemic outbreaks and biothreat events. therefore, laboratories are constantly under pressure to expand testing and meet the demands of their clinical staff and their diverse patient populations. to do this, laboratories must provide clinically relevant, high-quality, cost-effective naats that are preferably fda approved/cleared. as we move forward, we request that the fda work closely with such organizations as the infectious diseases society of america and the american society for microbiology, with laboratory directors, and with ivd device manufacturers so that this goal can be met. the labor-intensive, complex methods and platforms of yesterday have evolved into simpler, user friendly versions that can be applicable to all health care settings. we encourage the fda to allow the submission process to evolve in a similar manner. no longer are the old ''gold standards'' of culture applicable, and new ways to evaluate these tests must be considered [44] so that accurate performance characteristics can be determined in a cost-effective manner. the latter is especially true for low-volume but highly relevant assays, such as quantitative naats for transplantation monitoring. in a reasonable and clear regulatory environment, the ivd device manufacturers will succeed in bringing their naats through the approval process. the role of antimicrobial management programs in optimizing antibiotic prescribing within hospitals antimicrobial stewardship programs in health care systems excessive antibiotic use for acute respiratory infections in the united states lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults evaluation of multiple test 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luminex xtag respiratory virus panel is indicative of novel a/h1n1 (swine-like) influenza comprehensive evaluation of performance, laboratory application, and clinical usefulness of two direct amplification technologies for the detection of mycobacterium tuberculosis complex use of the amplified mycobacterium tuberculosis direct test in a public health laboratory: test performance and impact on clinical care evaluation and follow up of infectious tuberculosis at the university of ottawa updated guidelines for the use of nucleic acid amplification tests in the diagnosis of tuberculosis comparative evaluation of initial and new versions of the gen-probe amplified mycobacterium tuberculosis direct test for direct detection of mycobacterium tuberculosis in respiratory and nonrespiratory specimens comparison of the real-time pcr method and the gen-probe amplified mycobacterium tuberculosis direct test for detection of mycobacterium tuberculosis in pulmonary and nonpulmonary specimens evaluation of gen-probe amplified mycobacterium tuberculosis direct test by using respiratory and nonrespiratory specimens in a tertiary care center laboratory detection of mycobacterum tuberculosis by pcr amplification with pan-mycobacterium primers and hybridization to an m. tuberculosis-specific probe rapid diagnosis of pulmonary tuberculosis by using the roche amplicor mycobacterium tuberculosis pcr test use of roche amplicor mycobacterum tuberculosis pcr in early diagnosis of tuberculous meningitis application of the roche amplicor mycobacterum tuberculosis (pcr) test to specimens other than respiratory secretions xpert m. tuberculosis/ rif for point-of-care diagnosis of tb in high-hiv burden, resourcelimited countries: hype or hope? development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay principles of the xtag respiratory viral panel assay (rvp assay) xtag respiratory virus panel luminex molecular diagnostics enhanced viral etiological diagnosis of respiratory system infection outbreaks by use of a multitarget nucleic acid amplification assay wi: gen-probe/prodesse evaluation of the one-step multiplex real-time reverse transcription-pcr proflu-1 assay for detection of influenza a and influenza b viruses and respiratory syncytial viruses in children wi: gen-probe/prodesse wi: gen-probe/prodesse wi: gen-probe/prodesse verigene respiratory virus nucleic acid test il: nanosphere il: nanosphere homogeneous detection of unamplified genomic dna sequences based on colorimetric scatter of gold nanoparticle probes quality assurance in clinical virology laboratory surge response to pandemic (h1n1) 2009 outbreak discrepant analysis: how can we test a test? i sincerely thank the fda and the infectious diseases society of america for providing a forum for discussion and exchange of ideas relating to this timely and clinically relevant topic.financial support. this work was supported by us department of defense award w81xwh-08-1-0477. potential conflicts of interest. c. c. g. is a member of the scientific advisory boards of gen-probe/prodesse and luminex molecular diagnostics. c. c. g. has received research grants and honoraria from gen-probe/prodesse, luminex molecular diagnostics, and diagnostic hybrids and research grants from 3m molecular diagnostics and has been a consultant for nanosphere. key: cord-324707-9ld73wv1 authors: mitjà, oriol; corbacho-monné, marc; ubals, maria; tebe, cristian; peñafiel, judith; tobias, aurelio; ballana, ester; alemany, andrea; riera-martí, núria; pérez, carla a; suñer, clara; laporte, pep; admella, pol; mitjà, jordi; clua, mireia; bertran, laia; sarquella, maria; gavilán, sergi; ara, jordi; argimon, josep m; casabona, jordi; cuatrecasas, gabriel; cañadas, paz; elizalde-torrent, aleix; fabregat, robert; farré, magí; forcada, anna; flores-mateo, gemma; muntada, esteve; nadal, núria; narejos, silvia; gil-ortega, aroa n; prat, nuria; puig, jordi; quiñones, carles; reyes-ureña, juliana; ramírez-viaplana, ferran; ruiz, lidia; riveira-muñoz, eva; sierra, alba; velasco, césar; vivanco-hidalgo, rosa maria; sentís, alexis; g-beiras, camila; clotet, bonaventura; vall-mayans, martí title: hydroxychloroquine for early treatment of adults with mild covid-19: a randomized-controlled trial date: 2020-07-16 journal: clin infect dis doi: 10.1093/cid/ciaa1009 sha: doc_id: 324707 cord_uid: 9ld73wv1 background: no therapeutics have yet been proven effective for the treatment of mild-illness caused by sars-cov-2. we aimed to determine whether early treatment with hydroxychloroquine (hcq) would be more efficacious than no-treatment for outpatients with mild covid-19. methods: we conducted a multicenter, open label, randomized controlled trial in catalonia (spain) between march 17, and may 26, 2020. eligible covid-19 cases were non-hospitalized adult patients with recently confirmed sars-cov-2 infection and less than five days of symptoms. patients were assigned to receive hcq (800 mg on day 1, followed by 400 mg once daily for 6 days) or no antiviral treatment (not-placebo controlled). study outcomes were the reduction of viral rna load in nasopharyngeal swabs up to 7 days after treatment start, patient disease progression using the who scale up to 28 days, and time to complete resolution of symptoms. adverse events were assessed up to 28 days. results: a total of 293 patients were eligible for intention-to-treat analysis: 157 in the control arm and 136 in the intervention arm. the mean age was 41.6 years (sd 12.6), mean viral load at baseline was 7.90 (sd 1.82) log(10) copies/ml, and median time from symptom onset to randomization was 3 days. no significant differences were found in the mean reduction of viral load at day 3 (-1.41 vs. -1.41 log(10) copies/ml in the control and intervention arm, respectively; difference 0.01 [95% ci -0.28;0.29]) or at day 7 (-3.37 vs. -3.44; d –0.07 [-0.44;0.29]). this treatment regimen did not reduce risk of hospitalization (7.1%, control vs. 5.9%, intervention; rr 0.75 [0.32;1.77]) nor shortened the time to complete resolution of symptoms (12 days, control vs. 10 days, intervention; p = 0.38). no relevant treatment-related aes were reported. conclusions: in patients with mild covid-19, no benefit was observed with hcq beyond the usual care. since the emergence of the novel sars-cov-2 coronavirus in december 2019, various drugs have been proposed as antiviral agents for treating the coronavirus disease-2019 (covid19) , including the aminoquinolines chloroquine and hydroxychloroquine (hcq) [1] . at the time this work started, the us fda and eu ema had given emergency approval for the use of chloroquine and hcq in covid-19 patients [2, 3] . chloroquine and hcq have been extensively used for treating malaria and various autoimmune diseases, although other therapeutic effects, including antiviral effects, have been increasingly recognized [4, 5] . in-vitro studies showed that both drugs can block the viral replication of sars-cov-2 in cell cultures [6, 7] , but a high-level assessment suggested that calculated extracellular lung concentrations are well below the in vitro efficacy values and therefore the drug has low potential for in vivo activity at standard dosing regimens [8] . as of june 20, 2020, publicly available clinical data on the effectiveness of chloroquine and hcq for treating covid-19 were limited to two small randomized clinical trials [9, 10] and six observational studies [11] [12] [13] [14] [15] [16] . several studies were seriously flawed in important methodological respects and lacked internal validity [9, [11] [12] [13] 15] . a randomized trial with 150 patients found that hcq administration did not result in a significantly higher pcr negative conversion (85% vs. 81%) by 28 days [10] . however, the trial design raised concerns about the long delay between the onset of symptoms and the initiation of treatment (median 16.6 days) because antiviral therapy needs to be initiated early to have an impact on viral shedding. two large observational studies of hospitalized patients with covid-19 treated with hcq at physician's discretion found no significant reduction in the risk of death/intubation compared with no specific treatment [14, 16] . because the metrics for each trial were chosen rapidly due to the emerging threat, the measured outcomes were different from one study to the next. the clinical characterization and management working group established by who recently agreed on a minimal outcome set to facilitate study design and data sharing, including viral burden (i.e., quantitative viral rna or cycle threshold from nasopharyngeal swabs), clinical outcome (i.e., progression scale: ambulatory, hospitalized, death) and survival (i.e., all-cause mortality) [17] . we assessed the efficacy and safety of hcq initiated early for treating outpatients with mild covid-19 using the who core outcome set. [18] . trained physicians identified from that registry and selected for study participation non-m a n u s c r i p t 5 hospitalized patients of any kind (health worker, household contact, etc.) recently diagnosed. reasons for non-enrollment were recorded. adult patients aged 18 years or more were eligible if they had mild symptoms of covid-19 (i.e., fever, acute cough, shortness of breath, sudden olfactory or gustatory loss, or influenza-like-illness) for less than five days before enrollment, were non-hospitalized, and had a positive pcr test for sars-cov-2 in the baseline nasopharyngeal swab. patients were excluded if they had moderate-to-severe covid-19 disease (e.g., required hospitalization), any condition that might preclude following the study procedures safely (e.g., mental disability), known allergy or hypersensitivity to study drugs, known retinal and severe liver or renal diseases, history of cardiac arrhythmia, known qt prolongation or other diseases that could be exacerbated by study drugs (e.g., psoriasis), active treatment with medications that are contraindicated with study drugs, or known hiv infection. females who were pregnant (verbally declared or positive pregnancy test) or breastfeeding were also excluded. the study protocol and subsequent amendments were approved by the institutional review board of hospital germans trias pujol, and the spanish agency of medicines and medical devices. written informed consent was obtained from all patients. this trial was a secondary study of the barcelona postexposure prophylaxis study against sars-cov-2 (bcn pep cov-2 study) registered in clinicaltrials.gov, nct04304053. participants were randomized (1:1) using a computer-generated random-number list to either the control arm (no treatment aside from usual care) or the intervention arm (hcq -dolquine ® , 800 mg on day 1, followed by 400 mg once daily for six days). initially, the protocol included the use of hcq and cobicistat-boosted darunavir (drvc) combined treatment, but it was adapted to hcq alone after the recommendation of the pharmaceutical company not to use drvc for the treatment of covid-19 due to lack of activity in-vitro [19, 20] and the negative results in human clinical trials of closely related hiv protease inhibitors [21] . the study medications were dispensed by the hospital pharmacy and provided free of charge to the patients at the first home visit by dedicated outbreak field teams of trained nurses aided by trained paramedical staff. random allocation was done remotely by a member of the study team not involved in participants' enrollment. masking was not possible because a placebo could not be prepared due to the emergency nature of the trial. laboratory technicians were unaware of participants' treatment allocation, treatment response, and previous pcr results at all time points. participants were assessed on day 1 (baseline, hcq was started), and days 3, 7, 14, and 28. on day 1, patients were home visited for baseline assessment and patient enrollment. outbreak field teams verified the selection criteria for eligibility, obtained patients' signed informed consent, assessed specific symptoms associated with covid-19, and collected relevant epidemiological information from a structured interview. disease progression, safety, and self-reported treatment compliance were monitored by the clinical trials unit (ctu) of hospital germans trias pujol at days 3 and 7 (home visits), 14 and 28 (phone visits). compliance was assessed using self-reports in a telephone interview (e.g., number of doses taken between interviews). adverse events (ae) were defined as any new symptom or worsening of pre-existing symptoms and were followed until complete resolution of symptoms or up to day 28 after enrollment. serious adverse events (sae) were defined m a n u s c r i p t 6 as any medical event that required hospitalization or caused patient death; saes were graded for causality and expectedness and reported immediately to the contract research organization of the study sponsor and the trial pharmacovigilance consultancy (asphalion, barcelona) for independent adjudication of relatedness. study data were recorded electronically by the ctu during phone interviews, and on paper case record forms by the outbreak field teams during home visits and then entered into an electronic database by the data entry team of the sponsor. data validation and cleaning were done by trial researchers with the support of a trial data management consultancy (trial form support, barcelona). for each patient, serial oral and nasopharyngeal swab samples were planned to be obtained on days 1 and 3. however, preliminary analyses revealed a possible delay for a significant viral load reduction beyond day 3; therefore, we amended the study protocol to extend the collection of nasopharyngeal swabs to an additional sample on day 7 (a 3-day window was allowed for patients who could not be assessed on day 7). the presence of sars-cov-2 was investigated from nasopharyngeal swabs, and viral load was quantified in all rt-pcr positive cases (all time points collected). details on laboratory methods for sars-cov-2 identification and quantification are provided in supplementary material. the primary outcome was the reduction of viral rna load in nasopharyngeal swabs at days 3, and 7 after treatment start. the secondary outcomes were clinical progression measured by a simplified version of the who progression scale [17] (1, not hospitalized with or without resumption of normal activities; 2, hospitalized, requiring supplemental oxygen; 3, hospitalized, requiring invasive mechanical ventilation; and 4, death), and time from randomization to complete resolution of symptoms within the 28-days follow-up period. resolution of symptoms was assessed sequentially using a symptoms questionnaire designed to gather information on the type of symptom and last day experienced; complete resolution was considered when no covid-19-related symptoms were reported at all. safety outcomes included ae that occurred during treatment, sae, ae of special interest (i.e., cardiac), and premature discontinuation of therapy. ae were classified according to the national cancer institute common terminology criteria for adverse events, version 4.0. all unexpected sae were notified through eudravigilance. we estimated that a sample size of 280 patients would provide the trial with 80% power to detect a difference of 0.5 log 10 in the mean reduction of sars-cov-2 viral load at a two-sided significance level of α = 0.05, assuming an expected standard deviation of 1.5 [23] . a 0.5 log 10 copies/ml difference in reduction was chosen to represent the minimal threshold for a biologically relevant change for our analyses [24] . considering the open-label design and the possibility of side effects caused by the study medication, the primary efficacy analysis was performed on the intention-totreat (itt) population. sensitivity analyses were performed with the per-protocol (pp) population. safety was assessed in the safety population, which included all participants who received any therapy, including usual care. efficacy was determined by comparing the mean reduction of the viral load from baseline to days 3 and 7, with the use of a mixed effects regression model taking into account the randomization group and repeated measures within each individual. the viral load was provided in logarithmic scale; a c c e p t e d m a n u s c r i p t 7 specimens with undetectable viral load at a given follow-up assessment were assigned a value of 3 log 10 copies per ml (i.e., lower limit of detection) for the purpose of statistical analysis. the secondary clinical outcome regarding between-group differences in disease progression were assessed using risk ratio (rr) for the predefined events. the time to clinical improvement was analyzed using kaplan-meier survival functions and hazard ratios (hrs), calculated using a cox proportional hazards regression model based on the assumptions of proportional risks. kaplan-meier estimates were compared using the log-rank test. the significance threshold was set at a twosided α level of 0.05 unless otherwise indicated, and all analyses were conducted in r version 3.6.2 [25] . between march 17 and april 28, 2020, we assessed for eligibility 753 confirmed covid-19 patients. figure 1 summarizes the recruitment and follow-up flowchart of study participants. four-hundred (53.1%) of 753 did not meet the selection criteria and were therefore not enrolled. additionally, 60 (8.0%) participants were finally excluded from itt analysis because of negative rt-pcr at baseline, missing rt-pcr at all follow-up visits, or consent withdrawal, yielding an itt population of 293 covid-19 patients. during follow-up, 23 participants had a protocol deviation (8 were screening failure due to history of more than five days since start of symptoms, 1 was severely ill at baseline, 3 were taking contraindicated medication, 8 were lost-to-follow-up, and 3 had treatment compliance under 80%) and were excluded from pp population. the two study arms had similar characteristics at baseline (itt population), including age, gender, comorbidities, frequency of symptoms, and nasopharyngeal viral load ( table 1 ). the mean age of patients was 41.6 years (sd 12.6), and 201 (68.6%) of them were women. the median time from symptom onset to enrollment was 3 days (iqr 2-4). a total of 53.2% of the patients (156 of 293) reported chronic health conditions. fever, cough, and sudden olfactory loss were the most common presenting symptoms. the mean viral load in the nasopharyngeal swab at baseline was 7.90 (sd 1.82) log 10 copies/ml. most patients were healthcare workers (254 [86.7%] of 293). for the primary outcome of reduction of the viral load in nasopharyngeal swabs, there were no significant differences between the control arm and the intervention arm at day 3 or 7. the mean differences in viral load from baseline to day 3 were -1.41 and -1.41 log 10 copies/ml in the control and intervention arm, respectively (difference [d] 0.01 [95% ci -0.28; 0.29]) (table 2 and figure 2 ). the comparative analysis of the reduction of the viral load followed a similar trend at day 7: -3.37 and -3.44 in the control and intervention arm, respectively (d -0.07 [-0.44; 0.29]). the sensitivity analysis in the pp population also showed no difference between groups (table s1, supplementary material). sub-group analysis comparing the viral loads of patients treated with hcq plus drvc did not reveal differences compared with hcq alone (table s2) . m a n u s c r i p t 8 the clinical outcome of risk of hospitalization was similar in the control arm (7.1%, 11/157) and the intervention arm (5.9%, 8/136; rr 0.75 [95% ci 0.32; 1.77]) ( table 2) . no patients required mechanical ventilation or died during the study period. median time from randomization to the resolution of covid-19 symptoms was not significantly different in the control arm (12.0 days, iqr 6-21) and the intervention arm (10.0, iqr 4-18; log-rank-test for survival analysis p = 0.38; figure 3 ). in the safety population 16/184 (8.7%) patients in the control group and 121/169 (72.0%) in the intervention group experienced at least one ae during the 28 days of follow-up ( table 3 ). the most frequent treatment-related aes among participants given hcq were gastrointestinal (e.g., diarrhea, nausea, and abdominal pain) and nervous system disorders (e.g., drowsiness, headache, and metallic taste). twenty sae were reported, 12 in the control arm and 8 in the intervention arm, none of them related to hcq (table s3 ). the results of this randomized controlled trial convincingly rule out any meaningful virological or clinical benefit of hcq in outpatients with mild covid-19. we found that hcq initiated within five days from symptom onset (median 3 days) was not effective in reducing viral shedding compared with no antiviral therapy. the quantification of the viral load in the upper respiratory tract provides strong evidence on the capacity of the treatment to affect the pathogen burden. furthermore, this treatment regimen did not reduce the risk of hospitalization-though the trial was underpowered for this outcome-nor shortened the time to complete resolution of symptoms. the much higher proportion of participants with aes in the hcq arm suggests poor tolerability of the treatment; however, no major aes related to the study drug were observed. of participants who were treated with hcq, 70% self-reported mild-to-moderate side-effects that were mainly gastrointestinal. only eight patients presented a sae within 28 days of hcq treatment initiation, all related to disease progression. no cardiovascular events nor syncope/palpitation/dizziness suggestive of arrhythmia were reported. this finding is particularly important because it does not corroborate the concern for harm associated with hcq therapy, particularly cardiac disease [26] . our study has several limitations. first, clinical assessments on day 7 were not originally scheduled and therefore the number of patients analyzed for viral positivity at this time point was lower compared to day 3. while who has recommended a measure of viral burden in covid-19 clinical trials, they have neither set up the optimal time for measurement nor the minimal threshold for significant reduction between arms. we recommend the time for viral load reduction assessment to be long enough to capture a relevant decrease-ideally, 7 days or longer-and the significant reduction threshold to be set at 0.5 log 10 decrease or greater. second, we had originally chosen to combine hcq with the hiv protease inhibitor drvc because in silico molecular docking studies had predicted that drvc might have therapeutic effect on sars-cov-2 [27] and the better safety profile compared to other hiv protease inhibitors. however, in-vitro results showing no activity that became available after the start of our study, prompted the decision to drop drvc [19, 20] . the m a n u s c r i p t 9 concomitant administration of drv in some participants may have slightly increased plasma levels of hcq, thereby leading to increased hcq effect because drvc is a weak inhibitor of the metabolic enzyme of hcq, cyp2d6. therefore, we do not expect the use of drvc might have reduced the effect of hcq. third, owing to the urgency, the trial could not be masked with a placebo, which may affect the rate of ae declared (aes are less often reported in a control, non-placebo group). nevertheless, it did not affect the attrition numbers in the control arm. moreover, to minimize the detection bias of the primary outcome (i.e., the viral load), the laboratory staff remained unaware of participants' allocation. finally, the regional nature of the trial and overrepresentation of healthcare workers (>80%) may limit the generalization of our findings. therefore, cautiousness should be taken when extrapolating our data to other countries or settings. hcq and chloroquine have garnered unprecedented attention as potential therapeutic agents following inconclusive clinical trials in combination or not with azithromycin [9, 12] , uncontrolled case series [14] , and public figure endorsements [28] . while there is a growing body of scientific data against using hcq for treating covid-19 that includes a concern for harm, particularly cardiac disease, the potential for the treatment of mild covid-19 with hcq has been explored in this trial to provide definite evidence. our results indicate no impact on viral burden up to 7 days nor symptoms resolution or hospitalization rate up to 28 days following diagnosis. the added value of our study is the randomized-controlled design and the use of the agreed minimal outcome set for covid-19 clinical trials, including rt-pcr to conclusively determine the viral burden. our findings provide the scientific community and policymakers with essential insights on the inefficacy of hcq as a therapeutic candidate for sars-cov-2, at least in similar settings and conditions to ours. specimens with negative pcr (undetectable viral load) were assigned a value of 3 log 10 copies per ml for the purpose of statistical analysis. **estimated using a mixed effects regression model. none of the estimated mean differences and risk ratios were statistically significant. d: mean difference. rr: risk ratio. sd: standard deviation. se: standard error. m a n u s c r i p t 16 12 (6.6%) 9 (5.4%) injury, poisoning and procedural complications 0 (0.0%) 1 (0.6%) metabolism and nutrition disorders 1 (0.5%) 2 (1.2%) musculoskeletal and connective tissue disorders 0 (0.0%) 1 (0.6%) nervous system disorders 3 (1.6%) 63 (37.5%) psychiatric disorders 0 (0.0%) 2 (1.2%) renal and urinary disorders 0 (0.0%) 1 (0.6%) reproductive system and breast disorders 0 (0.0%) 1 (0.6%) respiratory, thoracic and mediastinal disorders 0 (0.0%) 2 (1.2%) skin and subcutaneous tissue disorders 0 (0.0%) 11 (6.5%) vascular disorders 0 (0.0%) 1 (0.6%) *none of the serious adverse events (sae) were adjudicated as related to hcq by the pharmacovigilance consultants. spanish council for scientific research (csic) germans trias i pujol research institute (igtp) centre of epidemiological studies of hiv/aids and sti of catalonia (ceeiscat) xarxa sanitària i social santa tecla, tarragona, spain 13. gerència territorial de barcelona discovering drugs to treat coronavirus disease 2019 (covid-19) covid-19: chloroquine and hydroxychloroquine only to be used in clinical trials or emergency use programmes covid-19: us gives emergency approval to hydroxychloroquine despite lack of evidence new insights into the antiviral effects of chloroquine hydroxychloroquine: from malaria to autoimmunity remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) connecting hydroxychloroquine in vitro antiviral activity to in vivo concentration for prediction of antiviral effect: a critical step in treating covid-19 patients efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study observational study of hydroxychloroquine in hospitalized patients with covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state a minimal common outcome measure set for covid-19 clinical research catalan ministry of health. catalan epidemiological surveillance system lack of antiviral activity of darunavir against sars-cov-2 lack of evidence to support use of darunavir-based treatments for sars-cov-2 a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 centers for disease control and prevention (cdc). cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel. 2020. available at temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study estimating the benefit of an hiv-1 vaccine that reduces viral load set point r: a language and environment for statistical com-puting risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease 2019 (covid-19) molecular modeling evaluation of the binding abilities of ritonavir and lopinavir to wuhan pneumonia coronavirus proteases therapeutic management of covid-19 patients: a systematic review we thank gerard carot-sans, phd, for providing medical writing support during the revisions of the subsequent drafts of the manuscript, the personnel from the fights aids and infectious diseases foundation for their support in administration, hr and supply-chain management, eric ubals (pierce ab) and òscar palao (opentic) for website and database management, óscar camps and openarms non-governmental organization for nursing home operations, anna valentí and the human resources department of the hospital germans trias i pujol for telephone monitoring. we are very grateful to marc clotet and natalia sánchez who coordinated the joemcorono crowdfunding campaign. we thank the institutional review board of hospital germans trias pujol and the spanish agency of medicines and medical devices (aemps) for their prompt action for consideration and approvals to the protocol. this work was mainly supported by the crowdfunding campaign joemcorono (https://www.yomecorono.com/) with the contribution of over 72,000 citizens and corporations. the study also received financial support from laboratorios rubió, laboratorios gebro pharma, zurich seguros, synlab barcelona, and generalitat de catalunya. laboratorios rubió also contributed to the study with the required doses of hydroxychloroquine (dolquine®). a c c e p t e d m a n u s c r i p t 11 a c c e p t e d m a n u s c r i p t 12 m a n u s c r i p t 14 a c c e p t e d m a n u s c r i p t 19 figure 3 key: cord-318458-jadk2qbm authors: jung, chan-young; park, haeyong; kim, dong wook; lim, hyunsun; chang, jung hyun; choi, yoon jung; kim, seong woo; chang, tae ik title: association between body mass index and risk of covid-19: a nationwide case-control study in south korea date: 2020-08-25 journal: clin infect dis doi: 10.1093/cid/ciaa1257 sha: doc_id: 318458 cord_uid: jadk2qbm background: increased body mass index (bmi) has been associated with higher risk of severe coronavirus disease 2019 (covid-19) infections. however, whether obesity is a risk factor for contracting covid-19 has been hardly investigated so far. methods: we examined the association between bmi level and the risk of covid-19 infection in a nationwide case-control study comprised of 3,788 case patients confirmed with covid-19 between january 24 and april 9, 2020 and 15,152 controls matched by age and sex, who were aged 20 years or more and underwent national health insurance service (nhis) health examinations between 2015−2017, using data from the korean nhis with linkage to the korea centers for disease control and prevention data. our primary exposure of interest was bmi level categorized into four groups; &18.5 (underweight), 18.5-22.9 (normal weight), 23-24.9 (overweight), and ≥25 kg/m (2) (obese). results: of the entire 18,940 study population, 11,755 (62.1%) were women, and the mean (sd) age of the study participants was 53.7 (13.8) years. in multivariable logistic regression models adjusted for sociodemographic, comorbidity, laboratory and medication data, there was a graded association between higher bmi levels and higher risk of covid-19 infection; compared to normal weight individuals, the adjusted ors in the overweight and obese individuals were 1.13 (95% ci, 1.03-1.25) and 1.26 (95% ci, 1.15-1.39), respectively. this association was robust across age and sex subgroups. conclusions: higher bmi levels were associated with higher risk of contracting covid-19. the ongoing coronavirus disease 2019 outbreak has led to an unprecedented worldwide health, economic, and social crisis. in south korea, the first covid-19 case was confirmed on january 20, 2020 (1) . with proactive containment efforts, comprehensive contact tracing, and extensive testing of symptomatic or suspected individuals for covid-19, south korea was able to flatten the curve of new covid-19 infections by mid-march [1, 2] . however, there are growing concerns of ongoing sporadic community infections spreading via asymptomatic patients or persons of unknown viral transmission route. given current limited availability of point-of-care covid-19 testing, identifying and prioritizing testing in individuals at higher risk of severe covid-19 has been a key emerging issue. from early reports, elderly patients, especially with comorbidities such as hypertension, diabetes, and cardiovascular disease, had the highest rates of hospitalization [3, 4] . as the prevalence of obesity itself and obesity-related non-communicable diseases increases in older adults, it is possible to speculate that obese patients may be more susceptible to develop a more serious illness. in fact, several recent studies found that obesity was associated with a more severe course of covid-19, which included higher risk of intensive care unit admission, tracheal intubation for mechanical ventilation, and death among patients hospitalized for covid-19 [5] [6] [7] [8] [9] . therefore, obese patients, whom comprise around 40% of the us population and around 13% of the world population [10] , should be considered as high risk and deserve extra attention and precautions to help improve outcomes in patients requiring hospitalization for severe meanwhile, whether obesity is a risk factor for contracting covid-19 infection is another important issue. it is crucial to identify individuals who are susceptible to a c c e p t e d m a n u s c r i p t infections in order to contain further covid-19 transmissions, particularly when testing capacity is limited, and in the absence of effective vaccines or antiviral drugs. however, recent studies that have included information regarding body mass index (bmi) of patients with covid-19 have largely focused on the severity and outcomes of infection, and to date there is a paucity of studies that examine the association between bmi and the risk of covid-19 infection [11, 12] . to address these gaps in knowledge, we sought to determine the association between bmi categories and the risk of covid-19 infection in a nationwide population-based case-control study. we obtained data from the korean national health insurance service (nhis) database, which is linked to national health screening examination information and korea centers for disease control and prevention (kcdc) data. the nhis covers compulsory health insurance for all citizens in korea and provides cost-free annual or biennial health screening examinations to all insured individuals. since korea has a single-payer national health system, all medical records of covered inpatient and outpatient visits as well as the results from the national health examinations are centralized in the nhis database, which includes diagnostic codes, procedures, prescriptions, medical costs, and personal information (e.g., age, sex, residential area, income level, and disability) [13, 14] . the kcdc database provides details on all epidemiological investigations for each individual infected with covid-19, which include date of laboratory confirmation, residential area, and exposure history [15] . m a n u s c r i p t in constructing the study population of this nationwide case-control study, we first identified all 10,237 patients confirmed with severe acute respiratory syndrome coronavirus 2 infections by a positive result on polymerase chain reaction test of a nasopharyngeal or oropharyngeal sample between january 24, 2020 and april 9, 2020, in south korea. we then excluded 481 patients aged 19 years and younger, and 5,945 patients who did not undergo nhis health examinations between january 1, 2015 and december 31, 2017. we further excluded 23 patients who had missing data for any of the study variables, (see following methods subsections), and thus a total of 3,788 confirmed cases were included. for each case patient, we randomly matched 4 controls by age (5 year intervals) and sex, who underwent nhis health examinations during the same period. individuals who died before april 9, 2020, or had missing information on key study variables were not selected as controls. therefore, the final study population comprised 18,940 participants, which included 3,788 case patients and 15,152 matched controls (supplementary figure 1) . the institutional review board of nhis ilsan hospital approved this study and waived the requirement for informed consent as only deidentified data was used. baseline data on sociodemographic information such as age, sex, income level, and residential area was collected in the year prior to the index date. we defined the index date as the end of the study period (i.e., april 9, 2020), for both case patients and matched controls. comorbidities (e.g., diabetes, ischemic heart disease, congestive heart failure, cerebrovascular disease, hemiplegia, dementia, peripheral vascular disease, liver disease, table 1 ), which were ascertained by the presence of at least two or more diagnostic codes up to five years prior to the index date. the charlson comorbidity index (cci) score was also calculated as a proxy of disease burden and illness severity [16] . use of angiotensinconverting enzyme (ace) inhibitors, angiotensin-receptor blockers, or dipeptidyl peptidase-4 (dpp-4) inhibitors were defined as the prescription for these medications identified up to five years before the index date. all anthropometry and laboratory results were collected at every health examination visit between 2015 and 2017, in which results were averaged over the three years. bmi was calculated as weight in kilograms divided by height in meters-squared. blood pressure was measured using standardized methods while the participant was sitting on a chair after a fiveminute rest. serum lipid, glucose, and creatinine levels were measured from specimens collected while fasting. estimated glomerular filtration rate (egfr) was estimated by the chronic kidney disease epidemiology collaboration equation for creatinine [17] . our primary exposure of interest was bmi, which was presented as the average of all measurements in each health examination visit between 2015 and 2017. in the study population, the mean (standard deviation, sd) and median (inter-quartile range, iqr) number of bmi measurements that contributed to each bmi value per individual were 1.7 (0.7) and 2.0 (1.0-2.0), respectively, which were equal between case patients and matched controls. given a possible non-linear relationship with covid-19 infection risk, bmi was treated as a categorical variable and divided into four categories: <18.5 (underweight), 18.5-22.9 (normal weight), 23-24.9 (overweight), and ≥25 kg/m 2 (obese). obesity was defined as ≥25 kg/m 2 based on a who cut-off point for asian populations [18] . the normal weight bmi category for sensitivity analyses, we additionally assessed the optimal cut-off point of bmi value for predicting covid-19 diagnosis, based on the area under the receiver operating characteristic curve (auroc). to compare predictive performance of bmi, we also calculated the net reclassification improvement (nri) and integrated discrimination index (idi) between adjusting models with and without bmi. data from descriptive analyses were summarized using means (sd), medians (iqr), or numbers (proportions) as appropriate, and were compared using independent-sample ttests or chi-square tests, respectively. all analyses were conducted using sas version 9.4 (sas institute inc., cary, nc). p <0.05 was used as the threshold for statistical significance for any tests. a c c e p t e d m a n u s c r i p t clinical characteristics of the 3,788 case patients with covid-19 and 15,152 matched controls who met the eligibility criteria for the study are shown in table 1 . the mean (sd) age of the study participants was 53.7 (13.8) years, among whom 62.1% were women, 87.0% were urban residents, and 13.7% had at least one or more comorbidities based 7) kg/m 2 , respectively. overall, case patients were more likely to reside in rural areas, have lower income levels, higher prevalence of comorbidities, and be prescribed with dpp-4 inhibitors. in contrast, matched controls were more likely to be current smokers, have higher systolic blood pressure, lipid levels, and egfr. in logistic regression models that were adjusted for socio-demographic, comorbidity, laboratory and medication data, there was a graded association between higher bmi levels and higher risk of covid-19 infection (figure 1 ). specifically, compared to normal weight individuals, we observed a 13% and 25% higher risk of covid-19 in overweight and obese individuals; adjusted ors in model (3) were 1.13 (95% ci, 1.03-1.25) and 1.25 (95% ci, 1.14-1.38), respectively (model 3 in table 2 ). it should be noted that these associations remained largely unchanged despite additional adjustments for medications known to potentially influence ace2 expression including ace inhibitors or angiotensin-receptor blockers, and dpp-4 inhibitors (model 4 in table 2 ). in contrast, underweight individuals tended to trend toward increased risk of contracting covid-19 infections, but this was not statistically significant. a c c e p t e d m a n u s c r i p t we then examined the association between bmi category and risk of covid-19 across clinically relevant subgroups. in subgroup analyses, a stepwise increase in risk of covid-19 infection associated with incrementally higher bmi was largely consistent across all age categories and sex (figure 2 ). of note, the magnitude of adjusted ors in obese individuals was much stronger in gradually younger age categories, which were 1.53 (95% ci, 1.18-1.99), 1.26 (95% ci, 1.10-1.44), and 1.19 (95% ci, 1.02-1.39) in the 20-39, 40-59, and ≥60 years age categories, respectively (table 3) . next, we determined the optimal cut-off points for predicting covid-19 diagnosis based on the auroc and found that optimal threshold levels of bmi were different across the whole population and various subgroups; these were lowest a c c e p t e d m a n u s c r i p t in this nationwide population-based case-control study, we found a graded association between higher bmi levels and higher risk of covid-19 infections. in particular, obese individuals showed the highest susceptibility to covid-19, even after adjusting for potential confounding factors including socio-demographic, comorbidity, laboratory, and medication data. this association was robust across age and sex subgroups. these findings suggest that bmi could be an important consideration in estimating an individual's risk of acquiring covid-19, and in devising strategies that could contain further spread of covid-19 at a population level. while obesity is a well-recognized risk factor for various cardio-metabolic diseases, its role in infection must not be ignored [19] . during the 2009 h1n1 influenza a virus pandemic, obesity emerged as a novel major predisposing factor in transmission and severe course of viral disease [20] [21] [22] . obesity not only prolonged the duration of influenza virus shedding, but was also linked to higher risks of hospitalization and mortality [23] [24] [25] . furthermore, impaired immune responses to vaccination in obese individuals have also been reported in several types of viral infections such as influenza, hepatitis b, and tetanus [26] [27] [28] . by analogy to other viral infections, obesity may play an important role in covid-19 transmission, but this hypothesis has hardly been investigated so far. in the present study using a national cohort of 18,940 case patients and matched controls, we found a significant a c c e p t e d m a n u s c r i p t relationship between incrementally higher bmi and the risk of covid-19 infection, in which obese individuals with a bmi of more than 25 kg/m 2 was associated with a 26% higher risk of contracting covid-19 than normal weight individuals. more importantly, these associations were consistent even after adjusting for socio-demographics, co-morbidities, cardio-metabolic components, and medications, which suggest that obesity may be an independent risk factor in acquiring covid-19 infections. to our knowledge, although several observational studies have reported a higher risk for severe covid-19 associated with obesity, this is the first case-control study to confirm the relationship between obesity and the risk of covid-19 acquisition. from the preventive perspective, our study findings are particularly informative in that obesity is not only a risk factor for severe course of covid-19 disease, but also for acquiring the virus itself. thus, not only should bmi be a major consideration in informing treatment strategies, but also in preventive strategies for covid-19. of note, the infectious disease society of america had recently developed recommendations for diagnostic testing prioritization, with priorities given to critically ill patients, health care workers, immunocompromised, and elderly patients [29] . based on our findings, future recommendations could include individuals with higher bmi levels. moreover, although the results of prediction testing in this study may be insufficient to provide concrete guidance for diagnostic testing prioritization and preventive strategies, this study could serve as a starting point for future investigations that could potentially look into the causal relationship between bmi and risk of contracting covid-19 infections. due to a paucity of data at this time, the underlying mechanisms responsible for the association between obesity and covid-19 infection are unclear, but several plausible explanations could be contemplated. first, covid-19 is known to have a high affinity for human ace2, which acts as the putative receptor of entry of covid-19 into host cells [30] . notably, ace2 expression levels are actually higher in adipose tissue than in other organs a c c e p t e d m a n u s c r i p t including the lungs, heart, and the kidneys. obese individuals with more adipose tissue therefore have more ace2-expressing cells and a larger amount of ace2, and thus greater vulnerability to covid-19 [31, 32] . second, by analogy of other viral agents such as human adenovirus ad-36, influenza a virus, human immunodeficiency virus, and cytomegalovirus [32, 33] , adipose tissue can also act as a reservoir and host entry point for covid-19. finally, metabolic inflammation associated with obesity potentially impairs immune responses to viral infections. for example, obesity appears to confer an inadequate immune response to viral infections such as h1n1 influenza or hepatitis b, and a greater vulnerability to overwhelming sepsis following viral illness [26, 34, 35] . all of these possible mechanisms suggest that studies into the pathogenesis of covid-19 should not merely consider the microbiologic aspects, but also the metabolic aspects of the viral disease. the strengths of this study include its availability of detailed patient-level information on socio-demographics, comorbidities, anthropometry, and laboratory data from a large national cohort; utilization of age, sex matched population cohort; and vigorous adjustment for potential confounders of obesity and related infection risk. however, several limitations of our study bear mention. first, potential selection bias cannot be excluded given our restriction of analyses to participants who underwent nhis health examination between 2015 and 2017, capturing only a third of total confirmed covid-19 cases between january 24 and april 9, 2020, in south korea. this proportion of complete health check-ups was particularly lower compared to that of the entire population, which was approximately 77% during the same period. second, rather than changes in bmi over time, the averaged bmi was assessed in this study. however, the likelihood of bmi substantially changing during the five years of follow up in our study was deemed unlikely. third, our findings may not be generalizable to populations outside of south korea, given the social factors, environmental exposures, national healthcare policies, and chronic disease burden including obesity that may be distinct a c c e p t e d m a n u s c r i p t from other countries. for instance, the small sample size of case patients with a bmi of over 30kg/m 2 , which is a frequently defined obesity threshold in western countries, makes it difficult to further categorize our bmi groups to include higher levels. in conclusion, higher bmi was associated with higher risk of covid-19 infections. as the covid-19 pandemic evolves, continued investigation into the interplay between metabolic health, especially obesity, and the risk of covid-19 is warranted in order to inform control strategies for covid-19. while awaiting further evidence supporting causal relationship between obesity and the risk of covid-19 infections, individuals with a higher bmi could be potentially classified as high risk and thus, prioritized in covid-19 testing. a c c e p t e d m a n u s c r i p t 0. m a n u s c r i p t m a n u s c r i p t center for disease control and prevention. the updates on covid-19 in korea: for press release. 2020. available at covid-19 in south korea -challenges of subclinical manifestations host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease covid-19 in critically ill patients in the seattle region -case series factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in new york city: prospective cohort study high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation prevalence of obesity among adult inpatients with covid-19 in france phenotypic characteristics and prognosis of inpatients with covid-19 and diabetes: the coronado study world health organization. global health observatory (gho) data: overweight and obesity accessed 2 bmi and future risk for covid-19 infection and death across sex, age, and ethnicity: preliminary findings from uk biobank risk factors for sars-cov-2 among patients in the oxford royal college of general practitioners research and surveillance centre primary care network: a cross-sectional study thirty years of national health insurance in south korea: lessons for achieving universal health care coverage background and data configuration process of a nationwide population-based study using the korean national health insurance system information technology-based tracing strategy in response to covid-19 in south korea-privacy controversies coding algorithms for defining comorbidities in icd-9-cm and icd-10 administrative data a new equation to estimate glomerular filtration rate appropriate body-mass index for asian populations and its implications for policy and intervention strategies obesity and infection populations at risk for severe or complicated influenza illness: systematic review and meta-analysis a novel risk factor for a novel virus: obesity and 2009 pandemic influenza a (h1n1) weight and prognosis for influenza a(h1n1)pdm09 infection during the pandemic period between 2009 and 2011: a systematic review of observational studies with meta-analysis obesity increases the duration of influenza a virus shedding in adults hospitalized patients with 2009 h1n1 influenza in the united states factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california obesity is associated with impaired immune response to influenza vaccination in humans obesity as a predictor of poor antibody response to hepatitis b plasma vaccine reduced tetanus antibody titers in overweight children covid-19 prioritization of diagnostic testing a pneumonia outbreak associated with a new coronavirus of probable bat origin obesity and impaired metabolic health in patients with covid-19 risk of covid-19 for patients with obesity specific biological features of adipose tissue, and their impact on hiv persistence diet-induced obesity dramatically reduces the efficacy of a 2009 pandemic h1n1 vaccine in a mouse model changes in human dendritic cell number and function in severe obesity may contribute to increased susceptibility to viral infection 3) between bmi and risk of covid-19 infection across subgroups stratified by age and sex. study participants, no (%) odds ratio (95% ci) case patients controls bmi model 3: all covariates in model (2) plus systolic blood pressure, low-density lipoprotein cholesterol level, triglyceride level, high-density lipoprotein cholesterol level, fasting glucose level, and estimated glomerular filtration rate model 4: all covariates in model (3) plus use of angiotensin-converting enzyme inhibitors or angiotensin-receptor blockers, and use of dipeptidyl peptidase-4 inhibitors. abbreviations: bmi, body mass index a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-316970-n2dly3oa authors: kerbaj, jad; cazorla, cecile; de greslan, thierry; serie, mathieu; gourinat, ann-claire; marot, benoit title: covid-19: the new caledonia experience date: 2020-05-16 journal: clin infect dis doi: 10.1093/cid/ciaa600 sha: doc_id: 316970 cord_uid: n2dly3oa new caledonia is a french associated territory in the south pacific ocean. while covid-19 is expanding over the world, we seem to be well preserved with a total of 18 documented cases. we report the measures implemented on our island that probably helped containing an epidemic spread. european and american countries took the situation lightly, comparing it to an influenza epidemic (4;5;6). the pandemic spread very quickly all over europe, and the world, and important lockdown and management measures were put in place to try and contain it. at this time, 20 th of april 2020, 2.4m people got reported all over the world with more than 165.000 deaths (7). new caledonia is a french associated territory in the south pacific ocean, composed of a main island and 4 inhabited small islands, with a population number around 300.000. it has one international airport. this french overseas territory has its own competence in health policy. a clinical screening of all passengers arriving from sars-cov-2 high risk areas was started on the 28 th of january in the international airport. the high risk areas were updated regularly following the pandemic spread and were defined by areas with active viral circulation. the airport control was reinforced by thermal cameras on the 6 th of march. every passenger with fever or cough was hospitalized in the centre hospitalier territorial cht (the main island's hospital, reference center in infectious diseases) and tested by sars-cov-2 reverse transcriptase polymerase chain reaction (rt-pcr). on february 10, screening by sars-cov-2 rt-pcr started for all patients hospitalized for an influenza like illness or a severe acute respiratory infection (sari). the hospital's laboratory collected also samples from the influenza sentinel network and tested them for sars-cov-2. a c c e p t e d m a n u s c r i p t anosmia. they were hospitalized in the infectious diseases ward of the cht for surveillance and isolation. they spent less than 18 hours in their hotel room and had very few contacts before diagnosis. the sanitary and health department traced the contacts of these patients before they sought care. all contacts were quarantined in a center that was arranged for this purpose. since the 19 th of march, government of new caledonia mandate a 14 days quarantine for all visitors and residents returning to the territory. travelers were transferred to a government designated hotel under strict surveillance, and all international flights were stopped on the 21st of march. all close contact to person confirmed with covid-19 infection were isolated in a quarantine facility for 14 days. inside flights or boat transfers to the other islands were stopped right after the first documented case (on the 18 th of march). on the 23 rd of march, officials put the whole territory under complete lockdown. schools and every non-essential business were closed. restaurants, bars, night clubs were closed, meetings were unauthorized. people were permitted to go out for a one hour period every day within 1km of their homes. after the detection of the first cases, testing centers were implemented all over the main island, with the raise of 2 screening tents in front of the main hospital. nasopharyngeal and pharyngeal samples were collected in every health care facility. the swabs were all sent to the microbiology laboratory of the cht for rt-pcr testing. we decided to test every person having influenza like illness (fever, cough, anosmia, chills). symptomatic persons who traveled or were in close contact with someone positive were hospitalized and immediately screened for sars-cov-2. additionally, patients with negative rt-pcr test and typical symptoms of pneumonia, were ct scanned. around 2.000 people have to be repatriated in new caledonia after the closure of international flights mostly from france, australia, and new zealand. these resident will be authorized to reach their the cht was reorganized to prepare for the potential influx of patients. we emptied our infectious diseases ward after the first covid-19 case (28 beds), then very quickly the pneumology ward (28 beds). the patients were diverted to other wards or other hospitals and clinics. in the icu we emptied a 10 beds ward. we planned to open very quickly if needed more than 112 beds in medicine wards and 49 beds in icu (figure 1 ). all persons detected positive for sars-cov-2 (even with mild or no symptoms) were hospitalized and isolated until they got asymptomatic and had 2 negative tests for sars-cov-2 at 48 hours intervals. on the 24 th of april 2020, we are on day 20 without any new documented case. some islands like iceland or japan have been very successful in managing the epidemic and were able to flatten the curve without being overwhelmed by patients. iceland has quickly considered all travels outside the island as high risk, has done a large population screening and important tracking of sars-a c c e p t e d m a n u s c r i p t 6 cov-2 infections, associating these measures with quarantine, self-isolation and social distancing (8) . japan has done an early detection and early response to infection clusters, with an enhancement of intensive care units (9) . multiple reasons could explain the low actual viral spread in new caledonia. the fact that we have one international airport helped in the management of all travelers. few people that arrived by boat were focused to one harbor, controlled, and placed in quarantine. our insularity and hot weather/ humidity could have had in impact on a lower contagiousness (10;11). the surveillance, quarantine measures, the hospitalization of all detected covid-19 positive patients and the rapid lockdown had probably an impact on stopping the spread. our large testing policy helped us to have a good idea on the viral spread. preparedness and anticipation seem very important factors to contain this epidemic spread. as iceland and japan, we implemented epidemic control strategies very soon after our first documented case and we tried to exclude new imported cases from general population. the difficult part will probably start now, after 4 weeks of lockdown, we will start to put in place measures of decontainment. our population is still non-immune and a low viral undetected circulation could evolve in an epidemic. also a viral introduction cannot be excluded even with the most drastic measures: few international flights are still necessary for freight or sanitary evacuation to france. a change of habits is needed, especially social habits, within a heterogeneous population where the western way of life rubs shoulders with a more traditional one. important distances should be kept between people in enterprises or schools, drastic hand hygiene policy must be taken and masks should be given to all population at risk to prevent a rapid viral spread. this issue is even more important concerning the gatherings of people linked to the melanesian cultural calendar and way of life. the opening of the international airport should be also rethought for the long term. this situation could persist for months or years (until a vaccine or effective treatment are put in place); and has to be compatible with economic and social viability. a c c e p t e d m a n u s c r i p t a review of the 2019 novel coronavirus (covid-19) based on current evidence an interactive web-based dashboard to track covid-19 in real time. the lancet infectious diseases 2019 novel coronavirus of pneumonia in wuhan, china: emerging attack and management strategies it averages between 27,000 and 70,000 per year. nothing is shut down, life & the economy go on. at this moment there are 546 confirmed cases of coronavirus, with 22 deaths coronavirus: 38 days when britain sleepwalked into disaster, the times the french response to covid-19: intrinsic difficulties at the interface of science, public health, and policy. the lancet public health spread of sars-cov-2 in the icelandic population why does japan have so few cases of covid-19? embo disponible sur stefan flasche & rachel lowe. effective transmission across the globe: the role of climate in covid-19 mitigation strategies the effects of temperature and relative humidity on the viability of the sars coronavirus a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-315180-itvc86cv authors: hollingsworth, t déirdre title: counting down the 2020 goals for 9 neglected tropical diseases: what have we learned from quantitative analysis and transmission modeling? date: 2018-06-15 journal: clin infect dis doi: 10.1093/cid/ciy284 sha: doc_id: 315180 cord_uid: itvc86cv the control of neglected tropical diseases (ntds) has received huge investment in recent years, leading to large reductions in morbidity. in 2012, the world health organization set ambitious targets for eliminating many of these diseases as a public health problem by 2020, an aspiration that was supported by donations of treatments, intervention materials, and funding committed by a broad partnership of stakeholders in the london declaration on ntds. alongside these efforts, there has been an increasing role for quantitative analysis and modeling to support the achievement of these goals through evaluation of the likely impact of interventions, the factors that could undermine these achievements, and the role of new diagnostics and treatments in reducing transmission. in this special issue, we aim to summarize those insights in an accessible way. this article acts as an introduction to the special issue, outlining key concepts in ntds and insights from modeling as we approach 2020. neglected tropical diseases (ntds) are a diverse group of infections identified by the world health organization (who) as diseases that predominantly infect low-income populations in tropical countries, causing a large burden of morbidity and some mortality, and thus perpetuate the cycle of poverty [1] . in 2012, the who declared ambitious targets to reduce the burden of these diseases by eliminating them as a public health problem by 2020 [1] . in support of these aspirations, a diverse consortium of donors, pharmaceutical companies, government agencies, and others made large commitments of funding, donated treatments, and other activities for 10 of these diseases in the london declaration on ntds [2] . large morbidity gains have been made over recent years [3] , and there are active discussions on how to exploit the likely synergies between the goals for ntds and universal health coverage (uhc), a sustainable development goal (sdg; target 3.8) [4] , in particular how to extend these gains to the hardest-to-reach or conflict-affected communities [5] . of these 10 diseases, guinea worm is targeted for eradication; the remaining 9 infections are targeted for elimination as a public health problem in some settings. the adjustment of strategies to achieve control of the 9 infections, informed by mathematical modeling, is the focus of this special issue. infectious disease modeling has an increasing role in public-health policy, with resulting challenges and successes [6] . appropriate analyses can provide thorough investigation and interpretation of data, as well as identify where the knowledge gaps are most acute. models can also be used to rigorize our thinking on the processes of infection and transmission and test hypotheses about the likely dynamics and epidemiology. although there has been ongoing research into modeling of ntds [7, 8] , this research has sometimes been limited by the extent of biological knowledge and data on which to base these models. the availability of more extensive data, together with strong partnerships between researchers in different fields, including by the ntd modelling consortium [9] , has led to marked improvements in these efforts. researchers have made contributions not only in informing treatment strategies but also in informing diagnostic development and the applicability of new tools or treatments and in understanding the natural history of disease. however, in comparison with other infectious diseases, we still have limited epidemiological data on ntds; thus, although we have performed formal model comparisons [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] , there remain large uncertainties in processes and parameters that could have an impact on the dynamics, as highlighted below. this means that we need to be cautious about overstating our results, even when the policy need is acute. this presents us with the challenge of correctly calculating and communicating the uncertainties in these complex systems while still giving an accessible message to end users. this article acts as an introduction for a special issue that aims to increase the accessibility of the results so far by summarizing insights from ntd models and identifying key themes for the control of these diseases. it should be noted that in this special issue and in this article we have focused on epidemiological analyses and modeling and have not extended our focus to geospatial, spatial dynamic, or health economic modeling, all of which have an important part to play in developing policy for infectious diseases. we focus on the role of interventions to reach the 2020 goals for ntds. the authors of this issue are aiming to increase the repeatability of our science. the code for the models used in this special issue were previously published alongside more technical articles [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] as supplementary information or on our website (www.ntdmodelling.org). the release of raw code is not a complete answer to accessibility and reproducibility, but it is a step in the right direction [20] . the diseases are usually divided into 2 groups based on the methods used to control them, and we have summarized our results in this way. the first group includes those diseases that are mainly controlled by intensified disease management (idm) or increased detection, screening, and treatment of infection. the second group includes those diseases that are mainly controlled by mass drug administration (mda). although there are major epidemiological differences between diseases in these groups, they share some common uncertainties in informing control, which are discussed in each section. leprosy, the gambian form of sleeping sickness (human african trypanosomiasis), visceral leishmaniasis in the indian subcontinent, and chagas disease are four london declaration ntds which are characterized as idm infections. they have long, variable periods between infection and symptomatic disease and, generally, late diagnosis. control strategies are focused on reducing time to diagnosis and case finding with accompanying vector control, where appropriate. these diseases are characterized by long, uncertain incubation periods and an unknown degree of transmission by asymptomatic individuals (figure 1 ). the potential role of asymptomatic individuals in transmission is well known in epidemiology. the close link between symptoms and infectiousness for smallpox and severe acute respiratory syndrome (sars) has been calculated to be crucial in controlling these diseases [21] . that analysis explicitly considers the relative contribution of asymptomatics in terms of their contribution to the number of onward transmissions that an individual would be responsible for during a new outbreak, or the basic reproduction number, r 0 , in a way that is implicitly included in many models, but less elegantly presented. we could adopt a similar framework for these ntds. first we would need to separate asymptomatics into presymptomatic and nonsymptomatic (figure 1 , yellow and blue), highlighting the problems in language around asymptomatics [22] . for the area under this curve is proportional to the expected number of onward transmissions due to different stages of infection in a wholly susceptible population. if there are many nonsymptomatic people for each symptomatic individual, they may collectively contribute substantially to transmission even if their individual contribution is low (multiple yellow areas for single blue, green areas). for the symptomatic individuals, the relative infectiousness and duration of the symptomatic phase will determine the population-level impact of diagnosing and treating cases earlier (covering more of the green or even blue area). vector control or other mass interventions could reduce both symptomatic and asymptomatic transmission by reducing all transmissions. nonsymptomatic individuals yellow, left hand plot (figure 1 ), infectiousness may rise and fall but is generally expected to be low, based on pathogen measurements. a key question is, of course, the relationship between these pathogen measurements and transmission, which is unknown and likely to be nonlinear. the contribution of a single nonsymptomatic individual to transmission is proportional to the area under the infectivity since infection curve (yellow area, figure 1 ), which might be large or small when compared with symptomatic infection. a similar calculation has contributed to the debate in human immunodeficiency virus (hiv) around the relative roles of the early, brief period of high infectivity when compared with the much longer period of asymptomatic infection with lower transmission rates [23] [24] [25] . unfortunately, every aspect of infectivity and duration of infection ( figure 1 ) is highly uncertain for these 4 ntds. of course, it is not only the contribution of each nonsymptomatic individual to transmission that is important, but also the proportion of the infected population who fall into this group ( figure 1 , yellow vs blue). it is also crucial to note that this contribution will change through the course of an epidemic and an intervention and will be dependent on the type of intervention being applied, as nicely illustrated for visceral leishmaniasis in this issue [26, 27] . if the symptomatic phase is highly infectious and of sufficient duration ( figure 1 , right-hand plot, green and red), interventions to identify cases early in the symptomatic period are likely to be highly effective. although chagas may not follow the increasing infectivity over time pattern but instead have rather high infectivity during early acute infection, diagnosis is so rare that postsymptomatic infections are often treated as asymptomatic infections, which may contribute substantially to transmission [28] . in summary, the balance between infectivity, duration of infection, and frequency of asymptomatic versus symptomatic infection may undermine any attempt to control a disease solely by increased case finding. this can be mitigated by reducing all infectivity through vector control or other transmission-reducing interventions, which reduces the infectivity of all infected invidiuals (the height of the curve in figure 1 ). although this theoretical framework is useful, there is much modeling work to be done to populate a more concrete discussion of the relative roles of different phases of infections for these complex diseases. the details of each infection are, of course, very important and should be considered individually. leprosy, a directly transmitted bacterial infection, was one of the first ntds to have global targets for elimination as a public health problem, leading to large declines, although these have stabilized in the last decade [29] [30] [31] . leprosy exemplifies the problem with surveillance for a disease in which cases are identified if both the infected individuals seek care and the appropriate care is available for them and mathematical modeling provides methods for estimating the pool of undiagnosed infections [32] . in addition, modeling has highlighted the need for much earlier diagnosis and suggests that targeted case finding through household contact tracing, perhaps combined with postexposure prophylaxis, could hold great potential for control (table 1 ). in constrast with the global scope of leprosy, the gambian form of sleeping sickness (human african trypanosomiasis), which is transmitted by tsetse flies, is focused in western africa. it has a high case fatality rate and is targeted for elimination because it is thought to be an anthroponotic disease and current interventions have led to large drops in case numbers [33] . the main method of control is through screening of populations and treatment of infected individuals. a key question for sleeping sickness is the potential contribution of vector control as a complement to screening and treating. modeling suggests that it could have a large impact, reducing transmission not only from cases but also from the uncertain quantity of asymptomatic or presymptomatic individuals, or even animal hosts [34] . the modelers also highlight the potential to increase the impact of screening, both passively, by increased access to diagnostics, and actively, by targeting highrisk groups (table 1) , to reduce the duration of infection and therefore transmission (the area under the curve in figure 1 ) by all infected individuals. kala azar, or visceral leishmaniasis in the indian subcontinent, is a parasite transmitted by sandflies, predominantly in the poorest communities. it poses a number of challenges for control, which consists of improving case detection and indoor residual spraying [35] . cases are falling drastically, reducing the burden of disease, but there is debate around the drivers of this decline, the size and nature of any asymptomatic pool, and the risk of resurgence [35] . modeling acts as a tool to investigate some of the possible scenarios and evaluate different policy interventions in response to them [22, 27] . in particular, the recent modeling of the different stages of the control effort suggests that there should be some accounting for underlying transmission rates when selecting interventions and that post-kala-azar dermal leishmaniasis (pkdl), a late-stage potentially highly infectious state, could undermine control and should be studied more closely (table 1) . chagas disease is an anthropozoonosis caused by the protozoan trypanosoma cruzi, which is often contracted in childhood, when symptoms are rarely diagnosed; instead it is more commonly diagnosed through sequelle such as heart disease in adulthood [28] . there are huge complexities in the zoonotic life cycle in different settings, and modeling can be used to evaluate how different vector-control interventions are likely to affect transmission [36] . the modeling summarized in this issue highlights the value of vector control in reducing the infectiousness of all infected individuals, as well as the value of increasing diagnosis rates (table 1) . across the idms, the models demonstrate how key uncertainties in life history have the potential to undermine the impact of current control long term but that intelligent intervention design may be able to overcome them. a cornerstone of large-scale ntd control, specifically for lymphatic filariasis, onchocerciasis, soil-transmitted helminths, schistosomiasis, and trachoma, is mda, sometimes in combination with vector control. an mda program requires repeated distribution of treatments to large numbers of individuals, without diagnosis. they are therefore only considered when diagnosis of infection is difficult (eg, stool-based microscopy, night-time blood samples and microscopy), there is little care-seeking by infected individuals, and there is a treatment with an excellent safety profile with a straightforward or single treatment schedule ( figure 2 ). donation of the treatments for these 5 diseases by the pharmaceutical manufacturers has transformed the opportunities for reducing the burden of these diseases, but single or limited course treatments it has required additional investment to deliver the treatments, as well as data to determine when and where mda should be delivered. through the course of a successful mda, increasing numbers of treatments go to those uninfected at the time (figure 3 ). of course, these individuals are uninfected because they have been protected due to the ongoing mda program, which is a measure of the program's success. the issue of infectious asymptomatic individuals, which is a major concern for the idm diseases, is not such an issue as asymptomatic people are regularly treated as part of the mda. therefore, the key questions are who to treat (eg, which age group), how often to treat, and when treatments can be stopped [37] . a number of things can lead to the failure of an mda, all of which have been investigated using mathematical modeling, including in this issue (figure 3) . one of the important issues in program design, and which can undermine a program's success, is which parts of the population should be treated (figure 3 ). if the wrong group is treated, you may see reductions in burden in this group, but not in the population at large. this is discussed in 2 of the papers in this special issue. soil-transmitted helminths are transmitted through helminth eggs in feces contaminating the environment [38] , and schistosomiasis is caused by intestinal worms that are passed in feces or urine and contaminate the water. in the case of schistosomiasis, the eggs then go on to infect snails, the parasite is amplified and rereleased into the water, and humans are infected through contact with that water [39] . for both of these infections, current guidelines suggest that treatments should be targeted at children, with different frequencies according to prevalence. the modeling studies in this special issue suggest that the current guidelines might be altered slightly to optimize their impact, through either targeting adults or changing the thresholds for switching strategies [40, 41] (table 2) . the 2 other helminths considered here, lymphatic filariasis, transmitted by mosquitoes and a risk factor for elephantiasis, and onchocerciasis, transmitted by black flies and the cause of river blindness, are also targeted for elimination through mda, sometimes accompanied by vector control [42] . the lymphatic filariasis campaign has been particularly successful, with billions of treatments given and recent scale-back of treatment in areas where the targets have been met. the policy discussion is around how best to accelerate achievement of the goals using alternative treatment strategies and, in particular, when and where these strategies might be most appropriate [43] and how they might be combined with vector control to slow down nonadherence figure 3 . schematic of factors that could undermine the success of a mass drug administration program. monitoring and evaluation of programs is usually focused around a survey just prior to a round of treatment. if infection is not falling as quickly as expected, it could be due to any of the reasons outlined in the schematic, most of which cannot be detected by routine surveillance. the epidemic growth rate between rounds of mda [44] . if the bounce back rate is too fast, or the interval between treatments is too long, this can lead to all the gains from the previous round being lost (figure 3 ). for onchocerciasis, the programs are at the point of adapting their strategies to reach beyond the large morbidity gains achieved so far. the modeling work discusses the alternative strategies and the potential for mda combined with vector control to accelerate or achieve elimination ( table 2) . all of the articles on mda policies highlight the importance of the epidemiological setting, the appropriate group being targeted, and systematic nonadherence, where particular groups either do not have access to or are refusing treatment, and note that these are often poorly measured ( figure 3 ) [40, 41, [43] [44] [45] . the issue of systematic nonadherence has been highlighted in modeling studies for many years but has recently become a point of focus again [11, 46, 47] . for trachoma, a bacterial infection that can cause blindness and is transmitted through an uncertain combination of vectors and direct contact, the modelers highlight an additional aspect of mda, which is resistance to the drugs used for mass treatment (table 2 ) [48] . this is because the mda is a single dose of a broad-spectrum antibiotic, and so the concerns about rapid emergence of resistance have been present since the beginning, but current evidence suggests that the selection pressure from this single dose may not be as high as feared. they also highlight the risk of resurgence due to importation of cases, which is a particular concern because of the rapid epidemic growth rate of trachoma [45] . both of these issues are relevant for the other mda campaigns ( figure 3 ), but the longer time between generations and hence the slower epidemic growth rates for the helminths mean that both resistance and re-emergence are likely to be slower than for trachoma. however, as these campaigns have been running for decades in some cases, it is important to consider. in addition to these issues, there are outstanding questions around when and where to halt mda campaigns, which future modeling will inform. treatment has already been halted in some areas for the lymphatic filariasis campaign. issues of ongoing residual transmission, albeit with a likely slow growth rate, are being addressed and the decision to stop is being reevaluated [49] . in this article, we have discussed 2 main interventions for the control of ntds: idm and mda. these definitions are part of a shifting landscape that is dependent on a changing epidemiology, demography, and on the availability of new tools. for example, with an appropriate treatment with a good safety profile in uninfected and nonsymptomatic persons, a disease could move from case detection to mda or, when combined with the right diagnostic, to a screen and treat infection. similarly, as prevalence falls for mda diseases, if the right diagnostic becomes available, addressing these diseases could shift to a test-and-treat campaign or even case management. one of the roles of modeling is to evaluate the likely impact of new tools, treatments, and diagnostics; this is an active area of ongoing research that is not stressed in this issue. modeling of ntds is constrained by particularly limited data, as these articles highlight through presentation of uncertainty in predictions, sensitivity analyses, or scenario-based investigation. in contrast with many other infections, the dynamics of these diseases are also characterized by slow timescales, which mean that many qualitative behaviors are robust to these unknowns. however, it should be noted that these analyses should be viewed as a current state of our knowledge, and data from ongoing research have the potential to reduce some of these key uncertainties. across these diverse diseases, there are a number of common themes. • interventions should be tailored to the environment in which they are used, which requires more intensive data but should deliver greater gains. • reaching the right populations and ensuring uptake of screening, treatment, or mda is an essential part of any campaign, and models can indicate at what level of coverage or systematic nonadherence these campaigns are more likely to fail. • for a number of diseases, the relative contribution of sustained vector control transmission is an area of current evaluation. vector control has the potential to speed the gains due to other interventions and maintain the gains once the biomedical interventions have taken place, but it may only be needed in certain areas. despite the large number of biological unknowns or uncertainties for ntds, the slower dynamics allow us to develop our insights as data become more available. in summary, the modeling analyses in this special issue demonstrate that 2020 goals for ntds are likely to be met in a large number of areas. they also indicate what additional interventions are likely to be required in higher transmission areas or areas with particular epidemiological features. as such, this represents state-of-the-art modeling in this area and provides actionable information for policy development. acknowledgements. the author would like to thank the editors and staff of clinical infectious diseases for all their contributions in developing and delivering this special issue on behalf of all the authors in it. their support, patience, and clear guidance have been invaluable. the author also thanks members of the ntd modelling consortium for discussions regarding their contributions to the special issue and their contributions to tables 1 and 2. disclaimer. the views, opinions, assumptions, or any other information set out in this article are solely those of the author and should not be attributed to the funders or any person connected with the funders. financial support. this work was supported by the bill and melinda gates foundation in partnership with the task force for global health through the ntd modelling consortium (grant no opp1053230) and the children's investment fund foundation (uk). supplement sponsorship. this article appears as part of the supplement "reaching the 2020 goals for nine neglected tropical diseases, " sponsored by the ntd modelling consortium. potential conflicts of interest. the author reports no conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. accelerating work to overcome the global impact of neglected tropical diseases-a roadmap for implementation available at: http:// unitingtocombatntds.org/london-declaration-neglected-tropical-diseases the global burden of disease study 2013: what does it mean for the ntds? global health policy and neglected tropical diseases: then, now, and in the years to come neglected tropical diseases, neglected communities, and conflict: how do we leave no one behind? modeling infectious disease dynamics in the complex landscape of global health mathematical models for neglected tropical diseases: essential tools for control and elimination, part a mathematical models for neglected tropical diseases: essential tools for control and elimination, part b quantitative analyses and modelling to support achievement of the 2020 goals for nine neglected tropical diseases elimination of visceral leishmaniasis in the indian subcontinent: a comparison of predictions from three transmission models measuring and modelling the effects of systematic non-adherence to mass drug administration predicting lymphatic filariasis transmission and elimination dynamics using a multi-model ensemble framework forecasting the new case detection rate of leprosy in four states of brazil: a comparison of modelling approaches comparison and validation of two computational models of chagas disease: a thirty year perspective from venezuela probabilistic forecasts of trachoma transmission at the district level: a statistical model comparison modelling the elimination of river blindness using long-term epidemiological and programmatic data from mali and senegal comparison and validation of two mathematical models for the impact of mass drug administration on ascaris lumbricoides and hookworm infection a comparison of two mathematical models of the impact of mass drug administration on the transmission and control of schistosomiasis data-driven models to predict the elimination of sleeping sickness in former equateur province of drc a guide to reproducible code in ecology and evolution: british ecological society factors that make an infectious disease outbreak controllable uniting mathematics and biology for control of visceral leishmaniasis hiv-1 transmission, by stage of infection no hiv stage is dominant in driving the hiv epidemic in sub-saharan africa hiv treatment as prevention: debate and commentary-will early infection compromise treatment-as-prevention strategies health-seeking behaviour, diagnostics and transmission dynamics in the control of visceral leishmaniasis in the indian subcontinent policy recommendations from transmission modeling for the elimination of visceral leishmaniasis in the indian subcontinent chagas disease back-calculating the incidence of infection of leprosy in a bayesian framework innovative tools and approaches to end the transmission of mycobacterium leprae global leprosy update, 2016: accelerating reduction of disease burden policy lessons from quantitative modeling of leprosy human african trypanosomiasis assessing strategies against gambiense sleeping sickness through mathematical modeling understanding the transmission dynamics of leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in bihar complementary paths to chagas disease elimination: the impact of combining vector control with etiological treatment optimisation of mass chemotherapy to control soil-transmitted helminth infection soil-transmitted helminth infections human schistosomiasis investigating the effectiveness of current and modified who guidelines for the control of soil-transmitted helminth infections are we on our way to achieving the 2020 goals for schistosomiasis morbidity control using current who guidelines? lymphatic filariasis and onchocerciasis are alternative strategies required to accelerate the global elimination of lymphatic filariasis? insights from mathematical models how can onchocerciasis elimination in africa be accelerated? modeling the impact of increased ivermectin treatment frequency and complementary vector control models of trachoma transmission and their policy implications: from control to elimination effectiveness of annual ivermectin treatment for wuchereria bancrofti infection the importance of patient compliance in repeated rounds of mass drug administration (mda) for the elimination of intestinal helminth transmission a comprehensive assessment of lymphatic filariasis in sri lanka six years after cessation of mass drug administration key: cord-318358-glbr8kxh authors: naik, george o a title: covid-19 and the raas date: 2020-06-20 journal: clin infect dis doi: 10.1093/cid/ciaa818 sha: doc_id: 318358 cord_uid: glbr8kxh nan m a n u s c r i p t dear editor: further to thomas hanff et al [1] timely call for epidemiological and clinical investigations of covid-19 infectious disease, measurements of the renin angiotensin aldosterone system (raas) components, as sub-studies would be insightful of this pandemic. angiotensin-converting enzyme 2 (ace 2) participates in the coronavirus (sars-cov-2) cell entry. this infection down regulates ace 2. drugs that block raas also affect ace 2 expression: it is down regulated by renin inhibition (ri) and up regulated by angiotensin-converting enzyme inhibitors (aceis), angiotensin receptor blockers (arbs) (1) and mineralocorticoid receptor antagonists (mras) [2] . other likely regulatory factors are age, type ii diabetes and sex difference [3] . these interactions would directly affect the balance between the beneficial and deleterious angiotensins (angs), such as, ang (1-7) and ang (1-9) versus excess ang ii. such perturbations would also indirectly influence other raas components, and the coordination between circulating and local tissue expressions, as shown in figure 1 ace 2 is distributed throughout the body and is abundantly expressed in the lung, small intestine, and in blood vessels of many organs including the brain, heart, kidney and testis [4] . these organs and blood vessels are potential sites of infection. the downregulation of ace 2 would reduce the production of ang (1-7) and ang (1-9), and concurrently prevent the reduction of the ang ii, tilting the balance to ang ii accumulation that may lead to toxicity [1] , figure 1 (b). such dysregulation likely contributed to reported cases of acute respiratory distress syndrome (ards) [1] , a c c e p t e d m a n u s c r i p t inflammation, myocardial injury [5] , neurological incidences [6] and gastrointestinal manifestations [7] . other components and the crosstalk between the systemic circulation and local tissue renin angiotensin system would also be disrupted. changes in circulating ang ii concentration alter renin secretion through a negative feedback loop, figure 1 (a), as ang ii decreases renin secretion increases and consequently affect renin concentration and plasma renin activity (pra). renin converts angiotensinogen to angiotensin i (ang i) and pra is a measure of this rate. renin catalytic activity is enhanced when bound to its receptor (ppr) [8] . the inhibition of renin or ace reduces circulating ang ii with an increase in renin concentration. it is conceivable that circulating renin could bind to ppr, where expressed, and activate local tissue renin angiotensin system. ang ii changes would also affect aldosterone stimulation and angiotensin iv (ang iv) production. ang iv through its receptor at4 has opposite biological effects to ang ii via receptor at1. we therefore suggest the measurement [9] 1-7) ). ang ii is further transformed to ang iii, ang iv and ang v. a negative feedback loop controls ang ii concentration changes with renin secretion that responds in the opposite direction. ang ii stimulates the release of aldosterone. however, excessive ang ii is deleterious and is associated with hypertension, congestive heart failure and chronic kidney disease. ace 2 transforms ang i and ang ii, that is, ang i to ang (1-9), and ang ii to ang (1-7). ang (1) (2) (3) (4) (5) (6) (7) (8) (9) and ang (1-7) have protective effects balancing the deleterious ang ii, when in excess. a c c e p t e d m a n u s c r i p t figure 1 is there an association between covid-19 mortality and the renin-angiotensin system-a call for epidemiologic investigations mineralocorticoid receptor blocker increases angiotensin-converting enzyme 2 activity in congestive heart failure patients individual variation of the sars-cov2 receptor ace2 gene expression and regulation tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis association of coronavirus disease 2019 (covid-19) with myocardial injury and mortality neurologic manifestations of hospitalized patients with coronavirus disease prevalence and clinical characteristics of mild severity patients with digestive symptoms the (pro)renin receptor: an emerging player in hypertension and metabolic syndrome specific and non-specific measurements of tissue angiotensin ii cascade members key: cord-327324-4c4a4bfz authors: wilkinson, robert j title: tuberculosis and type 2 diabetes mellitus: an inflammatory danger signal in the time of covid-19 date: 2020-06-13 journal: clin infect dis doi: 10.1093/cid/ciaa747 sha: doc_id: 327324 cord_uid: 4c4a4bfz nan however it is increasingly appreciated dm itself is an inflammatory disorder that may therefore interact with infection in more complex ways [10] . transcriptomic profiling of whole blood is a technique that has been widely applied and proven insightful in many infectious and inflammatory disorders [11] . active tuberculosis has a transcriptomic signature dominated by a neutrophil-driven type 1 and 2 interferoninducible gene profile. the transcriptomic signature relates to disease extent and resolves during successful treatment [12] . the differentiation of active from latent tuberculosis and other conditions may be aided by transcriptomic profiling [13] . the exaggerated inflammation that characterizes hiv-tuberculosis-associated immune reconstitution inflammatory syndrome is triggered by toll-like receptor and inflammasome signalling [14] . transcriptomic signatures may predict progression of, or detect, subclinical tuberculosis [15] [16] [17] . in this issue of the journal eckold and colleagues present an analysis of the effect of dm the study has the advantage of power and of validation across populations. limitations are that hba1 c is a relatively insensitive test for dm. it is known that tuberculosis itself associates with transiently impaired glucose tolerance which may even be in the frank diabetic range, yet which may resolve during tuberculosis treatment [7] , but the proportion of cases of ih falling into this category was not investigated longitudinally. sex influences type 1 interferon and neutrophil transcript abundance and there were some very substantial gender differences between groups ranging from 18-90% males. this publication coincides with the world grappling with the covid-19 pandemic. are there inferences that can be made from this very detailed study of susceptibility to tuberculosis conferred by diabetes? male sex and diabetes have been identified in virtually every study as risk factors for severe covid-19 infection, associated in the largest study to date with an adjusted hazard ratio for in-hospital death of 1.99 (male sex) and 1.50 for controlled (hba1c < 58 mmol/mol) and 2.36 for uncontrolled dm [18] . the pulmonary innate immune response in covid-19 patients with severe disease implicates a macrophage subpopulation with an interferon-associated hyperinflammatory response analogous to that observed in tb patients [19] . type 1 and 2 interferon stimulation increases angiotensin converting enzyme 2 (ace2), the receptor for sars-cov2, expression on respiratory epithelial cells. in particular, lungs of patients with hiv-associated tb show a c c e p t e d m a n u s c r i p t 4 the highest expression of ace2 in epithelial cells, compared to lungs of patients with tb only and healthy lungs [20] . hiv-1 infection, prior to antiretroviral therapy (art) also induces a similar interferon signalling gene (isg) profile to that of tb patients, a pattern also implicated in the context of hiv-tb co-infection, even prior to tb symptom onset [16] . given these findings, it is plausible that individuals with hiv-1 and/or tb infection could be at increased risk of sars-cov2 infection not only via immunosuppression but also via increased ace2 expression, predisposing to enhanced viral uptake. this theory is diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies effect of glycemic control and type of diabetes treatment on unsuccessful tb treatment outcomes among people with tb-diabetes: a systematic review the impact of diabetes on tuberculosis treatment outcomes: a systematic review diabetes is a strong predictor of mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients from mwanza, tanzania hyperglycemia during tuberculosis treatment increases morbidity and mortality in a contemporary cohort of hiv-infected patients in rio de janeiro, brazil stress hyperglycaemia tuberculosis, hiv and the association with transient hyperglycaemia in peri-urban south africa consultation meeting on tuberculosis and diabetes mellitus: meeting summary and recommendations infections in patients with diabetes mellitus: a review of pathogenesis immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections uninfected african adults using whole blood rna expression signatures: a casecontrol study hiv-tuberculosis-associated immune reconstitution inflammatory syndrome is characterized by toll-like receptor and inflammasome signalling characterization of progressive hiv-associated tuberculosis using 2-deoxy-2-[18f]fluoro-d-glucose positron emission and computed tomography complement pathway gene activation and rising circulating immune complexes characterize early disease in hiv-associated tuberculosis a modular transcriptional signature identifies phenotypic heterogeneity of human tuberculosis infection opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients single-cell landscape of bronchoalveolar immune cells in patients with covid-19 sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is enriched in specific cell subsets across tissues rjw is supported by the francis crick institute which receives funding from wellcome (fc0010218), ukri (fc0010218) and cruk (fc0010218). he also receives support from wellcome (014803 and 203135), nih (r01ai145436) and edctp (ria2017t-2004) . the views in this article are the author's alone and do not reflect the views of these agencies. a c c e p t e d m a n u s c r i p t key: cord-325416-c0qj3yd9 authors: zachariah, philip; halabi, katia c; johnson, candace l; whitter, susan; sepulveda, jorge; green, daniel a title: symptomatic infants have higher nasopharyngeal sars-cov-2 viral loads but less severe disease than older children date: 2020-05-20 journal: clin infect dis doi: 10.1093/cid/ciaa608 sha: doc_id: 325416 cord_uid: c0qj3yd9 nan m a n u s c r i p t 2 dear editor: published data suggest lower rates of severe coronavirus disease 2019 in pediatric age groups [1, 2] . however, the relative contribution of infants and children to community transmission is not known. data from china suggest that children were infected early in the outbreak [3] . a report published in this journal described the presence of a high nasopharyngeal (np) viral load of severe acute respiratory coronavirus 2 (sars-cov-2) in a well infant [4] . whether this observation is generalizable to symptomatic infants, and how it compares to np viral loads in older children and adolescents is not known. studies in adults have demonstrated a positive correlation between viral load and covid-19 severity [5] . while some data have suggested a higher disease severity in infants [2, 6] , how np viral load correlates with severity across the pediatric age spectrum has not been firmly established. here we report np viral load among infants, children and adolescents who were hospitalized and discharged from our children's hospital from march 14 th to april 24 th , 2020. all patients were tested either in the emergency department or during inpatient hospitalization based on symptoms suggestive of covid-19. for each patient, we extracted age, time from reported symptom onset to the date of test, and severity of disease during hospitalization. severity of covid-19 disease was determined by: i) the need for respiratory support with non-invasive positive pressure ventilation and/or mechanical ventilation or, ii) icu admission. our report suggests symptomatic infants have higher np viral loads at presentation but develop less severe disease as compared to older children and adolescents. whether this is attributable to slightly earlier presentation to clinical care vs. host biology requires investigation. these data have implications for mitigating spread, especially in congregate settings (e.g. child care centers) or hospital units (e.g. neonatal intensive care units) that serve this group. financial disclosures: there are no financial disclosures to report on any of the authors screening and severity of coronavirus disease 2019 (covid-19) in children in madrid epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china detection of covid-19 in children in early a well infant with coronavirus disease 2019 (covid-19) with high viral load viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china covid-19 situation summary a c c e p t e d m a n u s c r i p t 4 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-324607-rpwccvqi authors: rojek, amanda m; moran, james; horby, peter w title: core minimal datasets to advance clinical research for priority epidemic diseases date: 2020-02-15 journal: clin infect dis doi: 10.1093/cid/ciz760 sha: doc_id: 324607 cord_uid: rpwccvqi the ebola virus disease outbreak in west africa has prompted significant progress in responding to the clinical needs of patients affected by emerging infectious disease outbreaks. among the noteworthy successes of vaccine trials, and the commendable efforts to implement clinical treatment trials during ebola outbreaks, we should also focus on strengthening the collection and curation of epidemiological and observational data that can improve the conception and design of clinical research. during the currently ongoing ebola virus disease (evd) outbreak in the democratic republic of congo, a clinical trial of potential treatments has commenced. this is a significant step toward improving outcomes for patients with the disease. ebola virus disease constitutes but one of the priority diseases that the world health organization (who), in their blueprint for action to prevent epidemics, suggests poses a severe public health risk and for which there are insufficient countermeasures [1] . the purpose of this priority list is to identify high-threat pathogens for which there is a need to prioritize and advance the development of diagnostics, vaccines, and therapeutics. any diagnostics, drugs, or vaccines that are developed as a result of this and other initiatives, such as the coalition for epidemic preparedness innovation, will need to be fully evaluated in diagnostic evaluation studies or phase ii and iii clinical trials. however, due to the very nature of the epidemic-prone infectious diseases that appear in the who list of priority diseases, evaluation in clinical studies is challenging, not least because the epidemiology is unpredictable but also because the pathogenesis and natural history of many of these diseases are not well defined. for example, during the influenza a(h1n1)pdm09 pandemic, case fatality rate (cfr) estimates varied widely from 0 to 13 500 per 100 000 laboratory-confirmed infections, with a heterogeneity of 99.97% (using i 2 estimate) [2] . a therapeutic trial designed with patient survival as a primary outcome measure would have grossly misjudged the required sample size if the trial was designed using the wrong cfr. therapeutic trials for the prevention of congenital zika syndrome will be hindered by the absence of consistently used criteria to define the outcome of congenital malformations [3] . for middle east respiratory syndrome coronavirus, a lack of systematic biological sampling means that disease pathophysiology and factors associated with more severe disease and viral clearance (a commonly used secondary outcome measure) are not well understood [4] . the need for well-defined core minimal datasets for emerging infectious diseases is not a new observation. a decade ago sheila bird and jeremy farrar [5] noted the need to define a core minimal dataset for human cases of avian influenza a/h5n1, yet there remains no systematic examination of the completeness of the core data needed to design and conduct trials for highpriority pathogens. table 1 identifies some key domains that could contribute to a core minimal dataset that informs clinical trial design for each priority pathogen. the benefit of this approach, when complemented by scoring or assessment of the available information, is that it allows for initial bench-marking and triaging of unmet data needs in order to prioritize further data gathering activities. importantly, a harmonized data collection initiative can also prospectively embed data-sharing agreements into data-collection protocols. this will allow valuable clinical information to be readily available to stakeholders, while identifying and protecting the interests of those collecting data in regions where outbreaks occur. accumulation and curation of the data will depend on a variety of sources and methodology types, but it is critical that high-quality clinical data are highlighted as an integral component. often lost to competing priorities for clinicians during outbreaks, standardized data collection regarding the presentation and natural history of disease, biomarkers of disease severity, and response to supportive care can be sporadic or missing. while these data have their most important benefits in improving patient management (through better recognition of disease complications and informing supportive care) and public health control, patient-based data are also used to determine key parameters for clinical trials, such as the inclusion criteria, the nature and rate of clinically relevant outcomes, and potential confounders. we suggest that adoption of clinical case registries (such as those used for rare cancers) provides a feasible option to produce standardized clinical data that have multiple clinical, public health, and research benefits [6] . compared with expensive and lengthy countermeasure development pipelines, improving the scale, relevance, and quality of observational data is likely to be an efficient and cost-effective strategy to improve global preparedness against epidemic and pandemic infections. disclaimer. the funders had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. temporal and geographical profile of previous outbreaks this is required for logistical planning, to ensure that local teams are sufficiently trained in research practices (such as good clinical practice) and trial-specific equipment is available. an agreed-upon case definition clinical characteristics of the disease are used to define enrollment criteria. analysis of strength of evidence for factors associated with increased disease severity or fatality stratification (or other statistical adjustment) on the basis of severity is often required when interpreting the clinical trial outcome. best available descriptions of the type and rate of clinical outcomes clinical outcomes will function as a trial outcome measures. understanding the natural course of illness will also help differentiate disease course from adverse events from treatment. assessment of confidence in estimates of clinical outcomes heterogeneity in patient outcomes between or within outbreaks creates uncertainty for power calculations and will affect selection of a statistical design for a trial. spurious heterogeneity may occur due to random error in small cohorts, or represent ascertainment, lead-time, measurement, or follow-up bias. real heterogeneity can occur due to improvements in care over an outbreak, pathogen evolution, or changes in host susceptibility and vulnerability but should be adjusted for. analysis of known or suspected covariates of outcome highlights possible confounders that will alter outcome independently of treatment and that will require adjustment if unequally distributed between treatment and control arms. the mean time from onset of symptoms to outcome allows for an estimation of the feasibility and logistics of medical intervention. agreed-upon standards of care for patient treatment determines if there is standardized supportive therapy to be adopted in all arms of a trial. this is especially important for multicenter research the performance characteristics of the favored diagnostic method determines whether a trial will be performed on an itt basis or following laboratory confirmation. mean time for laboratory diagnosis determines whether a trial will be performed on an itt basis or following laboratory confirmation. community priorities and expectations for trials determines the priorities of affected communities in terms of access to trials, acceptable methodology, and acceptability of treatments or vaccines. abbreviation: itt, intention-to-treat. annual review of diseases prioritized under the research and development blueprint informal consultation case fatality risk of influenza a (h1n1pdm09): a systematic review clinical trials of therapeutics for the prevention of congenital zika virus disease: challenges and potential solutions development of medical countermeasures to middle east respiratory syndrome coronavirus minimum dataset needed for confirmed human h5n1 cases a systematic review and meta-analysis of patient data from the west africa (2013-16) ebola virus disease epidemic key: cord-312797-hohzjx74 authors: hamelin, marie-ève; abed, yacine; boivin, guy title: human metapneumovirus: a new player among respiratory viruses date: 2004-04-01 journal: clin infect dis doi: 10.1086/382536 sha: doc_id: 312797 cord_uid: hohzjx74 the human metapneumovirus (hmpv) is a newly described member of the paramyxoviridae family belonging to the metapneumovirus genus. since its initial description in 2001, hmpv has been reported in most parts of the world and isolated from the respiratory tract of subjects from all age groups. despite the fact that prospective and case-control studies have been limited, the epidemiology and clinical manifestations associated with hmpv have been found to be reminiscent of those of the human respiratory syncytial virus, with most severe respiratory tract infections occurring in infants, elderly subjects, and immunocompromised hosts. additional research is needed to define the pathogenesis of this viral infection and the host's specific immune response. part of the pneumovirinae subfamily within the paramyxoviridae family. initial electron microscopic examination of hmpv isolates revealed morphological characteristics consistent with paramyxoviruses. pleomorphic, spherical, and filamentous particles could be observed (figure 1) [6] . spherical enveloped particles vary in size, with a mean diameter of 209 nm. the nucleocapsid has a length varying from !200 to ∼1000 nm and a diameter of 17 nm. hmpv isolates were initially found to grow on tertiary monkey kidney and llc-mk2 (rhesus monkey kidney) cells, with poor or no viral growth on other cell lines, including madin darby canine kidney, vero (african green monkey kidney), human laryngeal carcinoma (hep-2), human foreskin fibroblast, human rhabdomyosarcoma, transformed human kidney (293), human lung adenocarcinoma (a-549), and human colon adenocarcinoma (ht-29) [4, 7] . in addition, hmpv isolates did not exhibit hemagglutinating activity when tested with human, turkey, chicken, or guinea pig rbcs, which is consistent with other members of the pneumovirinae subfamily [4, 7] . intranasal inoculation of ferrets and guinea pigs with hmpv isolates did not cause any clinical symptoms [4] . also, experimentally infected birds (juvenile turkeys and chickens) did not show clinical signs or virus replication over a 3-week period. on the other hand, there was evidence of efficient hmpv repfigure 1 . negative-stain electron micrographs of human metapneumovirus (hmpv). center image shows 5 pleomorphic hmpv particles; note the projections along the periphery of the viruses. upper right and lower left insets show the nucleocapsid and the filamentous rodlike particle, respectively. staining was done with 2% phosphotungstic acid. bar markers represent 100 nm. from [6] ; reproduced with permission from the university of chicago press. lication in the respiratory tract of experimentally infected monkeys (cynomolgus macaques) associated with mild upper respiratory tract signs [4] . thus, in contrast to apv, hmpv is a respiratory pathogen of primates. the hmpv genome consists of a single negative strand of rna of ∼13 kb containing genes coding presumably for a nucleoprotein (n), phosphoprotein (p), matrix (m) protein, fusion (f) protein, transcription elongation factor/rna synthesis regulatory factor (m2), small hydrophobic (sh) surface protein, major attachment (g) glycoprotein, and major polymerase (l) subunit, arranged in the order 3 -n-p-m-f-m2-sh-g-l-5 , similar to apv [8, 9] . two major differences exist between hmpv and hrsv genomes: the gene order is different, and hmpv does not contain nonstructural genes (figure 2) [10] . the possible relevance of the absence of these viral proteins, which have been associated with ifn antagonism by hrsv [11] , deserves additional study. for hrsv, the g and f surface proteins are known as the major protective antigens [11] , and this also needs to be verified for hmpv. phylogenetic analyses have demonstrated that, among members of the pneumovirinae subfamily, hmpv was most closely related to apv serotype c, the avian metapneumovirus that emerged in the united states in the late 1990s. for instance, the amino acid sequence identity between hmpv strains and apv-c and hrsv representatives varies from 66% to 89% and from 23% to 43%, respectively, when comparing the n, p, m, and f genes [4, 12] . the sh and g amino acid sequences of hmpv show important variability and share only 28% and 21% identity with their respective apv-c counterparts [8, 13, 14] . it is possible that this important divergence may account for the specific host range of these 2 related metapneumoviruses. genetic analysis on a large number of hmpv isolates has identified 2 major groups and 2 minor genetic clusters within each group [6, 7] . however, additional investigations are still required to determine whether these genotypes represent different antigenic groups. recently, the complete nucleotide sequences of hmpv isolates belonging to these 2 major groups were determined [9] . nucleotide and amino acid sequence identities between the 2 hmpv groups were found to be 80% and 90%, respectively, which is similar to what has been reported with hrsv a and b genotypes. since its initial report by the dutch researchers in 2001, hmpv has been found in most parts of the world, with reports from north america (united states and canada), europe (united kingdom, france, germany, italy, spain, and finland), asia (hong kong and japan), and australia (table 1). the virus has also been identified in hiv-infected and nonimmunocompromised children from south africa [15] . the few seroprevalence surveys from the netherlands [4] , japan [16] , and israel [17] have indicated that virtually all children are infected by 5-10 years of age. in addition, studies have shown that hmpv is not a new pathogen, with serological evidence of human infection dating from 1958 in the netherlands [4] and viral isolation for the past 10-20 years in europe and canada [4, 7] . cases of severe hmpv infection in adults [7] and of reinfection in immunocompromised subjects [18] suggest that, despite universal infection in childhood, new infections can occur throughout life due to incompletely protective immune responses and/or acquisition of new genotypes. surveys have indicated that hmpv has a seasonal distribution overlapping hrsv circulation, with most cases reported during the winter/ early spring months (table 1) . although most studies have limited their surveillance to the typical respiratory virus period, our group has shown that 87% of hmpv isolates recovered in cell culture in quebec, canada, were recovered during the period of december through may [7] . in addition, we found that outbreaks of hmpv infection tend to peak in early spring over a 4-6 week period, slightly later than outbreaks of hrsv infection, which also are more spread out in time [19] . however, additional studies over multiple years are needed to better define the seasonality of hmpv infection. the role of hmpv as the cause of arti has been evaluated in many studies, mostly using molecular detection methods (table 1) . young hospitalized children have been best studied, and hmpv has been generally found in 5%-10% of arti cases in that population. however, in one study from italy, the number of hmpv infections varied widely over a 3-year period and were associated with 7%-43% of hospitalizations for arti [20] . the relative role of hmpv in respiratory syndromes of adults has been less studied. in one study from rochester, new york, hmpv was detected by serological tests and/or rt-pcr in 4.5% of young and elderly adults with arti [21] . interestingly, evidence of hmpv infection was also found by serological testing in almost the same percentage (4.1%) in asymptomatic adults, raising the question of the causative role of hmpv in arti in adults. recent studies by our group indicate that hmpv is rarely identified by rt-pcr in npas obtained from asymptomatic young children, with a rate significantly lower than that of subjects with arti (!1% vs. 6%) [19, 22] . it is interesting to note that the rates of detection of hmpv have been generally higher in retrospective than prospective studies (table 1) , an observation consistent with some selection bias. thus, to better define the role of hmpv in various respiratory conditions, large prospective studies using appropriate controls need to be conducted. in addition, testing of all clinical samples (i.e., not only those samples that are found to be negative for other viruses) must be performed. nevertheless, most studies have so far indicated that hmpv is a frequent cause of severe arti, especially in young children. table 2 summarizes the clinical findings of the first 28 hmpvinfected canadian subjects retrospectively identified through our virology laboratory. symptoms of both upper and lower respiratory tract infections have been associated with hmpv in young children, although most reports are biased towards description of the most severe symptomatology in hospitalized subjects. in young hospitalized children, the clinical features associated with hmpv infections are very similar to those of hrsv [19, [23] [24] [25] . diagnoses of bronchiolitis, with or without pneumonitis, have been most commonly reported. a substantial proportion (up to 50%) of infected children also has concomitant otitis media [19] . acute wheezing and asthma exacerbation have been associated with hmpv in some [23, 26] but not all [27] studies. compared with hrsv infections, hmpv cases tend to occur at an older age [19, 26, 28] . the clinical outcome after hrsv infection has been more severe than that after hmpv infection when looking at the proportion of patients with hypoxemia, those with pneumonia, and those admitted to the intensive care unit in studies from canada [19] and europe [25, 28] . in contrast, researchers from hong kong found that hmpv infection was associated with a longer hospital stay and more cases of pneumonia than was hrsv infection [26] . the latter group reported an estimate of 442 hmpvassociated hospitalizations per 100,000 children aged р6 years. children with underlying medical conditions may have moresevere hmpv disease, leading to hospitalization. in studies from north america, 25%-33% of hmpv cases occurred in children with underlying conditions, such as prematurity, cardiopulmonary problems, and immunosuppression [7, 29] . hmpv has been associated with flulike illnesses and colds in healthy adults (table 2) [7] . stockton et al. [30] identified hmpv in 9 (2.2%) of 408 nasal and throat swabs obtained from subjects with flulike illnesses (i.e., patients who tested negative for influenza and hrsv rna) seen by general practitioners in england. eight of the 9 infected patients in that study were adults, and 6 of these patients also had clinical evidence of lower respiratory tract involvement. falsey et al. [21] found a higher rate of hmpv illness among young adults, although older adults experienced more dyspnea and wheezing than did younger adults, and those with cardiopulmonary conditions were ill for nearly twice as long as younger adults. among 10 retrospectively identified, infected patients aged 165 years who were hospitalized at our institution, 4 developed pneumonitis and 2 died (table 2) [7] . of note, all 10 patients had у1 underlying illness, such as leukemia/lymphoma and chronic cardiovascular, pulmonary, or neurologic diseases. collectively, available data in-dicate that the clinical presentation of hmpv is very similar to that of hrsv, with more severe outcomes in infants, elderly subjects, and persons with underlying diseases [31] . the detection of hmpv in special settings merits further discussion. preliminary data suggest that, similar to hrsv, hmpv infection may have a more fulminant course in severely immunocompromised individuals. our group reported the death of a 17-month-old girl with acute lymphoblastic leukemia due to severe pneumonitis and respiratory insufficiency [18] . although no autopsy was performed, an npa obtained before death revealed the presence of hmpv only. of interest, this child had presented with a first episode of bronchiolitis caused by hmpv 1 year earlier, and the 2 viral isolates were of different lineages (groups), highlighting the possibility of rapid reinfections in immunocompromised hosts. similarly, hmpv was the sole pathogen identified in the npa obtained from a hematopoietic stem cell transplant recipient who died of progressive respiratory failure after an upper respiratory prodrome [32] . the presence of a copathogen may also lead to more-severe hmpv infections. a report from england identified hmpv in 21 (70%) of 30 bronchoalveolar fluid specimens obtained from infants with severe hrsv bronchiolitis requiring ventilatory support, raising the possibility that hmpv might be a determinant of hrsv disease severity [33] . unfortunately, no data regarding dual infections in less severe hrsv disease were available for comparison. in contrast, the rate of bronchopneumonia was not altered by the presence of a second respiratory virus in italian infants with hmpv infection [20] . finally, the possible synergistic effect between hmpv and a new coronavirus has been recently postulated during outbreaks of severe acute respiratory syndrome (sars) in canada and hong kong [34, 35] . in a study of 48 patients with probable sars in hong kong, chan et al. [34] found evidence of hmpv infections by nested pcr in 25 cell cultures (52%) inoculated with npa samples. in fact, hmpv was more frequently isolated than the sars coronavirus in the latter study. this report is surprising, considering the results of experimental infections in macaques, in which severe multifocal pulmonary consoli-dation was induced by the sars coronavirus only, with no exacerbation after subsequent infection with hmpv [36] . hmpv growth in cell culture is fastidious; this may be one reason for its late identification. most studies have reported reliable cytopathic effects only in tertiary monkey kidney or llc-mk2 cells [4, 6, 7] . the cytopathic effect is variable, with some strains inducing hrsv-like syncytia formation and others inducing focal rounding and cell destruction ( figure 3) . typically, the cytopathic effect is displayed more than 10-14 days after inoculation (mean time was 17 days in our laboratory). confirmation of hmpv cytopathic effect is achieved by rt-pcr testing of infected supernatants in the absence of commercially available antibodies. a recent report by chan et al. [34] indicated that hmpv could replicate more efficiently in human laryngeal carcinoma (hep-2) cells, despite the absence of cytopathic effect. in that instance, presence of hmpv was confirmed by direct testing of hep-2 cell culture supernatants by rt-pcr or subsequent passage on llc-mk2 cells to observe cytopathic effect. because of the unavailability of rapid antigen detection tests and because of the slow and restrictive viral growth, rt-pcr has become the method of choice for the diagnosis of acute hmpv infection. most pcr protocols reported to date have relied on amplification of the l, n, or f gene (reviewed in [37] ), with primer sequences mainly derived from the prototype strain 001 from the netherlands (genbank accession number af371337). because of the existence of 2 hmpv lineages with significant genetic variability within each group, hmpv detection may be underestimated when inadequate primers are selected for pcr amplification. we have recently reported that rt-pcr assays targeting the n and l genes, which code for 2 internal viral proteins, are best suitable for hmpv diagnosis [37] . rapid and sensitive hmpv assays based on the real-time pcr methodology have recently been described, allowing amplification and detection of this pathogen in р2 h [37, 38] . serological testing only permits a retrospective diagnosis. because infection is almost universal in childhood, a seroconversion or a у4-fold increase in antibody titers must be demonstrated to confirm recent infection. the few serological surveys for hmpv were based on an indirect immunofluorescence assay using hmpv-infected cells [4, 16] . a home-brew elisa method has also been developed using cell lysates of hmpv [21] . clearly, simpler elisa tests using viral proteins possibly derived from the 2 main groups are needed to conduct large serological surveys in many parts of the world. no vaccines, chemotherapeutic agents, or antibody preparations are currently approved for the prevention or treatment of hmpv infection. recently, wyde et al. [39] showed that ribavirin and a polyclonal intravenous immunoglobulin (ivig) preparation had equivalent in vitro activity against hmpv and hrsv. in contrast, no activity against hmpv was conferred by palivizumab, a humanized monoclonal antibody directed against the f protein of hrsv. despite the unavailability of animal studies and the toxicity related to ribavirin administration, the combined use of ivig and ribavirin could be envisaged for treating severe hmpv infection in immunocompromised patients, as has been reported for hrsv infection [40, 41] . furthermore, until an effective vaccine is developed, another interesting strategy could consist in the development of hightitered ivig preparations with activity against hmpv. the recent identification of a presumably old viral pathogen is an exciting development in the field of respiratory viruses. available data indicate that hmpv appears to be a significant cause of both upper and lower respiratory tract infections in young children. hmpv reinfections seem to be frequent and could also lead to devastating complications in elderly subjects and immunocompromised hosts, although more studies with adequate control groups are needed to confirm this. overall, the epidemiological and clinical features of hmpv infection appear to be similar to those of hrsv, although differences have been noted. it is important to mention that many fun-damental questions related to viral pathogenesis and the host's specific immune response still remain to be answered. the tucson children's respiratory study ii: lower respiratory tract illness in the first year of life etiology of community-acquired pneumonia: impact of age, comorbidity, and severity prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants. pediatr a newly discovered human pneumovirus isolated from young children with respiratory tract disease metapneumoviruses in birds and 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infection in young children and genetic heterogeneity of the viral isolates children with respiratory disease associated with metapneumovirus in hong kong asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus as a cause of community-acquired respiratory illness human metapneumovirus infection in the canadian population human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease human metapneumovirus in severe respiratory syncytial virus bronchiolitis human metapneumovirus detection in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada koch's postulates fulfilled for sars virus comparative evaluation of real-time pcr assays for detection of the human metapneumovirus molecular assays for detection of human metapneumovirus comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro prevention and treatment of respiratory syncytial virus and parainfluenza viruses in immunocompromised patients combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients evidence of human metapneumovirus in australian children human metapneumovirus and community-acquired respiratory illness in children human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children key: cord-328667-r5w09lb6 authors: schwartz, david a title: the effects of pregnancy on women with covid-19: maternal and infant outcomes date: 2020-05-11 journal: clin infect dis doi: 10.1093/cid/ciaa559 sha: doc_id: 328667 cord_uid: r5w09lb6 nan m a n u s c r i p t outbreaks of new and emerging viral diseases have always caused anxiety among persons and societies at risk for infection. in particular, this has been especially true for pregnant women, who fear not only for themselves but often even more so for their unborn infants. pregnant women, their fetuses and infants are generally the most vulnerable members of society during an infectious disease outbreak. the normal physiological, anatomical and immunological changes that accompany pregnancy may increase a pregnant woman's susceptibility to newly emergent viral pathogen as well as increase the severity of infection. these changes, including an adaptive immune state including local suppression of cell-mediated immunity, changes in the maternal cardiovascular and respiratory systems including increased oxygen consumption, heart rate, stroke volume, decreased lung capacity and other physiological changes, may increase the likelihood for severe maternal disease from an infectious disease, and especially with those that have respiratory transmission [1, 2] . recent history is replete with examples of new and reemergent viral diseases that have adversely affected pregnant women, often causing greater harm to them as a result of their condition than among infected but non-pregnant women. such infectious diseases as influenza, ebola virus, hepatitis e and varicella may have a more severe clinical course, increased complication rate, and higher casefatality rate among pregnant women than in non-pregnant individuals. the impact of emerging infections on the embryo or fetus is difficult to predict and varies depending on such factors as the agent, gestational timing of infection, and such host factors as the maternal-fetal interface. during the recent ebola virus epidemics non-vaccinated pregnant women were especially prone to excess morbidity and mortality, and the effects on their unborn infants were worse -only 2 newborns having the infection survived [3] . in contrast, some pregnant women might be asymptomatic or have only mild or nonspecific symptoms from an infectious disease, resulting in their escaping detection as having infection even when the embryo or fetus is severely affected. during the zika virus pandemic, infected pregnant women were typically asymptomatic, or at most had mild symptoms, which were unconcerning. however, the virus stealthily crossed the placenta without even inducing an inflammatory response, to produce a tragic spectrum of fetal malformations, neurological injuries, and even death [4] . an important aspect of the current covid-19 pandemic is its effect on pregnant women and their infants. there have now been numerous publications addressing the adverse effects of covid-19 on pregnant women, as well as examining the infection status and clinical characteristics of their newborn infants [5] [6] [7] [8] . however, there have been no data available to determine whether pregnancy itself has any consequences on the health of reproductive aged women with covid-19. in order to investigate this important question, li et al. [9] evaluated for the first time the effects of being pregnant on covid-19 disease and pneumonia using a case-control experimental design conducted at the maternal and child health hospital of hubei province, a 1900-bed tertiary medical center in wuhan, china. to accomplish this, the authors enrolled two cohorts of pregnant women with covid-19 and pneumonia -one consisting of 16 pregnant women with pneumonia and rt-pcr confirmed sars-cov-2 infection, and the other with 18 pregnant women with pneumonia who were clinically and radiologically suspected of having covid-19 but had negative rt-pcr test results. these 34 pregnant women with confirmed or suspected covid-19 pneumonia were compared with two cohorts of control cases. the first control cohort consisted of 29 non-pregnant women who were also infected with sars-cov-2 -this group included 11 non-pregnant women with rt-pcr confirmed a c c e p t e d m a n u s c r i p t infection and 18 non-pregnant women with clinical and radiographic evidence of the disease but who had negative rt-pcr testing. the second control cohort consisted of two groups of randomly selected women of reproductive age from different time periods with no pneumonia and not having covid-19 -121 women in each group -for a total of 242 women. their results showed that covid-19 typically caused mild respiratory symptoms in pregnant women, most of whom were asymptomatic upon hospital admission. when compared with nonpregnant women with covid-19 pneumonia, the infected pregnant women generally had absent or mild respiratory symptoms, and none developed severe respiratory compromise or required intensive care. among their findings was a higher incidence of premature delivery in pregnant women with confirmed covid-19, although this was not the result of severe maternal respiratory disease. in analyzing the neonatal outcomes from the cohort of pregnant women with covid-19, there was no evidence for intrauterine transmission of sars-cov-2. this latter observation was similar to a multitude of studies of pregnant women with covid-19 and their infants from china (5) (6) (7) (8) and is typical of intrauterine maternal-fetal transmission patterns of other respiratory rna viruses [10] . this important case-control investigation by li et al. significantly expands our knowledge of the effects of pregnancy and covid-19 infection and the infection status of neonates from infected mothers. the clinical outcomes of pregnant women with covid-19 from wuhan, china discussed in this article were all favorable, and are similar to reports of all (but one) pregnant women from that country. however, this should not be understood to indicate that infection with sars-cov-2 is not capable of causing severe and even life-threatening disease among pregnant women. in the single case of an obstetric near-miss event reported from china, a woman who developed severe pneumonia from covid-19 at 34 weeks gestation delivered a stillborn infant, had deterioration of cardiopulmonary status and subsequently developed multiple organ system dysfunction syndrome requiring extracorporeal membrane oxygenation [11] . in countries outside of china there are increasing reports of poor clinical outcomes arising from covid-19 disease among pregnant women. in new york city 2 pregnant women who were initially thought to be uninfected with sars-cov-2 developed severe postpartum medical complications requiring admission to intensive care and were found to have covid-19 [12] . a study of 43 pregnant women with covid-19 presenting to 2 new york city hospitals, 4 (9.3%) women developed severe disease, and 2 (4.7%) had critical disease requiring intensive case treatment [7] . cardiomyopathy occurring in pregnant women with sars-cov-2 infection has also been described [13] . covid-19 is now recognized to cause the death of pregnant women. there have recently been 8 cases of mortality among pregnant women from iran reported [14, 15] , none of whom had preexisting comorbid conditions that were above the baseline population risk. in response to the increasing seriousness of covid-19 as a threat to maternal and infant health, national registries of pregnant women with covid-19 have been established in many parts of the world -these include the priority study in the united states, ukss registry in great britain, nethoss in netherlands, itoss in italy, chopan in australia, and others. data obtained from these registries will be of immense significance in further understanding the effects of sars-cov-2 infection during pregnancy and its effects on clinical outcomes of mothers and their infants. the author has no potential conflicts to disclose. emerging infections and pregnancy pregnancy and susceptibility to infectious diseases maternal and infant death and the rvsv-zebov vaccine through three recent ebola virus epidemics -west africa, drc équateur and dr.c kivu: four years of excluding pregnant and lactating women and their infants from immunization viral infection, proliferation and hyperplasia of hofbauer cells and absence of inflammation characterize the placental pathology of fetuses with congenital zika virus infection an analysis of 38 pregnant women with covid-19, their newborn infants, and maternal-fetal transmission of sars-cov-2: maternal coronavirus infections and pregnancy outcomes clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records covid-19 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies maternal and neonatal outcomes of pregnant women with covid-19 pneumonia: a case-control study infections in pregnancy with covid-19 and other respiratory rna virus diseases are rarely, if ever, transmitted to the fetus: experiences with coronaviruses, hpiv, hmpv, rsv, and influenza clinical manifestations and outcome of sars-cov-2 infection during pregnancy the babies were delivered. no one realized the mothers had the virus. the new york times two cases of covid-19 related cardiomyopathy in pregnancy maternal death due to covid-19 disease mortality of a pregnant patient diagnosed with covid-19: a case report with clinical, radiological, and histopathological findings a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-296631-43z3ee8m authors: de feria, alejandro; ortega-legaspi, juan m title: ace inhibitors/arb use and covid-19. time to change practice or keep gathering data? date: 2020-07-04 journal: clin infect dis doi: 10.1093/cid/ciaa819 sha: doc_id: 296631 cord_uid: 43z3ee8m nan a c c e p t e d m a n u s c r i p t since the emergence of the coronavirus disease 2019 (covid-19) pandemic, there has been intensive research dedicated to elucidating the pathogenesis of the virus as well as the risk factors that portend poor outcomes in this disease. case series from early on in the pandemic showed that several risk factors including diabetes, coronary artery disease, and hypertension were more common in those suffering with severe forms of covid-19 disease [1] . it is now known that sars-cov-2, the coronavirus that causes covid-19, enters human cells via binding of the viral spike protein to the angiotensin-converting enzyme 2 (ace2) [2] . this mechanism of entry, in combination with the findings of the previously mentioned risk factors, raised concerns that angiotensin-converting enzyme inhibitors (acei) and angiotensin receptor blockers (arbs) could increase both the susceptibility and severity of sars cov-2 infection. soon thereafter, members of both the healthcare community and the medical press began to call for the discontinuation of these drug classes both preemptively and in the setting of covid-19 infection. while this mechanism of increased susceptibility to covid-19 was biologically plausible, many jumped to conclusions about discontinuing these agents prior to the performance of rigorous human studies. multiple studies since the beginning of the outbreak have returned with conflicting results of the effects of acei or arb use on outcomes with covid-19. this particular conundrum has come to the light of science in a short period of time with significant implications for public health. inevitably, the studies have relied on available data that has been retrospective and with marked limitations. a large study out of new york city of over 5,800 patients showed no positive association of acei and arbs for either a positive test result or severe illness [3] . an international, multicenter study which included electronic records from 169 hospitals in 11 countries on three continents again confirmed that advanced age (>65), heart failure, coronary disease, and hypertension (among other factors) increased risk for in hospital mortality with covid-19, but acei/arb therapy showed no harm [4] . it is important to note, however, that the aforementioned study was recently retracted due to concerns about the quality of the data. in contrast to these findings, early studies out of china suggested that arb therapy may improve clinical outcomes in covid-19 infection [5, 6] . a separate study out of the united kingdom also suggested that there may be a trend towards beneficial effects of acei/arb therapy [7] . a c c e p t e d m a n u s c r i p t the current study is unique in that it focuses on patients with "severe covid" disease and found that chronic ace inhibitor and arb use was associated with an increased risk of acute kidney injury, as well as a signal for a dosage effect. the authors also showed a potential interaction between acei/arb use with the occurrence of acute respiratory failure. while this was a well-designed retrospective cohort study, it faces all the limitations that challenged the previous observational studies that were mentioned. also, the study was limited by a relatively small sample size, and this may in part be due to the focus on the "severe covid" disease population. nevertheless, the study adds high-quality retrospective data and, importantly, provides an elegant analysis that identified potential groups of patients who may be at higher risk of poor outcomes in the setting of covid-19. it is evident that randomized controlled trials must be conducted before we can establish a cause-and-effect relationship. at this relatively early stage of the pandemic, the accumulation of data, though substantial, has yet to change practice recommendations [8, 9] . one might conclude that the currently limited knowledge in the matter has provided more questions than answers. nevertheless, studies like the one commented here are worthy investments as they enhance medical understanding of the disease which may impact clinical decision-making in the near future. neither author has any potential conflicts to disclose. clinical characteristics of coronavirus disease 2019 in china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor renin-angiotensin-aldosterone system inhibitors and risk of covid-19 cardiovascular disease, drug therapy, and mortality in covid-19 anti-hypertensive angiotensin ii receptor blockers associated to mitigation of disease severity in elderly covid-19 patients renin-angiotensin system inhibitors improve the clinical outcomes of covid-19 patients with hypertension ace -inhibitors and angiotensin-2 receptor blockers are not associated with severe sars-covid19 infection in a multi-site uk acute hospital trust addresses concerns re: using raas antagonists in covid-19. available at: http%3a%2f%2fwww.acc.org%2flatest-in-cardiology%2farticles%2f2020%2f03%2f17%2f08%2f59%2fhfsa-acc-aha-statement-addressesconcerns-re-using-raas-antagonists-in-covid-19 position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-330214-28ah3nig authors: zhao, jiao; yang, yan; huang, hanping; li, dong; gu, dongfeng; lu, xiangfeng; zhang, zheng; liu, lei; liu, ting; liu, yukun; he, yunjiao; sun, bin; wei, meilan; yang, guangyu; wang, xinghuan; zhang, li; zhou, xiaoyang; xing, mingzhao; wang, peng george title: relationship between the abo blood group and the covid-19 susceptibility date: 2020-08-04 journal: clin infect dis doi: 10.1093/cid/ciaa1150 sha: doc_id: 330214 cord_uid: 28ah3nig to explore any relationship between the abo blood group and the covid-19 susceptibility, we compared abo blood group distributions in 2,173 covid-19 patients with local control populations, and found that blood group a was associated with an increased risk of infection, whereas group o was associated with a decreased risk. the novel coronavirus sars-cov-2, causing the new infectious disease covid-19, has spread widely around the world. current clinical observation suggest that people's age and gender are two risk factors in the susceptibility to covid-19 1 . older people and men are more susceptible to infection and development of more severe disease. however, no biological markers have been identified to predict the susceptibility to covid-19 so far. landsteiner's abo blood types are carbohydrate epitopes that are present on the surface of human cells. the antigenic determinants of a and b blood groups are trisaccharide moieties galnac1-3-(fuc1,2)-gal-and gal1-3-(fuc1,2)-gal-, while o blood group antigen is fuc1,2-gal-. while blood types are genetically inherited, the environment factors can potentially influence which blood types in a population will be passed on more frequently to the next generation. susceptibility of viral infection has been found to be related to abo blood group. for example, norwalk virus and hepatitis b have clear blood group susceptibility 2,3 . it was also reported that blood group o individuals were less likely to become infected by sars coronavirus 4 . here, we investigated the relationship between the abo blood type and the susceptibility to covid-19 in patients from three hospitals in wuhan and shenzhen, china. non-covid-19 people from shenzhen city were used as comparison controls for the wuhan and shenzhen patients with covid-19, respectively 5-6 . statistical analyses were performed using 2-tailed χ 2 . data from different hospitals were meta-analyzed using random effects models, with calculation of odds ratio (or) and 95% confidence interval (ci). statistical analyses were performed using spss software (version 16.0) and stata software (version 13). the abo blood group in 3,694 people in wuhan displayed a percentage distribution of 32.2%, 24.9%, 9.1% and 33.8% for a, b, ab and o, respectively, while the 1,775 patients with covid-19 from wuhan jinyintan hospital showed an abo distribution of 37.8%, 26.4%, 10 .0% and 25.8% for a, b, ab and o, respectively. the proportion of blood group a among patients with covid-19 was significantly higher than that among the control group, being a c c e p t e d m a n u s c r i p t 6 37.8% in the former vs 32.2% in the later (p < 0.001). the proportion of blood group o in patients with covid-19 was significantly lower than that in control group, being 25.80% in the former vs 33.84% in the later (p < 0.001, table 1 ). these results showed associations between abo blood groups and covid-19 susceptibility. the covid-19 risk significantly increased for blood group a ( figure 1 shows the estimates of ors of the risk of abo blood groups for covid-19 on the pooled data from the three hospitals by random effects models. again, the results showed that blood group a was associated with a significantly higher risk for covid-19 (or 1.21; 95% ci 1.02~1.43, p = 0.027) compared with non-a blood groups, whereas blood group o was associated with a significantly lower risk for the infection (or 0.67; 95% ci 0.60~0.75, p < 0.001) compared with non-o blood groups. compared with other abo blood groups, ab blood group (or 1.48, 95% ci 0.97~2.24) and b blood group (or 1.09, 95% ci 0.98~1.22) seemed to have a relatively higher risk of infection, although the associations did not reach statistical significance. in this study, we found that abo blood groups displayed different association risks for the infection with sars-cov-2 resulting in covid-19. specifically, blood group a was associated with an increased risk whereas blood group o was associated with a decreased there may also be other mechanisms underlying the abo blood group-differentiated susceptibility for covid-19 that require further studies to elucidate. in the 285 patients from shenzhen, we also found that blood group ab had an increased risk of infection. this result needs to be confirmed given the small size of this cohort of patients. after submission of our study, another study by michael et al. using observational data on 1,559 sars-cov-2tested individuals came online, reporting that the odds for sars-cov-2 infection was significantly increased for blood group a and decreased for blood group o. this reproduced our findings of the associations between the abo blood group and the covid-19 status 12 . in summary, we report a link between covid-19 susceptibility and the abo blood group. specifically, people with blood group a have a higher risk whereas people with blood a c c e p t e d m a n u s c r i p t 15 figure 1 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study association of abo and rh blood group types to hepatitis b, hepatitis c, hiv and syphilis infection, a five year' experience in healthy blood donors in a tertiary care hospital human susceptibility and resistance to norwalk virus infection abo blood group and susceptibility to severe acute respiratory syndrome distribution of abo and rhd blood group among healthy han population in wuhan distribution of abo and rh (d) blood group and qualty analysis inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus. j nature the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars testing the association between blood type and covid-19 infection, intubation, and death. medrxiv a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 12 key: cord-331930-w2055c42 authors: tso, eugene y. k.; tsang, owen t. y.; choi, k. w.; wong, t. y.; so, m. k.; leung, w. s.; lai, j. y.; ng, t. k.; lai, thomas s. t. title: persistence of physical symptoms in and abnormal laboratory findings for survivors of severe acute respiratory syndrome date: 2004-05-01 journal: clin infect dis doi: 10.1086/383580 sha: doc_id: 331930 cord_uid: w2055c42 nan sir-we performed a cross-sectional study to assess the physical symptoms in and abnormal laboratory findings for survivors of severe acute respiratory syndrome (sars) at their first follow-up visit after discharge from princess margaret hospital (hong kong, china). sixty-two patients who experienced the onset of sars symptoms during the period from 18 march 18 2003 through 30 march 2003 were recruited. all patients had pneumonia and positive sars-associated coronavirus (sars-cov) seroconversion. the mean age (‫ע‬sd) was 37.07 ‫ע‬ 12.96 years, the ratio of male subjects to female subjects was 0.82, and the intubation rate was 9.6%. in this cohort, 90.3% of patients received treatment with ribavirin and corticosteroids [1] . the median interval (‫ע‬sd) between the onset of sars symptoms and the first follow-up visit was weeks. 6.59 ‫ע‬ 1.07 symptoms reported at the first followup visit included palpitation (45.1% of patients), exertional dyspnea (41.9%), malaise (40.3%), easy forgetfulness (30.6%), chest discomfort (22.5%), hand tremor (21%), dizziness (17.7%), depression (16.1%), myalgia (12.9%), headache (9.6%), diarrhoea (8.1%), cough (8.1%), insomnia (6.5%), and hair loss over the scalp (3.2%). no patient reported sputum production. patients described palpitation as a paroxysmal, fast heart beat or extra heart beat sensation. a sinus tachycardia with resting heart rate of 100-110 beats/ min was identified in 18% of patients complaining of palpitation. laboratory findings included the following mean values (‫ע‬sd): hemoglobin of patients. these findings included patchy shadows, linear atelectasis, ground glass appearance, reticular marking, and streaky opacities. there was no significant difference in the rate of exertional dyspnea between patients with and patients without abnormal chest radiograph findings ( ). for all patients, pcr of urine, p p .51 nasal, and throat swab samples was negative for sars-cov rna. however, for 1 female patient, pcr of a stool sample obtained 35 days after the onset of sars symptoms was positive for sars-cov rna. no person who had close contact with that patient after she was discharged from the hospital contracted sars. from what we have learned, some sars survivors still had physical symptoms up to 6 weeks after the onset of sars symptoms, although their complete blood counts, the results of their liver and renal function tests, and their erythrocyte sedimentation rates were largely normalized. the finding of abnormal lactate dehydrogenase levels may imply that patients still had not fully recovered from sarsrelated tissue damage at the first followup visit. we should not overlook the effect of therapy with ribavirin and corticosteroids, which might have contributed to the symptoms and to the abnormal laboratory values. physicians providing care to pa-tients with sars during the convalescent period should be aware of the possibility of such abnormal findings. outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong key: cord-325136-oyizfh2z authors: pham, quang thai; rabaa, maia a; duong, huy luong; dang, quang tan; tran, dai quang; quach, ha-linh; hoang, ngoc-anh thi; phung, cong dinh; ngu, duy nghia; tran, anh tu; la, ngoc quang; tran, my phuc; vinh, chau; nguyen, cong khanh; dang, duc anh; tran, nhu duong; thwaites, guy; van doorn, h rogier; choisy, marc title: the first 100 days of sars-cov-2 control in vietnam date: 2020-08-01 journal: clin infect dis doi: 10.1093/cid/ciaa1130 sha: doc_id: 325136 cord_uid: oyizfh2z background: one hundred days after sars-cov-2 was first reported in vietnam on january 23(rd), 270 cases were confirmed, with no deaths. we describe the control measures used by the government and their relationship with imported and domestically-acquired case numbers, with the aim of identifying the measures associated with successful sars-cov-2 control. methods: clinical and demographic data on the first 270 sars-cov-2 infected cases and the timing and nature of government control measures, including numbers of tests and quarantined individuals, were analysed. apple and google mobility data provided proxies for population movement. serial intervals were calculated from 33 infector-infectee pairs and used to estimate the proportion of pre-symptomatic transmission events and time-varying reproduction numbers. results: a national lockdown was implemented between april 1(st) and 22(nd). around 200 000 people were quarantined and 266 122 rt-pcr tests conducted. population mobility decreased progressively before lockdown. 60% (163/270) of cases were imported; 43% (89/208) of resolved infections remained asymptomatic for the duration of infection. the serial interval was 3·24 days, and 27·5% (95% confidence interval, 15·7%-40·0%) of transmissions occurred pre-symptomatically. limited transmission amounted to a maximum reproduction number of 1·15 (95% confidence interval, 0·37-2·36). no community transmission has been detected since april 15(th). conclusions: vietnam has controlled sars-cov-2 spread through the early introduction of mass communication, meticulous contact-tracing with strict quarantine, and international travel restrictions. the value of these interventions is supported by the high proportion of asymptomatic and imported cases, and evidence for substantial pre-symptomatic transmission. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan city, hubei province, china, in late 2019 [1] . on january 30 th , the who declared the outbreak a 'public health emergency of international concern', and on march 11 th a global pandemic. by may 1 st 2020, the virus had infected more than 3 million people and killed over 200 000. sars-cov-2 is antigenically different from known human and zoonotic coronaviruses and there is no known pre-existing population immunity [2] . it is highly transmissible through respiratory secretions expelled from an infected person, with a basic reproduction number (r 0 ) estimated between 2 and 3 in the absence of control measures [3] [4] [5] [6] . many infections are asymptomatic [7] , while others lead to symptoms of coronavirus disease (covid-19) of varying severity [5] . analyses of serial intervals suggest that contagiousness can occur both before and after the onset of symptoms as well as in those who never develop symptoms [8] . the subsequent exponential rise in infections has threatened to overwhelm even the world's best developed health systems and cause major loss of life. methods to control the virus and reduce the impact of covid-19 have thus become a global priority. the preparedness, timing, and nature of the response to sars-cov-2 have varied substantially between countries. many affected countries have resorted to extreme social distancing measures through so-called 'lockdowns', where populations isolate themselves within their homes, reducing all but essential contact with others. as first observed in hubei province in china, and subsequently in other countries, these measures slow transmission and reduce disease incidence [9] [10] [11] , but at significant social and economic cost. however, 'lockdowns' represent a combination of potentially independent interventions (for example, closing schools and universities, suspending public transport, banning public gatherings, closing non-essential businesses), the effects of which in isolation are uncertain. determining their relative contributions to sars-cov-2 control is critical to understanding how they might be safely and incrementally lifted, or partially reinstated. such a c c e p t e d m a n u s c r i p t 6 information may be acquired from studying the measures employed by countries that have so far controlled the virus. vietnam is a low-middle income country that shares borders with china, the lao people's democratic republic, and cambodia. it is the 15 th most populous country on earth, with 97.3 million people, and it was one of the first countries affected by sars-cov-2, recording its first case on january 23 rd 2020. yet, by may 1 st , 270 cases were confirmed, with no deaths [12] . here we present a descriptive study that aims to characterize and quantify measures used for sars-cov-2 and characteristics of the cases in vietnam during the first 100 days of the epidemic. our aim was to identify the measures most closely associated with successful sars-cov-2 control. clinical, epidemiological and policy data were provided by vietnam's national steering committee for covid-19 response. data from 270 sars-cov-2-confirmed cases to may 1 st 2020 included their age, gender, nationality, dates of symptom onset (if any), entry to the country and quarantine (if any), hospital admission and discharge, and the results of rt-pcr tests. imported cases were distinguished from those acquired domestically, with information on quarantine at or after entry to the country. imported cases were denoted g0; and among domestically-acquired infections, those acquired directly from g0 cases were denoted as g1, others were denoted g2+. intervention data consisted of daily time-series of the numbers in quarantine and rt-pcr tests performed. daily reports from the ministry of health and vietnam's national steering committee for covid-19 response listed key milestones in national sars-cov-2 control measures. apple mobility data [13] and google community mobility data [14] provided proxies of population movements, with additional information provided in the supplementary appendix. serial intervals were calculated from dates of symptoms onset of infector-infectee pairs identified by contact tracing and fitted to a normal distribution by maximum likelihood [8] . the estimated a c c e p t e d m a n u s c r i p t 7 distribution parameters (mean and standard deviation, together with their confidence intervals and variance-covariance matrix) were used to estimate the proportion of pre-symptomatic transmissions and three time-varying reproduction numbers [15] : between g0 and g1 (step 1), between g1 and g2+ (step 2), and between g0, g1, and g2+ combined (step 1 and 2 combined)(further details in the supplementary appendix). we used a logistic regression to investigate the link between the proportion of asymptomatic infections and age, gender, nationality (vietnamese versus non-vietnamese), and imported versus domestically-acquired infection. we used a gamma regression to investigate the link between the duration of hospitalisation and the same variables listed above, plus symptomatic versus asymptomatic. to correct for potential confounding effects between the explanatory variables, we used type-ii likelihood ratio tests [16] . all analyses were done with r 4.0.0 [17] on january 10 th , before the first case was confirmed in vietnam, the vietnam government reinforced temperature and health status screening at border gates for passengers arriving from wuhan, tracing and quarantining of suspected cases and their contacts, monitoring of suspected cases of respiratory infections in hospitals and the community, and initiated mass communication to the public on preventive measures (hand washing, contact avoidance and mask wearing). the epidemic timeline for vietnam, including the numbers quarantined and hospitalised, tests performed, cases confirmed, population movements, and the timing and nature of major government-led control measures are summarised in figure 1 . the control measures are summarised in table 1 and table s1 . to date, two waves of transmission have occurred: the first a c c e p t e d m a n u s c r i p t 8 began on january 23 rd and resulted in 16 cases (9 imported, 7 acquired in-country), and the second on march 6 th , leading to 254 cases (154 imported, 100 acquired in-country). the first confirmed cases of sars-cov-2 infection presented in hanoi and ho chi minh city during the lunar new year holiday (23-29 th january). cases were travellers from wuhan city or their contacts, and were identified by the public health laboratory network using improvised molecular diagnostics, including agnostic sequencing, prior to implementation of the who-approved assays [22] . amongst the cases were the first confirmed human-to-human transmissions outside of china [23] . entry of airline passengers into vietnam from wuhan city and elsewhere in china was monitored and progressively limited ( table 1) after further measures to prevent entry of infected international travellers (table 1) forty-three percent (89/208) of discharged cases never developed symptoms, and this was not significantly associated with age, gender, nationality, or origin of infection (imported or domestically-acquired). among all the symptomatic cases, 25·3% (38/150) developed symptoms in a government quarantine facility. among the imported cases who developed symptoms, 73·9% (68/92) did so after entry to the country ( figure 3a , see table s4 for the numbers of symptomatic in imported and non-imported cases). the median age of symptomatic and asymptomatic cases was 30 (iqr 24-49) and 31 (iqr 23-45), respectively (no significant effect of age on the probability to develop symptoms, figure 3c ). among the 150 with symptoms, 21 (14·0%) developed severe disease, of whom five required mechanical ventilation and two received extra-corporeal membrane oxygenation. no fatalities were recorded. the duration of hospitalisation was significantly shorter (p<0·0001) for asymptomatic (17 days, iqr 13-22) than for symptomatic cases (19 days, iqr [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . while gender, nationality, and origin of infection did not have any significant effect, the duration of hospitalisation of symptomatic cases increased with age (with a discharge rate decreasing by 1·24% for every year older, p = 0·0060) (figure 3b) . a c c e p t e d m a n u s c r i p t 11 from 33 infector-infectee pairs, the mean serial interval was estimated to be 3·24 days (95% confidence interval (ci), 1·38-5·10 days) with a standard deviation of the distribution of 5·46 days (95% ci, 4·14-6·78 days). an estimated 27·5% (95% ci, 15·7%-40·0%) of the distribution was below zero, suggesting these transmissions occurred prior to the onset of symptoms in the infector ( figure 3d ). from the (non-quarantined) imported cases (g0) and onward infected cases (g1 and g2+), we calculated the effective reproductive number r by date (figure 1f-h) . limited transmission amounted to a maximum r of 1·15 (95% ci, 0·37-2·36). r rarely exceeded 1 and a decrease of r is seen as more mitigating measures were implemented from the end of march before the nationwide lockdown. when analysing r from g0 to g1 (step 1) and from g1 to g2+ (step 2) separately, we found that r was drastically decreased for step 1 simultaneously with suspension of all international travel (march 18 th ), while for step 2 transmission continues with r slightly above 1 despite intense contact tracing and quarantine. only during the nationwide lockdown r was reduced to less than 1 ( figure 1f and g) . on january 23 rd 2020, vietnam was one of the first countries to report sars-cov-2 infection and the first to report human-to-human transmission outside of china [23] . yet 100 days later, it confirmed just 270 cases despite extensive testing, with no community transmission since april 15 th . in the three weeks prior to may 1 st , there were only two imported cases and no reported cases elsewhere in the country. the nature, timing, and success of the control measures introduced may have relevance to other countries seeking to control sars-cov-2 transmission. vietnam has experience in responding to emerging infectious diseases. in the last 20 years, it has confronted outbreaks of sars [26] , avian and pandemic influenza [27, 28] , hand-foot-and-mouth disease [29] , measles [30] , and dengue [31] . its outbreak responses are coordinated by the ministry days of an epidemic that is likely to continue for many months. it is therefore impossible to conclude definitively which of these control measures have resulted in the current control of sars-cov-2 in vietnam and whether they will continue to work in the future. there are, however, two distinctive features of vietnam's response. first, the government acted quickly, educating and engaging the public, placing restrictions on international flights, closing schools and universities, and instituting exhaustive case-contact tracing from late january, well before these measures were advised by who. second, they placed the identification, serial testing, and minimum 14-day isolation of all direct contacts of cases, regardless of symptom development, at the heart of the response. our findings suggest the latter measure was likely to be especially effective given nearly half of those infected did not develop symptoms. table s1 ). the colours shown in the phase column indicate the intensity of control measures taken over different periods (white, initial; light yellow, early; light orange, intermediate; orange, pre-epidemic; brown, epidemic/lockdown; dark orange, post-lockdown), and correspond to those used in figure 1 and table s1 . event january 3 strengthening of border control measures announced by the government table s3 . panel d: distribution of serial intervals for 33 infector-infectee pairs together with a normal distribution fitted to it. the shaded area shows the 95% confidence interval. the vertical black line shows the estimate of the mean serial interval, together with its 95% confidence interval (dashed vertical lines). the proportion of the distribution to the left of the red line is a proxy for the proportion of infections that occur before the onset of symptoms. a novel coronavirus from patients with pneumonia in china novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 clinical characteristics of coronavirus disease 2019 (covid-19) in china: a systematic review and meta-analysis estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship serial interval of covid-19 among publicly reported confirmed cases covid-19: a&e visits in england fall by 25% in week after lockdown the positive impact of lockdown in wuhan on containing the covid-19 outbreak in china the global community needs to swiftly ramp up the response to contain covid-19 covid-19 -bộ y tế -trang tin về dịch bệnh viêm đường hô hấp cấp covid-19 apple covid-19 reports on mobility trends google covid-19 community mobility reports improved inference of time-varying reproduction numbers during infectious disease outbreaks r: a language and environment for statistical computing estimate time varying reproduction numbers from epidemic curves fitdistrplus: an r package for fitting distributions epidemic curves made easy using the r package incidence multivariate normal and t distributions. 2020. available at severe acute respiratory syndrome coronavirus 2 shedding by travelers importation and human-to-human transmission of a novel coronavirus in vietnam outbreak investigation for covid-19 in northern vietnam the first infant case of covid-19 acquired from a secondary transmission in vietnam clinical description of a completed outbreak of sars in vietnam avian influenza a (h5n1) in 10 patients in vietnam a clinical virological and epidemiological analysis enterovirus 71-associated hand, foot, and mouth disease, southern vietnam sero-prevalence surveillance to predict vaccine-preventable disease outbreaks; a lesson from the 2014 measles epidemic in northern vietnam the estimates of the health and economic burden of dengue in vietnam sustainable model for public health emergency operations centers for global settings key: cord-307342-3gkiukh4 authors: clark, eva; chiao, elizabeth y; amirian, e susan title: why contact tracing efforts have failed to curb covid-19 transmission in much of the u.s date: 2020-08-06 journal: clin infect dis doi: 10.1093/cid/ciaa1155 sha: doc_id: 307342 cord_uid: 3gkiukh4 by late april 2020, public discourse in the u.s. had shifted toward the idea of using more targeted case-based mitigation tactics (e.g., contact tracing) to combat covid-19 transmission while allowing for the safe “re-opening” of society, in an effort to reduce the social, economic, and political ramifications associated with stricter approaches. expanded tracing-testing efforts were touted as a key solution that would allow for a precision approach, thus preventing economies from having to shut down again. however, it is now clear that many regions of the u.s. were unable to mount robust enough testing-tracing programs to prevent major resurgences of disease. this viewpoint offers a discussion of why testing-tracing efforts failed to sufficiently mitigate covid-19 across much of the nation, with the hope that such deliberation will help the u.s. public health community better plan for the future. m a n u s c r i p t 4 many countries that have successfully mitigated the covid-19 pandemic to date did so via stringent measures to limit personal movement and abate public interactions [1, 2] , but these approaches are unlikely to be acceptable from an economic, legal, or sociocultural perspective in the united states. partly for this reason, our nation rushed to espouse the idea of targeted, case-based covid-19 management [3] [4] [5] [6] , focusing on expanded testing and contact tracing, while disregarding several major obstacles that set us apart from countries that succeeded in mounting a timely, targeted response. indeed, expansive testing-tracing programs have largely succeeded in curtailing community spread in certain countries, most notably south korea, which is commonly referred to as the archetype for controlling covid-19 while avoiding strict lockdowns [1, 6, 7] . arguments were made that the initial set of "stay at home" orders implemented in many regions of the u.s. were intended to prevent hospital overflow and to essentially buy time to plan out a more precise strategy that would have less impact on daily life [8] , taking note of what worked best in other parts of the world that preceded us in the pandemic curve. here, we discuss some urgent public health considerations related to why heavy reliance on expanded testing-tracing efforts were largely unsuccessfully in many states in the u.s. that are now experiencing record-breaking surges in case counts. from the beginning of the pandemic, there has been a noticeable lack of unified national leadership and coordination, which has resulted in both the absence of a robust plan (or common goals) for local and state health departments and the dissemination of confusing mixed messages to the lay public [9, 10] . for the most part, the u.s. centers for disease control and prevention (cdc) has remained uncharacteristically silent during this national crisis [9] . in may 2020, at a time when many jurisdictions had already started relaxing their "stay at home" mandates, the cdc released a watered-down version of the original guidance a c c e p t e d m a n u s c r i p t 5 documents censored by the trump administration [11, 12] . the resulting guidance allowed for the potential re-opening of schools, restaurants, bars, and other institutions that were closed in many jurisdictions earlier in the pandemic, with limited specific direction for addressing sustained community transmission [11, 12] . notably, joint white house and cdc benchmarks for re-opening (described elsewhere [13]) were flouted by several states, including texas, georgia, and florida [14] . few states had come close to meeting even just one of the cdc benchmarks, when they started reopening under the impression that voluntary social distancing and expanded testing-tracing would be sufficient to curb the epidemic in regions with seemingly flattening rates of ongoing transmission [15] [16] [17] . had states been encouraged to heed cdc benchmarks, it may have been possible to avoid the major surges now being observed in these states [16, 18] . in addition to the lack of coordinated public health leadership, it has been surprising that despite being a resource-rich nation, the u.s. still struggles to achieve adequate and consistent testing rates [19, 20] . in areas experiencing surges, there have been reports of long lines, test shortages, and over a week long turnaround times, despite the fact that the past 5 months since the start of the epidemic should have provided ample time to increase supply chains for testing materials [20, 21] . it is a fundamental concept that health departments cannot trace cases that remain undetected. yet, even prior to the current surges, many putative cases, even those who were symptomatic, were unable to obtain timely sars-cov-2 testing and results, and very few jurisdictions had implemented widespread freely-available public surveillance testing [22, 23] . this ineptitude in deploying a cohesive testing strategy stems from many organizational and national leadership barriers, including an underfunded public health outpatient testing infrastructure, regional insufficiencies in testing supplies/reagents, and a lack of national guidance regarding the best strategy for a c c e p t e d m a n u s c r i p t 6 implementing surveillance testing [24] . reliable, widespread, no-cost surveillance testing should have been available nationwide early in the u.s. epidemic, as it is the basis on which the other tools in the public health toolbox are predicated. had widespread testing been available while community spread was still relatively low, contact tracing endeavors may have been able to quickly identify and eradicate hotspots and transmission chains within affected communities. however, that window of opportunity has passed and sustained transmission has led to rapidly growing caseloads and inability to keep up with contact tracing in many jurisdictions, despite some efforts to scale capacity [15, 17, [25] [26] [27] [28] . currently, the goal of contract tracing is still to identify the maximal number of sars-cov-2 infected and exposed individuals in order to enable transmission mitigation through isolation and intervention [29] . however, contact tracing is usually most successful during troughs of the epidemic curve, when such efforts are more manageable. in these situations, theoretically, if nearly every case can be isolated quickly, and the majority of their contacts quarantined, then the local epidemic could be quelled enough by these targeted tactics to permit loosening of more stringent public health measures. however, loosening stricter social isolation measures before adopting the infrastructure prerequisite to allow for timely and thorough contact tracing is generally unadvisable, especially in the context of our decentralized and fragmented public health and healthcare systems. in states where the virus is currently surging, implementation and sustainable management of testing-tracing efforts became virtually unfeasible as transmission increased, and capacity was exceeded in some jurisdictions [27, 28] . indeed, cdc guidance states that contact tracing is not usually recommended in communities with "sustained ongoing transmission" [29] ; however, "sustained ongoing transmission" has not been clearly defined for covid-19. this confusion may have contributed to the development of ineffective policies in states that have now a c c e p t e d m a n u s c r i p t 7 experienced dramatic increases in case counts and hospitalizations, like florida and texas [16, 18] , both of which were depending on attempts to conduct contact tracing in the midst of high levels of sustained ongoing transmission [26, 28, 30] . comprehensive testing and contact tracing plans require a high level of forethought, coordination, communication, and social acceptability to be effectively executed [7] . this is partly why many countries that have more synchronized public health systems with legal authority to provide strong oversight have generally fared better [1, 31, 32] . robust plans, backed by considerable resources (i.e., financial, personnel, legal, and technological), combined with high adherence to physical distancing and face covering recommendations, have been instrumental to covid-19 mitigation in many countries, including ones with regions that have very high population densities [1, 7, 32] . for example, south korea, which had prior experiences with both sars and mers, had modified legislation after prior outbreaks to allow for prompt responses to epidemics [7] . as a result, south korea was able to integrate the following rich information sources into their contact tracing efforts: patient interview data, medical records data, gps data from mobile phones, cctv footage, and credit card transaction data. they also published the pre-diagnosis movements of confirmed covid-19 cases [7] . similarly, taiwan merged data between health insurance records, medical records data, travel history, and data from both an app and a toll-free hotline set up for the public to report suspected cases [7, 31, 32] . such methods are likely to be considered quite invasive, and therefore, neither legally nor culturally acceptable in the u.s. [33, 34] , particularly during the current climate of civil unrest and the expanding backlash against public health measures that may be due partly to the politicization of certain recommendations [10, 35] . there are some comparatively less intrusive voluntary technologies that have been used to supplement contact tracing and augment local public health efforts in some countries a c c e p t e d m a n u s c r i p t 8 [7, 36] . generally, with voluntary technologies, users agree to data collection and sharing for contact tracing purposes, and the data are deleted once obsolete. in europe, efforts are underway to develop and utilize general data protection regulation (gdpr) compliant phone apps [37] . a voluntary app was used in iceland to help mitigate covid-19 spread, but successful solutions from small countries like iceland may not be generalizable to other regions for many reasons beyond regulatory and sociocultural differences [7] . it is not possible to infer exactly how effective voluntary technology use will be in the u.s., especially without the aid of other major preventive tactics, given high levels of community spread in some parts of the country [18, 38] . attempts to use opt-in mobile phone apps are ongoing in various parts of the u.s. (e.g., massachusetts, california, san francisco) [36, 39] . evaluating the success of such programs over time may be helpful in planning for future surges of covid-19. it is clear that public buy-in and engagement are crucial to ensuring cooperation with, adherence to, and sustainability of expanded testing-tracing programs [33, 36] . the u.s. has substantial regulations that preclude enforcement of compliance with contact tracing [34] . this implies that the public's participation will be voluntary [34, 36] , and therefore, less likely to provide accurate and comprehensive information, limiting the effectiveness of these endeavors. besides legal hurdles related to civil liberties, there are also relevant ethical considerations about access to and use of data about people's contacts and whereabouts that need to be weighed [36, 40] . vulnerable individuals, such as immigrants and victims of crime or domestic violence, may not be comfortable with sharing such information, even with health departments [40, 41] . because some corporations have decided to conduct testing and tracing of their employees [42] , individuals may be concerned that hiring or termination decisions will be based on test results. therefore, public messaging about expanded testingtracing must clarify how the data can legally be used and how they will be managed and a c c e p t e d m a n u s c r i p t 9 protected, especially if private companies will be contracted to aid with data collection efforts [40] . potential for misuse by law enforcement, immigration enforcement, and for-profit companies should be addressed unequivocally [41] . many of these urgent considerations necessitate national-level guidance and leadership. at present, most local health departments are left to manage the public health concerns of their own jurisdictions with little support, and most lack the resources needed to adequately fulfill this responsibility [7, 43] . despite the fact that there is a pandemic roughly every decade, contact tracing systems run by health departments are generally not designed to handle rapidly transmissible, pandemic-scale diseases. taking over a week to conduct contract tracing may be effective for some communicable illnesses (e.g., syphilis, according to the association of state and territorial health officials (astho), the enormous scope of conducting contact tracing for sars-cov-2 is most closely exemplified by the u.s. response to the west african ebola outbreak, which was the largest contact tracing endeavor ever implemented here [7] . during this response, 30,000 individuals were actively monitored, but there were no reported cases [7, 45] -by contrast, almost 4 million covid-19 cases already exist on u.s. soil to date [16] . even for ebola contact tracing, there were significant operational barriers including: resource limitations, barriers in coordination and communication between jurisdictions, challenges in quarantine enforcement, and difficulties related to provision of isolation housing [7] . covid-19 has logarithmically amplified these obstacles. the size of the public health workforce required to adequately implement sar-cov-2 testing-tracing efforts depends upon many factors, such as the catchment area population size and the true incidence and prevalence. larger numbers of staff may be necessary as social distancing measures are loosened (or public adherence decreases) and case counts a c c e p t e d m a n u s c r i p t 10 increase, or if technologic tools are not used for augmentation. smaller numbers of staff would likely be necessary if local, state, and national public health agencies were able to communicate and coordinate effectively. creation of a national contact tracing system could eliminate geographic restrictions for hiring and would increase procedural standardization. astho and other organizations have been advocating for a coordinated, national approach for expansion of contact tracing, and requests were made for support from the federal government to acquire an additional 100,000-300,000 contact tracers [7, 43] . such a national resource would reduce the burden on the current public health workforce and ultimately, could also set the stage for a more strategic and effective national system for responding to current and future pandemics. since the beginning of the covid-19 outbreak in the u.s., a paucity of timely, national guidance and strategic planning, in culmination with an overwhelmed public health system, has served as a substantial obstacle to rapid disease mitigation [9] . in just a few months, the covid-19 crisis has exposed the deficiencies in our public health infrastructure and has led us to mull over the palpable changes that would have prevented the current tugof-war between epidemiologic, political, and economic sacrifices. largely due to these deficits, we missed a pivotal opportunity to curtail the spread of this epidemic in much of the u.s. however, the course of an epidemic is dynamic, and if tough, decisive, and criticallyneeded policy decisions are made in the upcoming months to curb transmission, we may, once again find ourselves, in a relatively better position to consider effective strategies, though the disease may become endemic. at the very least, public health practitioners and scientists must acknowledge the complexities of real-world testing-tracing efforts, and promote new policies aimed at both mitigating sustained community transmission and bolstering contact tracing capacity in their jurisdictions. while this type of resource is worthy a c c e p t e d m a n u s c r i p t 11 of investment for the longer-term, if contact tracing is to be considered the principal solution across the nation under current circumstances, then indicators for when social distancing can be relaxed (or needs to be strengthened) should include actionable thresholds around local contact tracing capacity [46] . these thresholds are particularly relevant because of the sparse capacity that is the reality for many jurisdictions at this time [7, 28] . overall, this is a crucial moment for our public health system to reassess its unmet needs, to evaluate and address the reasons behind its shortcomings, and to cultivate change before public momentum fades and we fall back into a national complacency, abandoning the opportunity for re-hauling and re-imagining a politically-independent, well-resourced, and innovative public health system. dr. chiao reports grants from nih, outside the submitted work. all other authors have no relevant conflicts of interest. covid-19-we urgently need to start developing an exit strategy they've contained the coronavirus. here's how. the new york times reopening of america: more than half of states will lift coronavirus restrictions by the end of the week all 50 u.s. states have taken steps toward reopening in time for memorial day weekend. the washinton post very aggressive' contact tracing needed for u.s. to return to normal coronavirus contact tracing could stop covid-19 and reopen america a national plan to enable comprehensive covid-19 case finding and contact tracing in the social distancing could buy u.s. valuable time against coronavirus. the washington post reviving the us cdc complicating outbreak response, preparedness centers for disease control and prevention. child care programs during the covid-19 pandemic restaurants and bars during the covid-19 pandemic public health experts say many states are opening too soon to do so safely states moving forward with reopening are seeing increases in new coronavirus cases. the washington post covid-19 dashboard coronavirus cases spike across sun belt as economy lurches into motion. the new york times sun belt hospitals are feeling the strain from virus' surge -and bracing for worse. the washington post trump hopes for 2 million tests per week by end of may-the low end of experts' estimates of what's needed to reopen cities still lack testing capacity as cases surge, lines for coronavirus tests sometimes stretch miles in the summer heat. the washington post early detection of covid-19 through a citywide pandemic surveillance platform rapid sentinel surveillance for covid-19 thousands of coronavirus tests are going unused in us labs sun belt hospitals are feeling the strain from virus' surge -and bracing for worse texas is short of its contact tracing workforce goal by more than 1,000 people coronavirus contact tracing is 'not going well,' dr. fauci says, u.s. still needs more testing houston's surge of covid-19 cases overwhelms contact tracing efforts contact tracing: part of a multipronged approach to fight the covid-19 pandemic what florida contact tracing is like during the covid-19 pandemic. health news florida contact tracing assessment of covid-19 transmission dynamics in taiwan and risk at different exposure periods before and after symptom onset contact tracing, testing, and control of covid-19-learning from taiwan most americans are not willing or able to use an app tracking coronavirus infections. that's a problem for big tech's plan to slow the pandemic. the washington post covid-19 contact tracing we can live with: a roadmap and recommendations coronavirus recommendations ignored as case numbers rise. the washington post a scramble for virus apps that do not harm. the new york times review of mobile application technology to enhance contact tracing capacity for covid-19, 2020. 38. show evidence that apps for covid-19 contact-tracing are secure and effective covid-19: the us state copying a global health template for contact tracing success ethics of instantaneous contact tracing using mobile phone apps in the control of the covid-19 pandemic should immigration status information be included in a patient's health record? contact tracing poses 'pandora's box' for reopening businesses a c c e p t e d m a n u s c r i p t 12 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-321873-9l9zp6gm authors: zhou, bo; she, jianqing; wang, yadan; ma, xiancang title: the duration of viral shedding of discharged patients with severe covid-19 date: 2020-04-17 journal: clin infect dis doi: 10.1093/cid/ciaa451 sha: doc_id: 321873 cord_uid: 9l9zp6gm the 2019 coronavirus disease (covid-19) has drawn global intensive attention(1-3). most of studies paid attention to epidemiological, clinical, and radiological features of inpatients with covid-19(1-3). however, little studies have focused on clinical characteristics of discharged patients with severe covid-19, especially the duration of viral shedding. m a n u s c r i p t we enrolled 41 discharged patients with severe covid-19 on the 7 th floor ward in the west district, union hospital of tongji medical college from february 5 th to march 16 th , 2020. the patients were evaluated with real-time reverse transcriptase-polymerase chain reaction (rt-pcr) tests for covid-19 nucleic acid. the rt-pcr tests were performed on throat swabs following a previously described method 3 moreover, another study displayed that four patients with covid-19 who met the criteria for hospital discharge in china had positive rt-pcr test results 5 to 13 days later 6 , suggesting that recovered patients might still be virus carriers. thus, it is plausible that the patients with severe covid-19 might need longer duration of symptomatic and supportive treatments due to prolonged duration of viral shedding. additionally, there was no significant difference with viral shedding time and the total time from illness onset to discharge between male and female, and between <65 yrs and ≥65 yrs. based on the results, we did not observe shortening of viral shedding time in the above groups, so it implied that all patients with severe covid-19 should complete adequate course of treatment, regardless of sex and age. previously, increased age has been reported as an important independent predictor of mortality in covid-19 5 . the current study also found that the number of discharged patients with severe covid-19 in the group (<65 yrs) was more than in the group (≥65 yrs), indicating that the elder patients with severe covid-19 may have poor outcome, and we a c c e p t e d m a n u s c r i p t should pay attention to the patients with severe covid-19, especially ≥65 yrs. the study was limited to a small number of discharged patients with severe covid-19, not including mild or moderate infection, because we enrolled patients from the designated hospital for severe covid-19. besides, the estimated duration of viral shedding is limited by the frequency of respiratory specimen collection and relatively low positive rate of sars-cov-2 rna detection in throat swab. it is advisable to continue follow-up discharged patients even after discontinuing quarantine for further viral shedding information. in addition, further studies based on larger cohorts would help to understand the prognosis and the duration of the disease. m a n u s c r i p t a c c e p t e d m a n u s c r i p t clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with novel coronavirus-infected pneumonia in wuhan new coronavirus pneumonia prevention and control program clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study positive rt-pcr test results in patients recovered from covid-19 a c c e p t e d m a n u s c r i p t key: cord-299359-s8j78naz authors: sundaram, maria e.; mcclure, david l.; vanwormer, jeffrey j.; friedrich, thomas c.; meece, jennifer k.; belongia, edward a. title: influenza vaccination is not associated with detection of noninfluenza respiratory viruses in seasonal studies of influenza vaccine effectiveness date: 2013-09-15 journal: clin infect dis doi: 10.1093/cid/cit379 sha: doc_id: 299359 cord_uid: s8j78naz background. the test-negative control study design is the basis for observational studies of influenza vaccine effectiveness (ve). recent studies have suggested that influenza vaccination increases the risk of noninfluenza respiratory virus infection. such an effect could create bias in ve studies using influenza-negative controls. we investigated the association between influenza infection, vaccination, and detection of other respiratory viruses among children <5 years old and adults ≥50 years old with acute respiratory illness who participated in seasonal studies of influenza vaccine effectiveness. methods. nasal/nasopharyngeal samples collected from 2004–2005 through 2009–2010 were tested for 19 respiratory virus targets using a multiplex reverse-transcription polymerase chain reaction (rt-pcr) platform. vaccination status was determined using a validated registry. adjusted odds ratios for influenza and vaccination status were calculated using three different control groups: influenza-negative, other respiratory virus positive, and pan-negative. results. influenza was detected in 12% of 2010 children and 20% of 1738 adults. noninfluenza respiratory viruses were detected in 70% of children and 38% of adults without influenza. the proportion vaccinated did not vary between virus-positive controls and pan-negative controls in children (p = .62) or adults (p = .33). influenza infection was associated with reduced odds of vaccination, but adjusted odds ratios differed by no more than 0.02 when the analysis used influenza-negative or virus-positive controls. conclusions. influenza vaccination was not associated with detection of noninfluenza respiratory viruses. use of influenza-negative controls did not generate a biased estimate of vaccine effectiveness due to an effect of vaccination on other respiratory virus infections. the case vs test-negative control study design is the basis for observational studies of influenza vaccine effectiveness (ve) [1] [2] [3] [4] [5] [6] . cases and controls are recruited at the time of presentation of acute respiratory illness (ari) in clinic and hospital settings. individuals presenting with ari who test positive for influenza are considered cases, whereas those who test negative for influenza are considered controls. this study design is convenient to implement and inherently accounts for potential confounding due to differences in healthcareseeking behavior between vaccinated and unvaccinated individuals [1] [2] [3] . it has recently been suggested that influenza vaccination may increase the risk of non-influenza respiratory virus infection by decreasing temporary nonspecific immunity [7, 8] . one proposed mechanism involves activation of the innate immune response following influenza infection, leading to a temporary reduction in the risk of infection with a different respiratory virus. by reducing the risk of influenza infection, the influenza vaccine could paradoxically create an increased risk of infection with other noninfluenza respiratory viruses. if this phenomenon occurs, it could lead to biased estimates of influenza vaccine effectiveness in studies using laboratory-confirmed influenza cases and influenza-negative controls. in this scenario, the risk of noninfluenza viral illness would be higher in vaccinated than unvaccinated individuals, and an 'influenza-negative' control group would therefore have a higher proportion of vaccinated individuals compared to the source population. this could theoretically contribute to overestimation of true ve (ie, bias away from the null); therefore, a key assumption of the test-negative control design of influenza vaccine effectiveness studies is that the proportion of noninfluenza viral illness does not differ by influenza vaccination status [9] . the goals of this study were to determine if influenza vaccination is associated with detection of noninfluenza respiratory viruses and to determine if vaccine effectiveness estimates differ when different control groups are used in the analysis. to achieve these goals, we analyzed available data from members of a community cohort who saw a physician for acute respiratory illness and consented to participate in a study of influenza vaccine effectiveness over 6 influenza seasons. the vaccine effectiveness study enrolled individuals of all ages (with some variation by season), but this analysis was restricted to children <5 years old and adults ≥50 years old. for participants in these age groups, multiplex reverse transcription polymerase chain reaction (rt-pcr) testing was subsequently performed to detect other respiratory viruses, providing an opportunity to investigate the relationship between influenza vaccination and infection with other viral pathogens. young children and older adults are among the most vulnerable individuals to influenza infection and complications [10, 11] and calculating influenza vaccine effectiveness in these groups is therefore of high importance [12] . the marshfield clinic research foundation has conducted seasonal studies of influenza vaccine effectiveness in a wisconsin population cohort since the 2004-2005 season. the details of the seasonal studies have been reported elsewhere [2, 3] . briefly, patients with ari were recruited during each influenza season in primary care clinics, urgent care, emergency department, and an acute care hospital. symptom eligibility criteria varied by season but included fever/feverishness or cough during most seasons. individuals with illness duration ≥10 days (2004-2005 through 2006-2007) or >7 days (2007-2008 through 2009-2010) were excluded to minimize false negative rt-pcr results. after obtaining informed consent, a nasal swab (children <12 years old) or a nasopharyngeal swab (adolescents and adults) was obtained and placed in viral transport media for influenza testing. symptoms and onset date were assessed during the enrollment interview. real-time rt-pcr was performed each season to identify influenza cases. after testing was complete, aliquots of samples in viral transport media were frozen at −80°c. this study was reviewed and approved by the marshfield clinic institutional review board. during each season, all study participants (or parents) provided informed consent for influenza testing. multiplex rt-pcr testing to detect additional viruses was subsequently approved by the irb with a waiver of informed consent. archived samples were tested for the presence of respiratory virus nucleic acid using a multiplex respiratory virus panel (genmark dx esensor respiratory viral panel). this multiplex panel tested for respiratory syncytial virus (rsv) a and b, human rhinovirus, human metapneumovirus, parainfluenza viruses 1-4, influenza a and b (including subtypes of influenza a), coronaviruses oc43, nl63, hku1, and 229e, and adenoviruses b and e. nucleic acid was extracted from the swabs using the roche magnapure 2.0 system and was then amplified using rt-pcr with target-specific primers. target-specific signals were determined by voltammetry, a process that generates electrical signals from ferrocene-labeled signal probes. the multiplex assay has been validated for influenza and rsv a and b against singleplex assays approved by the centers for disease control and prevention (cdc) and found to have 97%, 93%, and 98% sensitivity and 97%, 99%, and 99% specificity for influenza a, influenza b, and rsv, respectively (unpublished data). cases were defined as laboratory-confirmed influenza based on a positive real-time rt-pcr test for influenza a (h3n2), a (h1n1), a (h1n1)pdm09, or type b. three different control groups were constructed for analytical purposes. control group 1 included all individuals testing negative for influenza (influenza-negative controls); group 2 included individuals negative for influenza but positive for at least one other respiratory virus in the multiplex panel (virus-positive controls); and group 3 included individuals negative for all respiratory viruses in the multiplex panel ( pan-negative controls). the first control group is the standard definition used in test-negative case-control designs, whereas the second and third control groups represent subsets of the first group. vaccination status and dates were determined by a real-time, internet-based registry used by all immunization providers serving the local population (www.recin.org). cases and controls were considered vaccinated if seasonal trivalent inactivated influenza vaccine (tiv) or live attenuated influenza vaccine (laiv) was received at least 14 days before ari symptom onset. children who were partially vaccinated according to the advisory committee on immunization practices (acip) definition (ie, received 1 of 2 recommended doses) were excluded [13] . separate analyses were conducted for children <5 years old and adults ≥50 years old, and only the first enrollment was included for each individual in a given season. vaccination status was compared for virus-positive controls and pan-negative controls using logistic regression. odds ratios describing the association between vaccination and influenza infection were determined separately for each of the 3 case-control groups by logistic regression. logistic regression models included influenza status as the outcome and vaccination status as the primary exposure variable with adjustment for potential confounders, including gender, influenza season, age (continuous), presence of a chronic disease conferring increased risk for influenza complications [14] , and interval in days from symptom onset to swab collection. sas 9.2 (sas institute, cary, nc) was used for all statistical analyses. over 6 influenza seasons, 1616 children and 1568 adults contributed 2010 and 1738 unique observations, respectively. influenza was detected in 251 (12.5%) children and 343 (19.7%) adults. among those without influenza, a respiratory virus was detected in 1411 (70.2%) of children and 659 (37.9%) of adults. in children without influenza, the most commonly detected single viruses were rsv (n = 435), human rhinovirus (n = 298), human metapneumovirus (n = 150), and parainfluenza (n = 113); 271 (13.5%) of swabs from children were positive for ≥2 noninfluenza respiratory viruses. in children, the most common coinfection was rsv with rhinovirus (n = 73). in adults without influenza, the most commonly detected single viruses were rsv (n = 170), human rhinovirus (n = 126), human metapneumovirus (n = 125), and coronavirus (n = 122); 47 (2.7%) of swabs from adults were positive for ≥2 noninfluenza respiratory viruses. in adults, the most common coinfection was rsv with coronavirus (n = 8). the virus-positive controlgroup and pan-negativecontrolgroup did not differ with regard to age, gender, vaccination status, or interval from symptom onset to swab in either children or adults ( table 1 ). the proportion of children with a high-risk health condition did not differ between control groups; there were more adults with a high-risk health condition in the pan-negative control group than in the virus-positive control group (table 1) . in univariate analyses, no association was found between influenza vaccination and single virus detection of rsv, adenovirus, human metapneumovirus, human rhinovirus, or coronavirus. single infection with parainfluenza virus was less common in vaccinated children (4.6%) than vaccinated children (6.7%; p = .03). among adults, there was a significant association in the opposite direction: parainfluenza was detected in 4.6% of vaccinated and 2.6% of unvaccinated adults (p = .04). for both children and adults, the adjusted odds of influenza infection were significantly lower in the vaccinated group relative to any of the 3 control groups, indicating significant vaccine effectiveness ( table 2 ). among adults, the adjusted odds ratio (aor) ranged from 0.53 to 0.58; among children the aor ranged from 0.50 to 0.56 depending on which control group was used. in both children and adults, the aor using 'influenza-negative controls' (ie, the current standard for ve studies) varied by no more than 0.02 compared to the aor using other virus-positive controls, and all aor confidence intervals overlapped. the adjusted odds of being vaccinated did not differ significantly in the pan-negative control group vs the other virus-positive control group, either in children (p = .96) or in adults (p = .89). results were similar when the analysis was repeated after excluding 14 children who received laiv. there were no adults in this analysis who received laiv. detection of a noninfluenza respiratory virus by multiplex rt-pcr was not associated with influenza vaccination status over a period of six influenza seasons. in both children and adults, the virus-positive control group and the pan-negative control group were similar in terms of age distribution, gender, and influenza vaccination status. there was no association between influenza vaccination and detection of rsv, adenovirus, human metapneumovirus, human rhinovirus, or coronavirus. only parainfluenza infection was significantly associated with influenza vaccination, but the association was in opposite directions for children and adults. the p values were not adjusted for multiple comparisons, and the inconsistency between children and adults suggest that this association is the result of a type 1 error. to assess the potential impact of noninfluenza respiratory viruses in vaccine effectiveness studies, we calculated the aor for influenza vaccination in cases and controls using 3 different control groups. among children, the aors were 0.52 and 0.50, respectively, using influenza-negative controls and virus-positive controls. this corresponds to an absolute difference of 2% in the vaccine effectiveness estimate, with broadly overlapping confidence intervals. nearly all vaccinated children in this analysis received tiv, and the results were similar when laiv recipients were excluded. among adults ≥50 years old, the adjusted odds ratios were nearly identical (0.56 and 0.57, respectively) when using influenza-negative controls and viruspositive controls, with overlapping confidence intervals. other studies have found evidence of an association between influenza vaccination and infection with a non-influenza respiratory virus. in 2008, a study of influenza vaccine effectiveness in australian children 6 to 59 months old found that influenza vaccination was associated with detection of other respiratory viruses [15] . nasal swabs from 289 children were tested by rt-pcr for influenza, rhinoviruses, rsv, parainfluenza virus 1-3, human metapneumovirus, and enteroviruses. adjusted vaccine effectiveness was 58% (95% ci, 9-81) using influenza-negative controls and 68% (95% ci, 26-86) using other virus-positive controls. the authors noted the higher point estimate of vaccine effectiveness using virus-positive controls, although there was substantial overlap in the confidence intervals for these 2 point estimates. the authors speculated that the difference between point estimates could be due to false negative rt-pcr tests in some children (ie, pan-negative controls) due to inadequate sample collection. they also mentioned the possibility that influenza vaccination could increase the risk of infection with other viruses, but they viewed this as biologically implausible. the relationship between influenza vaccination and infection with noninfluenza viruses was also investigated in a clinical trial of tiv in children [7] . in that study, tiv recipients (n = 69) had a 4-fold increase in risk of infection with noninfluenza respiratory viruses, compared to placebo recipients (n = 46); the incidence of seasonal influenza did not differ significantly between the tiv and placebo groups. the authors acknowledged that the association with noninfluenza viruses could be spurious, but they suggested that 'receipt of tiv could increase influenza immunity at the expense of reduced immunity to noninfluenza respiratory viruses, by some unknown biological mechanism' [7] . they identified temporary nonspecific immunity as a possible explanation for this observation. according to this proposed mechanism, unvaccinated children were more likely to acquire influenza infection, and the abbreviations: aor, adjusted odds ratio; ci, confidence interval; uor, unadjusted odds ratio. a all models adjusted for gender, influenza season, age (in years), presence of high-risk health condition, and interval (days) from symptom onset to swab collection. resulting innate immune response provided temporary, nonspecific protection against infection with other respiratory viruses. this was described further in a letter that raised concerns about bias in studies of vaccine effectiveness that use rt-pcr confirmed cases and influenza-negative controls [8] . these 2 studies had relatively small sample sizes and were limited to a single influenza season. however, one of the studies was a randomized, placebo-controlled trial, a study design that is less susceptible to bias and confounding compared to observational studies. we were unable to replicate the previously reported association in children or adults with a larger sample size over multiple influenza seasons. however, we cannot rule out the possibility that vaccination may alter susceptibility to non-influenza viruses in some circumstances. there is limited biological evidence to support an effect of nonspecific immunity across virus families or species and limited evidence to support the role of vaccination in such immunity. cross-reactivity of the t-cell response has been described before for different subtypes of influenza a [16] [17] [18] , although cross-reactivity generated by the innate immune system likely lasts only 10-14 days [19] and may not apply to interactions between viruses of different families or species. other researchers have also suggested that outbreaks of different respiratory viruses may interact or interfere with each other on a population level [20] [21] [22] . this mechanism may help to explain effects not seen in our analysis but which have been reported elsewhere [7, 8] . in conclusion, our findings do not support the hypothesis that influenza vaccination is associated with an increased risk of infection with noninfluenza respiratory virus. further research may aid in determining the biological plausibility of temporary nonspecific immunity generated by infection with specific respiratory viruses. finally, we found no difference in vaccine effectiveness estimates using test-negative vs other virus-positive control groups. the results of this analysis strongly support the validity of case vs test-negative control study designs that are currently used in multiple countries to estimate influenza vaccine effectiveness in the 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interference between outbreaks of respiratory syncytial virus and influenza infection interference between outbreaks of epidemic viruses does viral interference affect spread of influenza? acknowledgments. we gratefully acknowledge the staff of data managers, research coordinators, interviewers, laboratory technicians, and others who contributed to the vaccine effectiveness study and this analysis. we also thank alicia fry, mark thompson, and frank sifakis for manuscript review and feedback.financial support. medimmune, llc, provided funding for multiplex reverse-transcription polymerase chain reaction testing.potential conflicts of interest. m. e. s., j. k. m., and e. a. b. receive research funding from medimmune, llc. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-322448-s04e6po9 authors: gadsby, naomi j.; russell, clark d.; mchugh, martin p.; mark, harriet; conway morris, andrew; laurenson, ian f.; hill, adam t.; templeton, kate e. title: comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia date: 2016-04-01 journal: clin infect dis doi: 10.1093/cid/civ1214 sha: doc_id: 322448 cord_uid: s04e6po9 background. the frequent lack of a microbiological diagnosis in community-acquired pneumonia (cap) impairs pathogen-directed antimicrobial therapy. this study assessed the use of comprehensive multibacterial, multiviral molecular testing, including quantification, in adults hospitalized with cap. methods. clinical and laboratory data were collected for 323 adults with radiologically-confirmed cap admitted to 2 uk tertiary care hospitals. sputum (96%) or endotracheal aspirate (4%) specimens were cultured as per routine practice and also tested with fast multiplex real-time polymerase-chain reaction (pcr) assays for 26 respiratory bacteria and viruses. bacterial loads were also calculated for 8 bacterial pathogens. appropriate pathogen-directed therapy was retrospectively assessed using national guidelines adapted for local antimicrobial susceptibility patterns. results. comprehensive molecular testing of single lower respiratory tract (lrt) specimens achieved pathogen detection in 87% of cap patients compared with 39% with culture-based methods. haemophilus influenzae and streptococcus pneumoniae were the main agents detected, along with a wide variety of typical and atypical pathogens. viruses were present in 30% of cases; 82% of these were codetections with bacteria. most (85%) patients had received antimicrobials in the 72 hours before admission. of these, 78% had a bacterial pathogen detected by pcr but only 32% were culture-positive (p < .0001). molecular testing had the potential to enable de-escalation in number and/or spectrum of antimicrobials in 77% of patients. conclusions. comprehensive molecular testing significantly improves pathogen detection in cap, particularly in antimicrobial-exposed patients, and requires only a single lrt specimen. it also has the potential to enable early de-escalation from broad-spectrum empirical antimicrobials to pathogen-directed therapy. community-acquired pneumonia (cap) is a common infectious disease with an estimated incidence of 2-11 cases per 1000 adults in the developed world and a mortality rate of 2%-14% [1] [2] [3] [4] [5] . due to the range of pathogens responsible for cap, in moderate or severe infection, broad-spectrum antimicrobial cover should be initiated before de-escalating to narrowspectrum pathogen-directed agents once a microbiological diagnosis has been made [1, 5] . unfortunately, de-escalation is uncommon in practice because current diagnostic methods may identify a pathogen in only 30%-40% of patients with cap [4, 6, 7] . recent studies have highlighted the need for more timely and sensitive microbiological diagnostic methods in cap, particularly for bacteria and in the common scenario of antibiotic administration prior to sampling [4, 8, 9] . the development of multiplex real-time polymerase-chain reaction (pcr) assays currently enables a respiratory specimen to be rapidly screened for a wide range of viral and atypical bacterial pathogens in a small number of reactions [10] [11] [12] [13] [14] [15] . however, a similar approach has not yet been adopted for typical bacterial pathogens. it is difficult to interpret pcr results for typical respiratory bacteria in nonsterile samples such as sputum due to the potential for contamination with the same organisms from oropharyngeal flora. however, accurate molecular quantification of bacterial loads may aid in distinguishing infection from contamination in a way that is analogous to the use of quantitative cultures. quantitative molecular assays for typical respiratory bacteria have been used previously in patients with pneumonia, but these assays lack a number of key targets, use targets that have been shown to lack specificity, or may be only semiquantitative [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . however, our group recently developed a suitable assay that is capable of quantifying bacterial loads of 8 typical bacterial pathogens from lower respiratory tract (lrt) specimens [33] . this assay was extensively validated on clinical isolates, reference strains, and clinical specimens such as sputa and bronchoalveolar lavage fluids, and all bacterial targets could be reliably quantified, even when present in mixtures of different concentrations [33] . our aim in the study was to assess the utility of a comprehensive molecular diagnostic approach encompassing 26 respiratory bacterial and viral pathogens and including bacterial quantification in patients with cap. patients presented to 2 tertiary care hospitals in edinburgh, united kingdom, over an 18-month period between september 2012 and february 2014. consecutive cases with clinical and radiological evidence of cap were identified through daily retrospective electronic review of all respiratory samples received by our microbiology laboratory. inclusion criteria for the study included the following: adult (aged ≥18 years); lrt specimen obtained within 48 hours of admission to the hospital; new radiographic infiltrate as determined by radiologist or consultant respiratory physician; and 3 of more of the following signs or symptoms: new or worsening cough, new or worsening expectoration of sputum, hemoptysis, new or worsening dyspnea, pleuritic chest pain, fever, headache, or abnormalities on chest auscultation or percussion. exclusion criteria included the following: bronchiectasis, cystic fibrosis, or healthcareassociated pneumonia (hospitalized in an acute care hospital for 2 or more days within 90 days of the infection or resided in a nursing home or long-term care facility) [34] . radiology reports were reviewed for all patients, and only patients with radiological changes consistent with pneumonia were included. where there was any uncertainty, the images were reviewed by an experienced respiratory physician (a. t. h.). clinical data including comorbidities, outcomes, and antibiotic prescriptions were collected retrospectively from patient hospital records using a standard pro forma as part of a clinical audit of pneumonia management (approved by the quality improvement team, royal infirmary of edinburgh). drug administration prior to hospitalization, including antibiotic prescription, was obtained from the electronic primary care prescription records on admission and later verified by a pharmacist. lrt specimens were cultured according to national standard protocols to detect common respiratory bacteria [35] . isolates were identified using standard biochemical methods and/or matrix-assisted laser desorption/ionization time-of-flight (bruker, coventry, united kingdom). specimens with any named bacterial species identified by culture were considered to be culture positive. following routine culture, lrt specimens were centrifuged, and mucopurulent material was stored at −80°c for up to 15 months. as part of the study protocol, specimens and associated clinical data were collected and anonymized before molecular testing in accordance with local ethical approval (south east scotland scottish academic health sciences collaboration human annotated bioresource reference no. 10/s1402/33). thawed specimens were homogenized by vortexing with sterile glass beads, and a 200-μl aliquot was treated with lysozyme at 37°c followed by proteinase k at 56°c for 1 hour each. total nucleic acid was then extracted using the automated nuclisens easymag (biomérieux, basingstoke, united kingdom) instrument with an off-board lysis protocol, including the addition of phocine herpes virus as an internal extraction control. extracts were stored at 4°c for up to 5 days or longer at −80°c. fast multiplex real-time pcr was performed using a combination of multiplex assays developed or adapted in-house for the following [10] [11] [12] [13] [14] [15] 33] . as previously described [33] , bacterial loads for s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus, e. coli, k. pneumoniae, p. aeruginosa, and a. baumannii were expressed as colony forming units (cfus) per milliliter of purulent material and calculated from standard curves generated by target gene plasmid dilution series on each pcr run. quality control reactions (quantitative real-time pcr for gapdh human gene and qualitative real-time pcr for gb phocine herpes virus internal control [ic]) were carried out on all specimens [33] . quantitative gapdh pcr was used as an indication of the cell content of each specimen because the gapdh gene is present in human dna. negative (no template) controls were included on every extraction and pcr run to check purity of reagents. runs were accepted if the negative controls were negative and the standard curves were linear; runs that failed quality control were repeated. qualitative results were accepted if the internal control was positive or if at least 1 other target was positive. quantitative results were accepted if the internal control quantification cycle (cq) value fell within the range ±1 log cq difference to negative extraction controls. outside of this range, quantitative results were not accepted due to the potential for partial pcr inhibition leading to inaccuracy of measurement. the prescribed empirical therapy for each patient was compared with what antimicrobial(s) would have been appropriate for pathogen-directed therapy, based on the molecular identification result. appropriate pathogen-directed therapy was determined using uk national guidelines adapted for local antimicrobial susceptibility patterns (supplementary table 1 ) [1] . categorical variables were compared using fisher exact test or χ 2 test. continuous data were assessed using the shapiro-wilk w test for nonnormality and analyzed using the mann-whitney u test or t test. the mcnemar test was used to compare paired proportions. the bonferroni method was used to correct for multiple comparisons during significance testing. to control for potential confounding factors that affect associations with the outcome of infection, we used logistic regression analysis. for the analysis of the outcome of infection, a combined outcome measure of 30-day mortality and/or intensive care unit (icu) admission was used because of the infrequent occurrence of either event in our cohort. data were analyzed using statsdirect software, version 2.8 0 (altrincham, united kingdom). a total of 323 hospitalized adult patients with cap were included in the study cohort; 55% were male and the median age was 67 years (table 1) . critical care admission (icu or highdependency unit) was required for 18.6% of patients, with 12.4% admitted specifically to the icu. total 30-day mortality was 6.2%. a total of 323 lrt specimens were received from 323 patients; 310 (96.0%) were sputa and 13 (4.0%) were endotracheal aspirates. culture-based microbiological testing identified a bacterial pathogen in 127 (39.3%) patients. all specimens were positive by pcr for the human gene target gapdh, with an average load of 1.24 × 10 5 ± 1.18 × 10 5 gene copies per reaction; all specimens met quality control criteria for qualitative molecular testing. bacteria were detected by molecular testing in specimens from 262 (81.1%) patients (table 2) . when a cutoff of ≥10 5 cfu/ml was applied for assays where bacterial load was quantified, bacteria were detected in specimens from 231 (71.5%) patients. including respiratory virus results, overall pathogen detection by molecular testing rose to 86.7% (n = 280; table 2 ). h. influenzae and s. pneumoniae were the most frequently identified bacteria in patients with cap, detected in 130 (40.2%) and 115 (35.6%) patients, respectively. detection of more than 1 bacterial species occurred in 102 (31.6%) patients. h. influenzae or s. pneumoniae were present in 94 (92.2%) codetections. viruses were detected in 98 (30.3%) patients (table 2) ; rhinovirus was most commonly detected (12.7%, n = 41) followed by influenza a/b (7.1%, n = 23). however, viruses alone were found in only 18 (5.6%) of our patients, with 80 (81.6%) viruses codetected with bacteria ( table 2 ). the most common combination was rhinovirus with h. influenzae and/or s. pneumoniae in 30 (37.5%) cases. pcr detected significantly more h. influenzae, s. pneumoniae, m. catarrhalis, s. aureus, e. coli, and k. pneumoniae than standard culture-based methods (supplementary table 2 ). of 127 culture-positive specimens, 125 (98.4%) were also pcr positive for the same species of bacteria; the 2 discrepant specimens were h. influenzae culture positive but culture negative by pcr. significantly, pcr was able to detect bacteria in 143 culture-negative specimens. specimens from 27 (8.4%) patients grew bacteria that were not included in our pcr assays (supplementary table 3 ). bacterial load quantifications from 29 (9.1%) specimens were excluded from analysis due to the detection of partial inhibition by the ic assay. a higher bacterial load was detected by pcr in culture-positive specimens in comparison to culture-negative specimens. the mean combined bacterial load was 8.77 × 10 8 cfu/ml in culture-positive vs 7.03 × 10 7 cfu/ml in culturenegative specimens (p < .0001). significant differences between culture-positive and culture-negative specimens were also observed when h. influenzae (p < .0001) and s. pneumoniae (p < .0001) were considered individually. neither culture-positivity nor pcr-positivity was associated with the outcome of infection (p = .2; odds ratio [or] = 1.5; 95% confidence interval [ci], .8-2.9; and p = .8; or = .9; 95% ci, .4-2.6, respectively). polymicrobial detection was also not associated with the outcome of infection (p = .4; or = 1.3; 95% ci, .7-2.5). the bacterial load of s. pneumoniae was associated with the outcome of infection on univariate analysis, with a higher load present in patients with 30-day mortality and/or icu admission (4.33 × 10 8 cfu/ml vs 1.29 × 10 8 cfu/ml; p = .009). however, the curb-65 score (curb-65; confusion of new onset, blood urea nitrogen >7 mmol/l , respiratory rate ≥30 breaths per minute, blood pressure <90 mmhg systolic or ≤60 mmhg diastolic, age ≥65 years) was also associated with outcome on univariate analysis (p = .01). when logistic regression was used to correct for curb-65 score in patients with s. pneumoniae infection, bacterial load was no longer significantly associated with outcome (p = .09; supplementary table 4 ). of 316 patients with information available on antimicrobial administration prior to sampling, 268 (84.8%) had received antimicrobials either while in the hospital or in the community during the 72 hours before an lrt specimen was obtained. prior antimicrobial administration was significantly associated with a patient having a culture-negative lrt specimen (p < .0001; or = 9.1; 95% ci, 4.1-22.4). of the 268 patients who had received prior antimicrobials, 77.6% (n = 208) had a bacterial pathogen detected by pcr, but only 32.1% (n = 86) were culture positive (p < .0001). the mean combined load of all detected bacteria per patient was significantly higher in patients who had not received antimicrobials prior to sampling (5.40 × 10 9 cfu/ml) compared with those who had received antimicrobials (2.67 × 10 8 cfu/ml; p = .0001). records of antimicrobial treatment during hospitalization were available for 99.1% (n = 320) of patients. molecular testing had the potential to lead to de-escalation in number and/or spectrum of initial empirical antibiotic agents in 247 (77.2%) patients, escalation in number and/or spectrum of antibiotic agents in 19 (5.9%) patients, and no change in 54 (16.9%) patients ( table 3 ). the majority of the potential de-escalation events were related to switching of amoxicillin-clavulanate to narrower-spectrum agents such as amoxicillin and doxycycline in cases where s. pneumoniae or h. influenzae were detected by pcr and to the removal of clarithromycin in cases where atypical bacteria were not detected by pcr. this study demonstrates that the use of a comprehensive multibacterial, multiviral molecular testing approach approximately doubles pathogen detection in patients with cap from 39.3% to 86.7%, as well as provides valuable information about individual bacterial loads. testing can be carried out within 1 working day to enable reporting of results in a clinically relevant time-frame, requires only a single lrt specimen, and is not negatively impacted by antibiotic administration prior to sampling. only limited quantitative molecular bacterial testing has been carried out in a well-defined cap setting [21, 25, 28, 29, 31] . as our study focused on testing sputum by molecular methods, our high level of pathogen detection for typical bacteria may not be directly comparable to results from other recent studies in hospitalized adult cap. by combining several methodologies such as serology, culture, antigen detection, and pcr on multiple sample types, etiology was determined in 38%-76% of cases [4, 7, 21, 31, [36] [37] [38] [39] [40] [41] . atypical bacteria were uncommon in our cohort (<5% patients), but it was not an epidemic period for m. pneumoniae [42] . viruses were detected in approximately one third of patients, which is in agreement with the 13%-56% range reported in previous studies; influenza was also detected at comparable levels (<10%) [4, 7, 21, 31, 36, 38, 39, 43 ]. although we were not able to test controls, viral detections by pcr from the upper respiratory tract have been found to be rare in asymptomatic adults compared with those with cap in a recent study [43] . one limitation of our study was that it was restricted to cap patients who could produce a sputum specimen. also, we did not include blood culture or urinary antigen tests. sputum is a pragmatic, although imperfect, choice of specimen type for the microbiological investigation of cap. microscopy for sputum quality is not routinely carried out locally; however, only mucopurulent material was tested, and the cellular content was quantified by human gapdh gene pcr to rule out salivary specimens. however, sputum that is expectorated from the lower airways will always be at risk of oropharyngeal flora contamination. despite the limitations, national cap guidelines [1, 5] recommend sputum investigation for patients admitted to the hospital with moderate to severe cap, both to aid microbiological diagnosis in order to guide therapy as well as for surveillance. alternative lower respiratory specimen types such as bronchoalveolar lavage fluids may be expected to be less at risk of contamination, but these require a semi-invasive procedure that is certainly not routine in nonintubated patients. a key strength of our study was the availability of quantitative bacterial load outputs in addition to qualitative detection results. quantification of bacterial dna load may be important in distinguishing infection from oropharyngeal contamination in sputum. most molecular work done to date has focused on s. pneumoniae, and a cutoff of 10 4 -10 5 gene copies/ml is typically described as a significance threshold [20, 21, 29, 44] . a cutoff of 10 5 cfu/ml is also generally agreed upon for sputum culture as determined through years of experience. based on these data, we applied a significance threshold of ≥10 5 cfu/ ml for all bacterial loads, and this did not significantly decrease overall pathogen detection. however, it remains unclear whether a single molecular cutoff is relevant for all bacterial species and whether cutoffs for sputum culture are relevant to sputum pcr. quantitative molecular tools for many bacterial species are new [32, 33] , and there is a clear need for further exploration of their role. determination of thresholds for significant and nonsignificant detection are key targets for future work. we found a higher mean bacterial load by pcr in culturepositive specimens in comparison to culture-negative specimens. however, the culture-negative group was more frequently antibiotic exposed, and antibiotic exposure was also associated with lower bacterial loads. as pcr is able to detect dead as well as viable bacteria, it is not clear for how long bacterial loads might be detectable after initiation of appropriate antibiotics in patients with cap and if monitoring of bacterial load by pcr would be useful in these patients. with the quantitative molecular tools now available for the relevant bacteria, these issues can also be further investigated. a recent review has noted that interventions to reduce excessive and increase effective antibiotic prescribing in hospitalized patients can have a positive impact on antimicrobial resistance, hospitalacquired infections, and clinical outcomes [45] . however, studies in the pneumonia setting have been small and either focused on atypical bacteria and viruses [46] or nonquantitative molecular detection of bacteria [30] , demonstrating 11% and 67% antibiotic modification, respectively. the results of comprehensive molecular testing in our study were not available to the treating physicians due to the anonymized study protocol. however, we estimated that de-escalation and/or reduction in the number of agents might have occurred in three quarters of our cap patients. this was based on significantly improved bacterial detection capability, high rates of broad-spectrum empirical antibiotic usage in our cohort, and knowledge of local antimicrobial resistance patterns. clearly, a large number of additional factors influence antimicrobial selection (eg, severity of illness, concurrent infection at sites other than the lower respiratory tract, drug allergy, antimicrobial susceptibility testing, inflammatory markers), and we do not suggest that sputum pcr testing alone will be sufficient. however, the lack of positive microbiological identification is a significant issue, and it is highly likely that enhancing the detection of pathogens and reporting of bacterial loads would have a major impact on the clinical decision-making process. therefore, our study illustrates the feasibility of providing the physician with significantly more information on which to base treatment decisions than is currently available and suggests that comprehensive pcr testing including bacterial load quantitation should be one of the inputs to future prospective studies in this area. supplementary materials are available at http://cid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. bts guidelines for the management of community acquired 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in adults the etiology of community-acquired pneumonia in australia: why penicillin plus doxycycline or a macrolide is the most appropriate therapy viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation aetiology of community-acquired pneumonia among adults in an h1n1 pandemic year: the role of respiratory viruses increased reports of mycoplasma pneumoniae from laboratories in scotland in 2010 and 2011-impact of the epidemic in infants respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia use of a rapid test of pneumococcal colonization density to diagnose pneumococcal pneumonia interventions to improve antibiotic prescribing practices for hospital inpatients impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection acknowledgments. the authors thank the staff of the royal infirmary of edinburgh medical microbiology laboratory for their help with data and specimen collection.financial support. this work was supported by the chief scientist office (grant number etm/250).potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-314025-h9gj814e authors: lai, mary y. y.; cheng, peter k. c.; lim, wilina w. l. title: survival of severe acute respiratory syndrome coronavirus date: 2005-10-01 journal: clin infect dis doi: 10.1086/433186 sha: doc_id: 314025 cord_uid: h9gj814e background. the primary modes of transmission of severe acute respiratory syndrome (sars) coronavirus (sars-cov) appear to be direct mucus membrane contact with infectious droplets and through exposure to formites. knowledge of the survival characteristics of the virus is essential for formulating appropriate infection-control measures. methods. survival of sars-cov strain gvu6109 was studied in stool and respiratory specimens. survival of the virus on different environmental surfaces, including a laboratory request form, an impervious disposable gown, and a cotton nondisposable gown, was investigated. the virucidal effects of sodium hypochlorite, house detergent, and a peroxygen compound (virkon s; antec international) on the virus were also studied. results. sars-cov gvu6109 can survive for 4 days in diarrheal stool samples with an alkaline ph, and it can remain infectious in respiratory specimens for >7 days at room temperature. even at a relatively high concentration (10(4) tissue culture infective doses/ml), the virus could not be recovered after drying of a paper request form, and its infectivity was shown to last longer on the disposable gown than on the cotton gown. all disinfectants tested were shown to be able to reduce the virus load by >3 log within 5 min. conclusions. fecal and respiratory samples can remain infectious for a long period of time at room temperature. the risk of infection via contact with droplet-contaminated paper is small. absorbent material, such as cotton, is preferred to nonabsorptive material for personal protective clothing for routine patient care where risk of large spillage is unlikely. the virus is easily inactivated by commonly used disinfectants. in the early spring of 2003, a mysterious outbreak of severe pneumonia occurred in southern china and rapidly spread throughout the world. the causative agent was later found to be a novel coronavirus and was designated "severe acute respiratory syndrome (sars) coronavirus" (sars-cov) [1] [2] [3] . as of 31 december 2003, a total of 8096 cases had been reported, of which 774 were fatal [4] . altogether, 1706 health care workers were affected. more than 20% of the patients with sars were themselves health care workers, which could be explained by the unique shedding pattern of sars-cov, with viral loads reaching a peak ∼2 weeks after onset of disease, when patients were in hospital care [5] . this shedding pattern of sars-cov also highlights the importance of control of nosocomial spread of the disease. soon after the isolation of sars-cov in our laboratory, we were able to perform a survival study of the virus, and partial results were reported on the world health organization communicable disease surveillance and response web site on sars [6] . here, we provide a full report of our study of the survival characteristics of sars-cov in different clinical sample matrices, as well as on various environmental surfaces in the laboratory and hospital. the risk of acquisition of sars-cov attributed to the inanimate environment is also discussed. we also report the virucidal effect of 3 common liquid disinfectants on sars-cov. viruses and cell line. sars-cov strain gvu6109 was used in the present study. gvu6109 was isolated from a lung tissue specimen obtained from a patient during the sars outbreak in 2003. the virus was inoculated into the vero e6 cell line, which was grown in minimum essential medium (mem) with 2% fetal calf serum at 37њc. all virus culture experiments were performed in a biosafety level 3 laboratory. four different stool samples were used. the samples tested negative for sars-cov, norovirus, rotavirus, and other viral agents. stool sample a was a normal stool specimen obtained from a 6-month-old baby, and samples b and c were 2 normal stool specimens obtained from adults. sample d was a diarrheal stool specimen obtained from an adult. a 10% suspension of each stool specimen was prepared in pbs (ph, 7.4). after centrifugation at 1500 g for 20 min, the supernatant was collected, and the ph was checked with ph paper. stool sample a had a ph of 6-7, sample b had a ph of 7-8, sample c had a ph of 8, and sample d had a ph of 9. a total of 1.8 ml of each 10% stool suspension was spiked with 0.2 ml of virus stock gvu6109 (10 7 tcid 50 /ml). as a control, 1.8 ml of viral transport medium was also spiked with 0.2 ml of the virus stock. the samples were incubated in closed containers at room temperature (20њc) for 0.5 h, 1 h, 3 h, 6 h, 1 day, 2 days, 4 days, 5 days, 6 days, and 7 days. serial 10fold dilutions of a different stool suspension and control were prepared in earl's diluent. fifty microliters of each dilution was inoculated into 4 wells of a 96-well plate. one hundred microliters of vero e6 (10 5 cells/ml) was added to each well, and the plates were sealed and incubated at 37њc in 5% co 2 for 4 days. virus concentration (in tcid 50 /50 ml) for each stool suspension at a different time was calculated on the basis of the kärber method [7] . the whole experiment was repeated using a trivalent poliovirus vaccine to compare the effect of ph on a nonenveloped rna virus. survival of sars-cov in different respiratory specimens. a total of 0.3 ml of virus stock gvu6109 (10 7 tcid 50 /ml) was added to 2.7 ml of a nasopharyngeal aspirate (npa) specimen, throat and nasal swab (tns) specimens, and viral transport medium as a control. the respiratory specimens had been determined to be negative for respiratory viruses. they were then incubated in closed containers at room temperature or 4њc for 3 h, 6 h, and 1, 3, 4, 5, 7, and 10 days. the virus concentration (tcid 50 /50ml) for each sample at various time points was determined as above. survival of sars-cov on paper, impervious disposable gowns, and cotton nondisposable gowns. to simulate the event of large droplets that contain sars-cov falling on paper and on cotton and disposable gowns, experiments were performed to determine whether sars-cov survived on these surfaces. paper. a paper laboratory request form was cut into small pieces (area, cm), which were sterilized by autoclave at 1 ϫ 1 121њc for 15 min. stock virus gvu6109 (10 7 tcid 50 /ml) was serially diluted to 10 4 tcid 50 /ml with pbs. at each virus dilution, 5 ml was applied to the surface of each piece of sterilized paper. the sample was allowed to be absorbed at room temperature, and the paper pieces were left to stand for different durations (5 min, 1 h, 2 h, 3 h, 6 h, 1 day, and 2 days). each piece of paper was then placed into a vero e6 cell culture tube. for each virus dilution and at different intervals after absorption, 4 pieces of paper were inoculated into 4 cell culture tubes. all of the tubes were incubated at 37њc and were examined after 4 days. sterilized paper without virus suspension was also included in the study to check for any toxicity to cell culture. disposable gown. for the disposable gown, the whole process used for paper was repeated, except that a disposable gown was used after treatment by irradiating it under uv light for 1 h. the gown is part of the personal protection equipment used in our laboratory when handling specimens that are potentially contaminated with sars-cov. it is made of polypropylene material (35 g/m 2 ) coated with a polyethylene film (15 g/m 2 ), and the waist and neck are tied when the gown is used to provide full-body protection. cotton gown. for testing of the cotton gown, a large piece of cloth cut from an ordinary cotton laboratory coat was soaked in distilled water overnight and was then boiled for 1 h. the whole process was repeated 3 times to remove chemical residue that was found to be toxic to the cell culture. after drying, the cloth was cut into small pieces (area, cm). the pieces 1 ϫ 1 were then sterilized by autoclaving. the sterilized cotton cloth was then tested in the manner used for the paper and the disposable gown. different dilutions of sodium hypochlorite solution (1:50 and 1:100 of the stock solution, which contains 50,000 ppm of active chlorine); a household detergent containing sodium lauryl ether sulphate, alkyl polyglycosides, and coco-fatty acid diethanolamide (1:50 and 1:100; axe brand); and virkon s (1%; antec international) were made by dilution with distilled water. fifty microliters of stock virus gvu6109 (10 7 tcid 50 /ml) was added to 450 ml of different dilutions of the hypochlorite solution, household detergent, and virkon s and to viral transport medium as a control. after standing at room temperature for 5 min, 10 min, 20 min, and 30 min, serial 10-fold dilutions of different disinfectants or controls were made in earl's diluent. fifty microliters of each virus dilution was added to each of 4 wells of a 96-well plate. a total of 50 ml of mem was added to each well. after adding 100 ml of vero e6 cells to each well, the plates were sealed and incubated at 37њc in a 5% co 2 incubator. cytopathic effect was recorded at day 4, and residual virus tcid 50 was calculated from wells without showing cell toxicity. figure 1 shows the duration of sars-cov survival after incubation in stool specimens at different phs. the virus was not recoverable within 1 day after incubation in normal adult stool specimens or within 3 h after incubation in the baby stool specimen with a slightly acidic ph. however, the virus survived for 4 days in a diarrheal stool specimen with a ph of 9. poliovirus did not show these survival characteristics. poliovirus spiked in the same baby stool specimen survived for 14 days, and it survived for even longer in the diarrheal stool specimen (data not shown). the duration of survival for sars-cov in the stool suspension was retested in another 2 diarrheal stool specimens, with the same results (data not shown). the virus can remain alive in respiratory specimens, such as npa or tns specimens, for 17 days at room temperature and for 120 days at 4њc (figure 2). table 1 shows the duration of survival for sars-cov on different materials. even with a relatively high virus load in the droplet, rapid loss of infectivity was observed for paper and cotton material. inactivation on impervious surface took much longer. no cell culture toxicity was observed for the paper, disposable gown, or cotton nondisposable gown. a recent study showed evidence that sars-cov has contaminated a variety of environmental surfaces in some hospital settings [8] . the presence of sars-cov on surfaces is always a concern, although few studies in which live virus has been successfully isolated from an environmental surface have been reported. surfaces were usually contaminated with patient's droplets or by indirect transfer of virus from gloves that were contaminated with excreted virus. the increase in the isolation rate of methicillin-resistant staphylococcus aureus at the intensive care unit of a hong kong hospital during the sars outbreak suggested that increased cross-contamination could occur if gloves and gowns were worn all of the time [9] . it is important to gather evidence of the survival of sars-cov on surfaces so that appropriate infection-control measures can be taken. the present study demonstrates that sars-cov can survive in respiratory samples for 5 days at room temperature and for up to 3 weeks at 4њc. although normal fecal material seems to have a deleterious effect on its survival, the present study shows that the virus could have a prolonged survival when present in diarrheal stool. the virus can survive for 4 days at room temperature after being spiked in diarrheal stool with an alkaline ph. this observation lends evidence that fecal droplets containing sars-cov remain infectious for a period of time. this may explain the amoy gardens outbreaks, in which the drainage and sewage system was implicated in facilitating the spread of sars, as was pointed out in the sars expert committee study [10] and in a simulation study by yu et al. [11] . on the basis of quantitative data obtained from our own study [5] , stool samples contain a much higher viral load than do npa samples. the mean virus concentration may reach 10 5 tcid 50 /ml at 2 weeks after onset of disease in stool samples, compared with 10 2.2 tcid 50 /ml for npa samples. our present data show that, at a high concentration of virus (10 6 tcid 50 / ml), sars-cov can survive for 4-5 days at room temperature in both respiratory and diarrheal stool samples. from the point of view of infection control of sars, it is important to know that excreta from patients with sars (especially those who have diarrhea) may remain highly infectious for a considerably long period, and appropriate precautions must be taken to prevent formation of aerosols, because of probable airborne transmission of sars. during the sars outbreak in 2003, contamination of paper documents was a concern for health care workers, who frequently had to handle such documents in their daily work. the present study simulates a situation in which large respiratory droplets (volume, 5 ml; radius, ∼1 mm) that contain the virus fall onto paper. even with a higher concentration of virus (10 4 tcid 50 /ml) than would normally occur in npa samples (10 2.2 tcid 50 /ml), no virus infectivity remained after the paper was dried. paper contaminated with a higher concentration of virus (equivalent to that of fecal excreta [i.e., 10 5 tcid 50 /ml]) was not infectious after 3 h, and no viral infectivity was shown after 24 h, even with a concentration of 10 6 tcid 50 /ml. our study shows that the risk of infection through contact with a dropletcontaminated paper is small. standard infection-control measures, such as hand washing after touching any potential infectious material, are effective against nosocomial transmission of sars [12] . a previous study reported that coronavirus 229e and oc43 can survive for a few hours after drying on 3 different surfaces (aluminium, cotton gauze sponges, and latex gloves) [13] . in the present study, we compared the survival of sars-cov on 2 types of gowns: disposable gowns and cotton gowns. our results showed that, even with a high concentration of virus (10 5 tcid 50 /ml), the droplets will lose all infectivity after 1 h on cloth, compared with 24 h needed for the disposable gown. apparently, droplets will be absorbed more quickly on cotton material than on fluid-repellent material. the present data show that an ordinary cotton gown offers reasonable protection against small droplets containing sars-cov. our study also raises the possibility that any droplets that hang on a nonabsorbent disposable gown may pose a risk of contaminating the environment when health care workers wear the gown all of the time or when they try to remove the gown. a similar conclusion may also be drawn for gloves, although gloves were not tested in the present study. a specially designed disposable garment with a fluid-repellent lamination that has an outer fluid-absorbing sheet may offer better protection for the personnel. finally, our study shows that 3 common liquid detergents/ disinfectants are equally effective against the sars-cov. all demonstrated a minimum 10 3 -fold reduction in the initial virus titer within 5 min after incubation in solution [14] . the household detergents tested in this study were shown to be effective against the sars-cov with a lipid envelope and could be used for cleaning common items and surfaces that are not grossly contaminated with secretions or excreta. although we did not perform specific neutralizing steps for the 3 detergents/disinfectants, the fact that the wells that we examined to calculate the residual virus tcid 50 were free of cell toxicity highly suggests that nonneutralized disinfectants also have no effect on the virus during the 4 days of incubation. the sars-cov is a newly discovered virus. thus far, there have only been a few reports of its survival characteristics [15] . here, we demonstrate that this deadly virus can remain infectious for a long period in stool specimens. the samples that we spiked with sars-cov were incubated in closed containers during the entire period of incubation, simulating the conditions in a sewage drainage pipe. thus, our results showed that, in this situation, droplets may be a concern with regard to disease transmission, as occurred in the amoy gardens outbreaks. this has significant implications for sewage treatment in both domestic and hospital environments. fortunately, this virus is also susceptible to drying. we showed that, when viruscontaining droplets were dried, the virus was inactivated rapidly on paper and cotton cloth. transmission through dropletcontaminated paper and cotton gowns is unlikely, and common household detergents can be effective decontaminating agents for use in the laboratory and hospital. identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome. sars study group a novel coronavirus associated with severe acute respiratory syndrome. sars working group summary of probable sars cases with onset of illness from 1 viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome world health organization. first data on stability and resistance of sars coronavirus compiled by members of who laboratory network diagnostic procedures for viral, rickettsial and chlamydial infections severe acute respiratory syndrome coronavirus on hospital surfaces increase in methicillinresistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome sars in hong kong: from experience to action. report of the sars expert committee. hong kong: health, welfare, and food bureau evidence of airborne transmission of the severe acute respiratory syndrome virus effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) survival of human coronavirus 229e and oc43 in suspension and after drying on surfaces: a possible source of hospital-acquired infections principles and practice of disinfection, preservation and sterilization stability and inactivation of sars coronavirus potential conflicts of interest. all authors: no conflicts. key: cord-317948-svgguadm authors: xiao, ai tang; tong, yi xin; zhang, sheng title: profile of rt-pcr for sars-cov-2: a preliminary study from 56 covid-19 patients date: 2020-04-19 journal: clin infect dis doi: 10.1093/cid/ciaa460 sha: doc_id: 317948 cord_uid: svgguadm a novel coronavirus (covid-19) pandemic threatens the world. here, we first studied the dynamics profile of sars-cov-2 from 56 recovered covid-19 patients. we found virus shedding was up to 6 weeks after onset of symptoms. prolonged observation period is necessary for older patients. to date, an outbreak of infectious diseases--coronavirus disease 2019 (covid-19) associated with severe acute respiratory syndrome coronavirus -2 (sars-cov-2) continues in wuhan, china and threatens countries such as korea, italy, iraq and japan, etc. [1] [2] over 50 countries are fighting against the disease. as of march 4 th , 2020, more than 85,000 cases were diagnosed worldwide with a death rate around 2%. before this study, some studies reported cases of viral detection by rt-pcr at different timepoints throughout the disease course. [3] [4] [5] however, these reports monitored sars-cov-2 in the acute phase of infection. currently no study reported the viral dynamics of sars-cov-2 infection in a long observation period. understandings the profile of virus in patients' respiratory specimens can assist diagnosis and reflect the disease course. therefore, we collected clinical specimens from 56 covid-19 patients and reported the results of sars-cov-2 detection during the disease course. reading frame 1ab (orf1ab) and nucleocapsid protein (n), were tested during the real-time rt-pcr assay. if two consecutive negative results were achieved, the period between symptoms onset and the date of first negative rt-pcr test result was defined as viral nucleic acid conversion time. all data (test dates and results of rt-pcr assay) were collected up to the final follow-up date (march 3 rd , 2020). a total of 56 patients diagnosed as covid-19 were included in this study. according to the guideline, all included patients were mild to moderate [7] . no patient was transferred to icu. the median age was 55 years (interquartile range, iqr, 42-68; range, 25-83), comprising 34 (60.7%) men and 22 (39.3%) women. at the end of follow-up, all patients recovered and were discharged from hospital. the total number of sars-cov-2 rt-pcr assay from 56 patients was 299, with 5 tests per patient. the longest duration between rt-pcr test for sars-cov-2 was 42 days after onset of symptoms. the median duration between onset of symptom to nucleic acid conversion was 24 days (iqr, 18-31). the details of demographic characteristics and rt-pcr test results were shown in supplemental table 1 . the number of positive and negative results of rt-pcr tests were shown in figure 1a . in first 3 weeks after symptoms onset, majority results of rt-pcr for sars-cov-2 were positive. from week 3 after symptoms onset, number of negative rt-pcr results increased. all results of rt-pcr tests were negative in week 6 after onset. (supplemental table 2 ) the positive rate of rt-pcr test results was highest at week 1 (100%), followed by 89.3%, 66.1%, 32.1%, 5.4% and 0% at week 2, week 3, week 4, week 5 and week 6 respectively. (figure 1b) we divided patients into non-prolonged and prolonged shedding group according to nucleic acid conversion time (≤24 days or >24 days). as shown in supplemental table 3, patients with prolonged viral shedding tend to be older (p=0.011) and were more likely to have comorbidities as diabetes (p=0.016) and hypertension (p=0.006). test results kept declining in 6 weeks. (figure 1) the above findings suggested that sars-cov-2 viral replication has a relatively long period in infected patients. our study attempted to explore the correlation between clinical characteristics and viral shedding in covid-19 patients. we found that patients with prolonged viral nucleic acid conversion tend to be older and with more comorbidities. previous studies suggested that coronavirus is more likely to infect older individuals, for whom the immunopathogenesis and induction of a proinflammatory cytokine storm might be the culprit. [12] older patients with impaired immune function might have a prolonged period of viral elimination. 6 as a result of errors in sampling and testing, false negative result of rt-pcr for sars-cov-2 is very common in clinical settings. meanwhile, it is recommended by the current diagnosis and treatment guideline for sars-cov-2 from chinese national health committee that the criteria to discharge a patient included the relief of symptoms, improvement in radiography and two consecutive negative rt-pcr results for sars-cov-2. [7] in our study we found two consecutive negative rt-pcr test results followed by a positive result in 4 patients (patient number 20, 24, 37 and 56). evidence suggested that the outbreaks of covid-19 may be correlated to its rapid person-toperson transmission ability. [2] since specific treatment had not been validated for covid-19, traditional public health tactics-isolation, quarantine and community containment are critical to control the spread. [14] [15] [16] this preliminary study has found evidence of the dynamic profile of sars-cov-2 in non-icu covid-19 patients during disease course. according to the results in our study, we suggested prolonged observation and repeat confirmation of rt-pcr test from respiratory specimens for safe discharges and discontinuation of quarantine. all authors participated in the study design. atx and sz conceived the study, analyzed the data and drafted the manuscript. yxt helped critically revise the manuscript and collected data. all authors have agreed on the final version and meet the major criteria recommended by the icmje 12 figure 1 outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention sars-cov-2 viral load in upper respiratory specimens of infected patients viral load of sars-cov-2 in clinical samples detection of sars-cov-2 in different types of clinical specimens interim infection prevention and control recommendations for patients with confirmed coronavirus disease 2019 (covid-19) or persons under investigation for covid-19 in healthcare settings diagnosis and treatment of 2019-ncov pneumonia in china. (version 5) in chinese summary of probable sars cases with onset of illness from 1 geneva: world health organization a familial cluster of infection associated with the 2019 novel coronavirus indicating potential person-to-person transmission during the incubation period. the journal of infectious diseases a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. the lancet clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study clinical characteristics of coronavirus disease 2019 in china positive rt-pcr test results in patients recovered from covid-19 early transmission dynamics inwuhan, china, of novel coronavirus-infected pneumonia sars to novel coronavirus: old lessons and new lessons risk for transportation of 2019 novel coronavirus disease from wuhan to other cities in china we thank ms. cheng chen for english grammatic correction of this manuscript. this study was approved by the ethics committee of tongji hospital, tongji medical college, huazhong university of science and technology. all procedures followed in this study were in accordance with the 1964 helsinki declaration and later versions. oral consent was obtained from patients involved before enrollment when data were collected retrospectively. the database used and/or analyzed during the current study is not publicly available (to maintain privacy) but can be available from the corresponding author on reasonable request. funding no funding resources to declare for this study. all authors declare that there are no conflicts of interest.8 key: cord-320562-zvsf3va0 authors: dookie, navisha; padayatchi, nesri; naidoo, kogieleum title: tuberculosis elimination in the era of covid-19: a moving target date: 2020-09-14 journal: clin infect dis doi: 10.1093/cid/ciaa1400 sha: doc_id: 320562 cord_uid: zvsf3va0 nan consequences from covid-19 related disruption, with modelling studies predicting an additional 6.3 million cases of tb and 1.4 million deaths over the next five years. this translates to a five to eight-year setback in tb control, receding even further away from the 2030 target of tb elimination [2] . in this issue, liu and colleagues describe the impact of a temporary disruption in health services due to covid-19 mitigation measures on diagnosis and treatment of tb in the jiangsu province of china. the study tracked weekly tb notification data from january 2015 to may 2020 from the tuberculosis management information system for the province. the results highlight three significant consequences for tb control arising from the pandemic. firstly, their analysis of tb case notifications between january to may 2020, demonstrates a drastic decrease of 36% and 52% in overall notifications compared to the same period in 2019 and 2015, respectively. secondly, tb treatment completion rates reduced from 60.2% in 2019 to 51% for the same period in 2020. thirdly, they report significantly lower rates of screening for tb drug-resistance during 2020. this work contributes to the growing global corpus of reports demonstrating a decline in tb case notifications documented during the covid-19 pandemic [3] . a c c e p t e d m a n u s c r i p t 3 tb notification rates have decreased by 78% in india, 47.8% in shanghai china, 43% in uganda, 34% in nigeria, 33% in south africa, 33% in japan, 24% in south korea and 20% in taiwan in early 2020 compared to the same period over the last 2-5 years [4] [5] [6] [7] [8] [9] [10] [11] . most settings are now reporting an increase in tb notification rates as covid-19 rates decline and restrictive social measures are being lifted. it is therefore evident that the short-term infection control measures implemented to reduce covid-19 transmission does not directly influence tb incidence rates as tb has a long and varying incubation period. thus, the resulting decline in case notifications is likely an unintended consequence of limited access to health care services during the pandemic. all through history the burden of tb has been impacted by major disruption resulting from natural disasters, war and infectious disease pandemics. the 1889 russian and 1918 spanish influenza pandemics resulted in increased tb related mortality [12] . during the first and second world wars, a quarter of the resulting deaths were attributed to tb [13] . with the hiv pandemic in 1980, tb emerged as an opportunistic infection leading to widespread mortality [14] . resulting from limited access to diagnosis and treatment, could potentially escalate tb related mortality have a lasting impact on tb burden. tb-related mortality is projected to increase by an additional 20% over the next five years [18] . the marked decreased in tb case detection due to missed diagnoses has resulted in an accumulation of undetected tb, contributing to ongoing tb transmission, and higher rates of latent tb infection. latent tb infections could potentially take years to manifest into active tb infection. thus, disruption to health care services will likely result in short term escalation in tb mortality and postlockdown surge in tb case notification, followed by a sustained increase in tb incidence and mortality over the next several years, with long-term consequences on tb elimination. as a result of the strict physical distancing measures implemented, many individuals were deterred from seeking medical care. this was compounded by patient concerns of increased exposure to the sars-cov-2 virus at healthcare facilities. health facilities reported a decline in the number of patients collecting treatment on schedule during lock-down periods. this could have severe consequences for tb control, importantly the development of drugresistant tb. the re-allocation of laboratory infrastructure and health services to control covid-19 could have inadvertently impacted tb diagnosis and treatment [19] . in a survey conducted in 106 countries, 78% of tb programs reported widespread disruption to tb services as a result of covid-19 [20] . twenty high-burden tb countries further reported that tb program staff were engaged in covid-19 related activities, disrupting tb services [21] . routine immunization programs also faced disruption during this time, with some settings reporting a decrease in bcg immunization by 66.8% [22] . a further challenge threatening the diagnosis of tb is the overlap of symptoms between the two diseases. both affect the respiratory system with an overlap in symptoms such as cough, fever and difficulty in a c c e p t e d m a n u s c r i p t 5 breathing with varying severity and duration. these similarities could have potentially hindered the diagnosis of tb as patients presenting with respiratory symptoms are more likely to be prioritized for covid screening. what comes next for tb control? it is apparent that measures taken in response to the covid-19 pandemic will have far reaching consequences on the tb epidemic. in order to mitigate the consequences of covid-19 on tb and preserve the gains made over the last decade in the fight against tb, it is important for control programs to strengthen tb control strategies and prioritize delivery of tb services. such strategies should include intensified case finding, scale-up of contact tracing efforts, community engagement to maintain awareness of symptoms suggestive of tb and vaccine catch-up, the use of digital health tools for remote management of tb in settings still affected by covid-19 disruption and ensuring continuity of tb treatment and care, including uninterrupted access to drugs and diagnostics, for all tb patients. above all, prioritized funding and mobilization of resources will be required to overcome the formidable challenges imposed by the covid-19 pandemic. none of the authors has any conflicts to disclose. a c c e p t e d m a n u s c r i p t m a n u s c r i p t coronavirus disease (covid-19) weekly epidemiological update the potential impact of the covid-19 response on tuberculosis in high-burden countries: a modelling analysis collateral impact of the covid-19 pandemic on tuberculosis control in tuberculosis in the era of covid-19 in india impact of the covid-19 pandemic on the detection of tb in shanghai, china impact of shelter-in-place on tb case notifications and mortality during the covid-19 pandemic impact of covid-19 on tb care: experiences of a treatment centre in nigeria impact of covid-19 intervention on tb testing in south africa the covid-19 pandemic and the true incidence of tuberculosis in japan effect of covid-19 on tuberculosis notification the covid-19 pandemic and tuberculosis in taiwan influenza pandemics and tuberculosis mortality in 1889 and 1918: analysis of historical data from switzerland interpreting the decline in tuberculosis: the role of secular trends in effective contact the growing burden of tuberculosis: global trends and interactions with the hiv epidemic lowered tuberculosis notifications and deterred health care seeking during the sars epidemic in hong kong public health impact of the ebola outbreak in west africa: seizing opportunities for the future middle east respiratory syndrome coronavirus and pulmonary tuberculosis coinfection: implications for infection control potential impact of the covid-19 pandemic on hiv, tuberculosis, and malaria in low-income and middle-income countries: a modelling study covid-19 affects hiv and tuberculosis care global fund survey: majority of hiv, tb and malaria programs face disruptions as a result of covid-19 the tb response is heavily impacted by the covid-19 pandemic immunizations during covid-19 restrictions-insights from zindagi mehfooz eir big data analysis accessed: 2 nd key: cord-318204-t024w7h6 authors: fang, ferric c; naccache, samia n; greninger, alexander l title: the laboratory diagnosis of covid-19-frequently-asked questions date: 2020-06-08 journal: clin infect dis doi: 10.1093/cid/ciaa742 sha: doc_id: 318204 cord_uid: t024w7h6 diagnostic testing has played and will continue to play a major role in the covid-19 pandemic. the ability to detect the sars-cov-2 coronavirus in respiratory secretions is essential to determine when an individual is infected and potentially infectious to others. viral detection is used for the identification, management and isolation of individual patients. viral detection is also used to determine when the virus has entered a community and how rapidly it is spreading. as communities attempt to re-open following periods of shutdown, the detection of both sars-cov-2 and specific antibodies recognizing the virus will become increasingly important as a means to assess infection and immunity in individuals and communities. here we discuss questions commonly asked by clinicians about covid-19 diagnostic testing. limitations in the availability of ppe and testing supplies, in addition to the operational difficulty of scaling np swab collections for asymptomatic screening, have led to the evaluation of alternative samples including nasal swabs, mid-turbinate swabs, oropharyngeal swabs, and saliva. saliva is particularly attractive as it requires neither swabs nor transport media, although collection and processing of saliva presents other challenges (20) . the comparability of these specimen types for a qualitative test depends on the viral load present at the time of infection. oropharyngeal swabs have shown less sensitivity compared to nasopharyngeal and nasal swabs (21) . nasal swabs and nasopharyngeal swabs may be comparable in sensitivity, but more studies are needed to compare these specimens across different patient populations. new specimen types for an eua test must receive a specific amendment for that specimen type before they can be reported. some studies have indicated that self-collected samples are comparable in sensitivity to those collected by health care providers, which can obviate the need to use ppe for testing (22) (23) (24) (25) . although sputum and bronchoalveolar lavage samples may have higher viral loads and can therefore provide greater test sensitivity than upper respiratory samples (26, 27) , particularly at later stages of illness, they entail a higher risk of aerosol generation or require an invasive procedure, so these sample types are obtained more selectively. a c c e p t e d m a n u s c r i p t (28, 29) . this may be a consequence of the variable distribution of the ace2 viral receptor protein in the respiratory tract (30) . interestingly, viral tropism for the lower respiratory tract was also seen during the h1n1 influenza epidemic (31) . is it worth repeating a test after a patient has tested negative? in view of the less than ideal sensitivity of an np swab to detect sars-cov-2 infection, it may be useful to repeat testing in a patient in whom the clinical suspicion is high (32) . in our experience, the yield of repeat testing from the same source is low (33) . however, the yield from repeat testing may be substantial in higher prevalence settings (34) . shedding culturable virus after about one week from symptom onset, but can continue to have detectable viral rna in their respiratory tracts for longer periods of time (35, 36) . more severely ill patients will remain pcr-positive for longer, sometimes extending for weeks-to-months (37) . sars-cov-2 rna can also be found in stool samples and typically continues to be detectable in the feces for weeks (38) . evidence indicates that human intestinal epithelial cells can support replication of a c c e p t e d m a n u s c r i p t 7 the virus (39) , and sars-cov-2 has been cultured from stool samples (40) . fecal-oral transmission of sars-cov-2 has not been demonstrated, but there is concern that fecal shedding might contribute to the spread of infection. does a positive specimen mean that a patient is infectious? not necessarily. although viral nucleic acids can be detected during convalescence (35) , culturable virus is believed to represent a better correlate of infectivity (41) . however, sars-cov-2 culture requires a bsl-3 facility and there are no authorized clinical assays utilizing sars-cov-2 viral culture at this time, so clinical monitoring is dependent on rna detection. negative? two negative pcr assays at least 24 hours apart are commonly used as a criterion to discontinue isolation (42) . however, a number of patients will revert to pcr-positivity after two negative samples (43) . this may reflect fluctuations in the quantity of viral rna shedding during recovery. when monitoring for viral load as inferred by real-time pcr cycle threshold (ct), cases that have reverted to positivity consistently exhibit high ct values indicative of a low viral load (44) . there is presently little evidence of true virological and clinical relapse, and the prognosis for these patients appears to be good. a c c e p t e d m a n u s c r i p t 8 is quantitative pcr (viral load) useful? there is some correlation between illness severity and viral load on presentation, as inferred by the real-time pcr cycle threshold (ct) (37, 45) . however, an isolated viral load estimate is of limited prognostic value, and even asymptomatic individuals may have high viral loads (46) . viral load trends may have greater value and might help to inform decisions to initiate a trial of antiviral therapy, but viral loads typically decline regardless of the clinical course (3, 6, 47) . older patients tend to have higher viral loads (6), just as they are at greater risk for more severe or critical illness (48, 49) . imply that only one of two pcr targets was detected, and confirmation with a different assay that detects alternative targets is recommended. given the high specificity of the pcr assays, most inconclusive results will ultimately be confirmed as positive. inconclusive or indeterminate results could also indicate that the internal controls failed and may indicate a technical issue, such as the presence of a pcr-inhibitory substance in the sample. different labs and tests may use different terminology, so it is worth contacting a given clinical laboratory to determine how they report low positive results. diagnostic platforms used for the detection of specific antibodies to sars-cov-2 proteins include rapid diagnostic tests (rdt) such as lateral flow assays (lfa), enzyme-linked immunosorbent assays (elisa), neutralization assays and chemiluminescent immunoassays (50) . only neutralization assays can provide information regarding the ability of antibodies to inhibit viral growth. the performance of various serologic tests is more variable than the rt-pcr assays for sars-cov-2 (8, (51) (52) (53) . this is particularly important because the positive or negative predictive value of a test is dependent not only on the intrinsic test accuracy but also on the prevalence of disease in the population. an insensitive test will have a poor ability to exclude the presence of disease when prevalence is high, but more germane to covid-19, a test with low specificity will have a poor ability to indicate the presence of disease when the prevalence is low. when only a few percent of the population have immunity to sars-cov-2, as is presently the case in most regions, a positive result from a serologic test with low specificity will be more likely to represent a false-positive. there are four kinds of human coronavirus that cause mild-to-moderate seasonal respiratory tract infections: 229e, nl63, oc43 and hku1. cross-reactivity with antibodies to seasonal coronaviruses is a theoretical concern for a sars-cov-2 serologic test, but for most of the commercial assays evaluated thus far, this does not seem to be the case (54, 55) . to rule-out cross reactivity, a new assay should be tested against a panel of serum samples that pre-date the emergence of sars-cov-2, ideally more than 500, to a c c e p t e d m a n u s c r i p t 10 accurately ascertain specificity. cross-reactivity between sars-cov-1 or mers-cov and sars-cov-2 is more likely (56) , but should be a limited concern. are serologic tests useful to diagnose acute covid-19? although the primary use of serologic tests is to determine prior exposure to sars-cov-2, the detection of specific antibodies may support the diagnosis of covid-19 in a patient with a high clinical suspicion but negative pcr tests (57-59). igm and igg directed against sars-cov-2 may appear as early as 3-6 days after the onset of symptoms. by three weeks, nearly all patients have seroconverted, and the antibodies persist for at least two months, with igg showing greater persistence (4-8, 59). the duration of antibody responses to sars-cov2 is unknown. antibody responses to the common respiratory coronaviruses decay after a few years (56) , and it is suspected that immunity to sars-cov-2 will be similar. antibody responses have been observed in nearly all patients with covid-19, although it is possible that some very mild or asymptomatic infections, or infections in immunocompromised patients, may not result in seroconversion (5, 60) . a c c e p t e d m a n u s c r i p t 11 does a positive antibody test mean that a patient is immune? it is likely that the detection of specific antibodies in a patient with a history of covid-19-like illness will be indicative of at least some degree of immunity (61) . experimental animals re-challenged with pathogenic coronaviruses exhibit resistance to re-infection (62) . however, a quantitative cutoff of antibody titer that correlates with protective immunity is undefined. as with other viruses, it is possible that a low titer of antibodies is not protective. also, it is not presently known whether neutralizing antibodies are the primary mechanism of immune protection. in fact, higher antibody titers are observed in patients with more severe illness (5, 6, 51) . as patients with mild covid-19 may recover despite low antibody titers, and patients with severe covid-19 have persistent illness despite the development of high antibody titers, one may question whether neutralizing antibodies are in fact protective. the reported therapeutic benefits of convalescent plasma might be due to constituents other than neutralizing antibodies (63) . moreover, the development of neutralizing antibodies is accompanied by t cell responses (64, 65) , which may contribute to protection. are quantitative serologies helpful? because sars-cov-2 is a new human pathogen, pre-existing adaptive immunity is non-existent, so acute and convalescent titers are not required to establish the diagnosis of covid-19. until specific cutoffs are identified as a correlate of protective immunity, qualitative serologic results are sufficient to provide clinical guidance. however, reporting of quantitative serological read-outs could offer clinicians more information about the potential for false positives and false negatives for values near the positivity threshold. a c c e p t e d m a n u s c r i p t 12 is there a correlation between age and antibody titer? older age correlates with a higher likelihood of severe illness from covid-19 and with the development of higher antibody titers (59), perhaps due to a higher antigen load. is point-of-care testing available? affordable point-of-care (poc) diagnostics for sars-cov-2 could facilitate the widespread testing and contact tracing strategies proposed for post-pandemic wave containment (66) . however, performance characteristics and usability are critical parameters for these tests, as they are typically deployed without the quality assurance apparatus of a high complexity laboratory. a poc antigen detection assay was recently authorized by the fda, but is known to be less sensitive than pcr, so its clinical role has yet to be defined. negative results using this assay should be confirmed by a more sensitive method in most instances. other assay technologies, including a crispr-based nucleic acid detection system (67), may be utilized in poc formats in the future if appropriate sensitivity can be achieved. a variety of biomarkers, including lymphocyte count, neutrophil-tolymphocyte ratio, crp, troponin t, d-dimer, ldh, procalcitonin, il-6 and ferritin are predictive of disease progression and mortality in covid-19 (table 1) (68, 69) . these laboratory tests play a vital role in identifying patients at risk for complications and to guide treatment interventions. early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application temporal dynamics in viral shedding and transmissibility of covid-19 diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 antibody responses to sars-cov-2 in patients with covid-19 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody detection and dynamic characteristics in patients with covid-19 performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in presumed asymptomatic carrier transmission of covid-19 a covid-19 transmission within a family cluster by presymptomatic infectors in china contact tracing assessment of covid-19 transmission dynamics in taiwan and risk at different exposure periods before and after symptom onset covid-19 illness in native and immunosuppressed states: a clinicaltherapeutic staging proposal infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing failure of the cobas® sars-cov-2 (roche) e-gene assay is associated with a c-to-t transition at position 26340 of the sars-cov-2 genome diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: a narrative review food and drug administration. emergency use authorizations comparison of four molecular in vitro diagnostic assays for the detection of sars-cov-2 in nasopharyngeal specimens comparison of commercially available and laboratory developed assays for in vitro detection of sars-cov-2 in clinical laboratories performance of abbott id now covid-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a new york city academic institution saliva as a non-invasive sample for the detection of sars-cov-2: a systematic revieew comparison of nasopharyngeal and oropharyngeal swabs for sars-cov-2 detection in 353 patients received tests with both specimens simultaneously consistent detection of 2019 novel coronavirus in saliva effect of throat washings on detection of 2019 novel coronavirus saliva as a non-invasive specimen for detection of sars-cov-2 patient-collected tongue, nasal, and mid-turbinate swabs for sars-cov-2 yield equivalent sensitivity to health care worker collected nasopharyngeal swabs detection of sars-cov-2 in different types of clinical specimens sars-cov-2 viral load in clinical samples of critically ill patients correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases diagnosis of the coronavirus disease (covid-19): rrt-pcr or ct sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract diagnosis of influenza from lower respiratory tract sampling after negative upper respiratory tract sampling infectious diseases society of america guidelines on the diagnosis of covid-19 occurrence and timing of subsequent sars-cov-2 rt-pcr positivity among initially negative patients clinical performance of sars-cov-2 molecular testing virological assessment of hospitalized patients with covid-2019 predicting infectious sars-cov-2 from diagnostic samples viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china prolonged presence of sars-cov-2 viral rna in faecal samples sars-cov-2 productively infects human gut enterocytes infectious sars-cov_2 in feces of patient with severe covid-19 sars-cov-2 shedding and infectivity discontinuation of transmission-based precautions and disposition of patients with covid-19 in healthcare settings pcr assays turned positive in 25 discharged covid-19 patients findings from investigation and analysis of re-positive cases viral dynamics in mild and severe cases of covid-19 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility clinical and virological data of the first cases of covid-19 in europe: a case series estimating clinical severity of covid-19 from the transmission dynamics in wuhan, china opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients the role of antibody testing for sars-cov-2: is there one? severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease test performance evaluation of sars-cov-2 serological assays evaluation of nine commercial sars-cov-2 immunoassays patients with common cold coronavirusaes tested negative for igg antibody to sars-cov-2 validation and performance comparison of three sars-cov-2 antibody assays a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of of disease profiling early humoral response to diagnose novel coronavirus disease (covid-19) early detection of sars-cov-2 antibodies in covid-19 patients as a serologic marker of infection longitudinal change of sars-cov2 antibodies in patients with covid-19 different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid-19 patients covid-19 and postinfection immunity: limited evidence, many remaining questions sars-cov-2 infection protects against rechallenge in rhesus macaques convalescent plasma in covid-19: possible mechanisms of action detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals laboratory diagnosis of emerging human coronavirus infections -the state of the art point-of-care testing for covid-19 using sherlock diagnostics hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis elevated levels of il-6 and crp predict the need for mechanical ventilation in covid-19 interpreting diagnostic tests for sars-cov-2 a c c e p t e d m a n u s c r i p t 15 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-315356-9zf2h0xr authors: apisarnthanarak, anucha; warren, david k.; fraser, victoria j. title: issues relevant to the adoption and modification of hospital infection-control recommendations for avian influenza (h5n1 infection) in developing countries date: 2007-11-15 journal: clin infect dis doi: 10.1086/522538 sha: doc_id: 315356 cord_uid: 9zf2h0xr the reemergence of avian influenza (h5n1 infection) has heightened concern for a potential human influenza pandemic. recommendations regarding preparation for a global avian influenza pandemic are available, and it is imperative that health care workers participate in preparedness planning and training. in developing countries, health care worker preparedness training should address the modes of avian influenza transmission and specify how to implement appropriate infection-control strategies to prevent and control the spread of avian influenza. we provide evidence for avian influenza transmission methods and identify prevention strategies relevant to infection control for hospitals in developing countries. pandemic influenza preparedness plans must include health care administrative support, mechanisms to rapidly create temporary isolation facilities, systems to restrict access to exposed health care workers, and plans to involve specialists to screen and identify cases early, to provide for continuous monitoring to ensure adherence to optimal infection-control practices, and to provide regular feedback to health care workers it is estimated that the next influenza pandemic will infect ∼20% of the world population. one in every 100 individuals with influenza is expected to be hospitalized, and 7 million people will potentially die within a few months [1] [2] [3] . the ongoing avian influenza (h5n1 infection) epidemic poses significant risks to both humans and animals [4] [5] [6] [7] . the potential exists for cross-species transmission to humans and subsequent reassortment of avian and human influenza viruses in coinfected individuals [8] . pandemic influenza planning and worldwide surveillance are key factors in mounting an effective global preparedness strategy for avian influenza [9] . although recommendations for hospital infection control for avian influenza are available [10] , adopting these recommendations has been an issue in developing countries. because infection-control resources, including infrastructure and personal protective equipment (ppe), may vary considerably in different settings, hospital policy-makers in developing countries need to make decisions regarding the use of infectioncontrol resources on the basis of disease prevalence, local priorities, personnel availability, and financial constraints. it is, therefore, important for health care workers (hcws), who in many cases serve as first responders (i.e., may triage and eval-uate an index case), to understand the dynamics of avian influenza transmission and to recognize the appropriate infection-control strategies recommended for the prevention and control of avian influenza. current evidence suggests that h5n1 viral infection occurs via bird-to-human transmission, possibly via environment-to-human transmission, and via limited, nonsustained human-to-human transmission [11] . to date, animal-to-human transmission is thought to be the predominant mode of avian influenza acquisition in humans [12, 13] . risk factors include plucking and preparing ill birds, handling cocks for fighting events and tourism, playing with poultry, consumption of duck blood or undercooked poultry, and exposure to live poultry within 1 week prior to the onset of illness [11, 12] . interestingly, interspecies transmission of avian influenza viruses has occurred in experimental settings from chickens to tigers, chickens to leopards, and chickens to domestic cats [14] [15] [16] . human-to-human transmission of avian influenza has occurred via intimate contact without the use of barrier precautions but has not occurred from casual, social contact [17] . serological studies of avian influenza among exposed hcws, household contacts, and poultry cullers suggest that human-to-human transmission to date has been inefficient and that antibodies may develop in asymptomatic, exposed persons [13, [17] [18] [19] [20] [21] . albeit rare, probable human-to-human transmission of h5n1 virus has been reported in several household clusters [8, 22] . although it is unclear what role aerosols play in the natural transmission of avian influenza, it is important to keep in mind that the main goal of infection control is to minimize the risk of transmission of avian influenza within health care facilities. the recommendation to routinely use n95 respirators during an influenza pandemic is complicated by several factors. first, n95 respirators require fit testing. once tested, any individual must use only the brand and size of respirator for which they have been certified. during a pandemic, it is likely that supplies of respirators may be limited, and supply chains may become disrupted. therefore, it will be difficult to provide the appropriate size of respirator for each hcw. also, the large number of n95 respirators marketed will make the stockpiling of appropriate numbers and sizes of respirators logistically difficult. the reproducibility of fit testing over time has been questioned [23] , and prolonged use of n95 respirators has been shown to cause headaches [24] and facial discomfort [25] and may result in hypoxemia [26] . poor compliance or incorrect use of an n95 respirator may negate any potential benefit that might be gained from their use. therefore, pandemic influenza plans in developing countries might have to take these realities into consideration and use a more risk-based stratification approach to address droplet and contact precautions versus airborne and contact precautions. the infection-control components of an avian influenza preparedness plan include: (1) basic infection-control measures, including hand hygiene; (2) use of ppe; (3) vaccination with seasonal influenza vaccines; (4) administration of prophylactic antiviral drugs; (5) surveillance and monitoring for hcw exposures; (6) evaluation of ill hcws; (7) precautions for household and close contacts; and (8) limited visitation, if not quarantine, of ill patients. the rationale for most pandemic influenza preparedness plans are based on several assumptions: (1) it is unlikely that appropriate vaccines will be readily available in adequate supplies; (2) populations at high risk for complications may increase tremendously, given the high attack rate and mortality among adults aged 15-35 years seen in the 1918 pandemic [27] ; and (3) containing illness among hcws during an influenza pandemic will be challenging even if there is excellent compliance with infection-control practices. in general, influenza attack rates during outbreaks among unvaccinated hcws are reported to be as high as 59% [28] . influenza attack rates often remain 110% among unvaccinated hcws, even when there are excellent infection-control measures and behavioral compliance [29] . viral shedding of influenza in adults can extend for 7 days after symptom onset and for weeks in infants and immunocompromised individuals. this makes environmental control of influenza even more difficult in health care facilities. thus, the initial specific protection of hcws will need to include available antiviral agents (e.g., oseltamivir and zanamivir) for chemoprophylaxis and treatment. a recently proposed university hospital-based preparedness plan has recommended the stockpiling of oseltamivir [29] . there are 4 potential strategies for using antiviral agents during an influenza outbreak [30, 31] : chemoprophylaxis for the entire influenza outbreak and season, postexposure chemoprophylaxis, treatment of ill patients, and a combination of chemoprophylaxis and treatment. several studies support the use of prophylactic viral agents [31] [32] [33] [34] [35] . furthermore, a model of the 1957-1958 asian influenza pandemic predicted that 8 weeks of antiviral prophylaxis for close contacts of index case patients would have reduced the attack rate from 33% to 2% [36] . thus, targeted prophylaxis for hcws and patients would likely mean administering 6-8 weeks of prophylaxis to all vulnerable groups. although attractive, this strategy is prohibitively expensive for most hospitals and other health care facilities in both developed and developing countries. furthermore, widespread, long-term use of antiviral prophylaxis could promote antiviral resistance. an alternative strategy that focuses on the treatment of ill hcws, with some targeted prophylaxis of heavily exposed hcws, would be more financially feasible. this strategy would provide for treatment of those hcws directly caring for patients with influenza during a pandemic. recent studies have reported that neuraminidase inhibitors administered within 48 h of symptom onset decrease the duration of illness and viral shedding, the incidence of hospitalization, antibiotic use, and mortality [35, 37, 38] . health care workers should be tracked and monitored for influenza-like illness (e.g., myalgia and fever). it is likely that such monitoring would identify most of the ill hcws and, thus, allow timely administration of antiviral therapy. despite these recommendations, health care facilities in developing countries may find it difficult to implement antiviral therapy because of the associated high cost. for preparedness planning in health care facilities in developing countries, some practical issues that are relevant to the adoption and modification of the above recommendations should be considered [39] . health care administrative support. the level of occupational protection of hcws in developing countries has not met the minimal standards of the us occupational health and safety administration. despite the logistical and economic challenges, efforts to promote implementation of effective infection control and occupational health strategies are overdue, and a global need for such strategies is now recognized. given the experience of severe acute respiratory syndrome (sars) outbreaks, which occurred in both designated "sars hospitals" and "non-sars hospitals" [40] [41] [42] , global preparedness plans should facilitate administrative, fiscal, and infrastructure support for routine occupational health and safety programs for hcws, appropriate infection-control expertise, and infrastructure in health care settings, available ppe for hcws, and epidemiological resources for the prevention and control of emerging infectious diseases. these expenditures should not be viewed as an increase in the cost of health care but as preventive health and safety measures that insure protection of hcws and, ideally, provide an anticipated return on investment to the health care institution. development of practical surveillance strategies. surveillance strategies should be developed in accordance with the current level of the influenza pandemic and the existing local infrastructure. such strategies should be continuously monitored and modified. during the sars outbreak in hong kong, there were very few private rooms in which to isolate patients. some hospitals controlled the outbreak by having designated sars teams and geographically separate wards for patient triage, patients with confirmed sars cases, and patients without sars [43] . during an avian influenza outbreak, some hospitals in thailand performed surveillance for avian influenza among all patients who were admitted to the intensive care unit with severe community-acquired pneumonia [44, 45] . the estimated annual cost for avian influenza screening in a thai hospital was $7375 [44] . these studies reported a low detection rate for avian influenza (3 cases among 5496 patients). therefore, cost-effectiveness studies of screening programs, based on the level of local avian influenza activity, are warranted before this strategy can be adopted in areas where h5n1 infection is endemic. involvement of specialists. as in developed countries, health care providers with the least experience are often the first to evaluate patients with unrecognized emerging infectious diseases in developing countries [46, 47] . this may lead to delayed recognition of disease and missed opportunities to interrupt disease transmission [46, 47] . several reports from developing countries emphasize the added value of specialists (e.g., infectious diseases, pulmonary, and emergency medicine specialists) in screening for suspected cases of emerging infectious diseases and early recognition of atypical cases in acute and ambulatory care settings [7, 21, 41, 48] . given the difficulty of rapidly diagnosing avian influenza [49, 50] , early involvement of specialists could promote the obtaining of adequate, lower respiratory tract specimens from an index patient before that patient receives antiviral medication. although the value of infection control and health care epidemiology expertise has been formally recognized in north america and europe [51] [52] , this is not the case in most acute-care facilities in developing countries. creation of a temporary isolation ward during an epidemic. rapid creation of a temporary isolation ward using existing functional hospital units is readily applicable to clinical settings in developing countries. such units should be divided into a clean zone, for changing into and out of street clothes and for recording the hcw's name and entry and exit times; an intermediate zone, for donning ppe that has been modified according to resource availability (e.g., scrubs and double-glov-ing); and a contaminated zone, for entering isolation areas [53] . exhaust fans could be installed above windows in each room if isolation rooms with negative pressure ventilation are unavailable. at least 1 m of distance should be placed between patients to reduce the risk of cross-transmission through respiratory droplets. although creation of a temporary isolation ward seems to be attractive during an epidemic, it is important to realize that the ward designated for temporary isolation should have separate entrance and exit pathways for hcws and patients, and a one-way entrance and one-way exit should be designated for patients, to minimize problems involving cross-contamination (figure 1). as is the case with any set of practices, infection-control practices may be difficult to orchestrate without effective communication that clearly outlines the objectives for these practices. this point was emphasized in recent reports of increased rates of methicillin-resistant staphylococcus aureus acquisition in hong kong and singapore during the sars outbreak [54, 55] . these studies suggested that rates of methicillin-resistant s. aureus transmission increased when hcws adopted the nonstandard practice of wearing gloves and gowns all of the time. several infection-control practices, such as proper hand hygiene and the correct use of ppe, may need to be highly monitored, with feedback provided to hcws in a timely manner, to optimize appropriate infection-control practices and to reduce the risk of disease transmission. avian influenza is a likely candidate for the next influenza pandemic. given the global experience from the sars epidemics, it is reasonable to suspect that health care facilities may serve as the nidus for large-scale community outbreaks. health care facilities in developing countries will benefit from adopting or modifying their strategic pandemic influenza plan and identifying local expertise to optimize control of outbreaks at their earliest stages. inluneza pandemics of the 20th century the economic impact of pandemic influenza in the united states: priorities for intervention presentation at the who consultation on priority public health interventions before and during the influenza pandemic fear of human pandemic grows as bird flu sweeps through asia human disease from 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to hospital employees avian influenza h5n1 and healthcare workers seroprevalence of anti-h5 antibody among thai health care workers after exposure to avian influenza (h5n1) in a tertiary care center three indonesian clusters of h5n1 virus infection in 2005 fitting characteristics of eighteen n95 filtering-facepiece respirators headaches and the n95 face-mask amongst healthcare workers effects of wearing n95 and surgical facemasks on heart rate, thermal stress and subjective sensations the physiological impact of wearing an n95 mask during hemodialysis as a precaution against sars in patients with endstage renal disease capturing a killer flu virus influenza in the acute hospital setting preparing for pandemic influenza: should hospitals stockpile oseltamivir? who guidelines on the use of vaccines and antivirals during influenza pandemics: global health security-epidemic alert and response oseltamivir (tamiflu) and its potential for use in the event of an influenza pandemic safety and 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care unit patients with communityacquired pneumonia surveillance for avian influenza in human beings in thailand severe acute respiratory syndrome-singapore mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of healthcare workers in a teaching hospital in singapore avian influenza a (h5n1) infection in eastern turkey in 2006 difficulty in the rapid diagnosis of avian influenza a infection: thailand experience requirements for infrastructure and essential activities of infection control and epidemiology in hospitals: a consensus panel report. society for healthcare epidemiology of america apic/chica-canada infection control and epidemiology: professional and practice standards. association for professionals in infection control and epidemiology and the community and hospital infection control association-canada rapid creation of a temporary isolation ward for patients with severe acute respiratory syndrome in taiwan increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome paradoxical increase in methicillin-resistant staphylococcus aureus acquisition rates despite barrier precautions and increased hand washing compliance during an outbreak of severe acute respiratory syndrome financial support. national center and genetic engineering and biotechnology, national science and technology development agency (bt-b-01-mg-13-5019) and thai research fund (to a.a.).potential conflicts of interest. d.k.w. is a consultant for and has received research support from 3m healthcare and is a consultant for enturia. a.a. and v.j.f.: no conflicts. key: cord-328267-tk0zc8il authors: liu, qiao; lu, peng; shen, ye; li, changwei; wang, jianming; zhu, limei; lu, wei; martinez, leonardo title: collateral impact of the covid-19 pandemic on tuberculosis control in jiangsu province, china date: 2020-08-28 journal: clin infect dis doi: 10.1093/cid/ciaa1289 sha: doc_id: 328267 cord_uid: tk0zc8il the covid-19 pandemic may impede global tuberculosis elimination goals. in jiangsu province, china, tuberculosis notifications dropped 52% in 2020 compared to 2015–2019. treatment completion and screening for drug resistance decreased continuously in 2020. urgent attention must be paid to tuberculosis control efforts during and after the covid-19 pandemic. the tuberculosis burden in china has seen dramatic reductions in the past three decades. 1, 2 between 1990 and 2010, there was a 65% and 48% decrease in the prevalence of smear-and bacteriologically-positive tuberculosis throughout china. 2 these reductions represent hundreds of thousands of saved lives over the time-period and maintaining these gains in the tuberculosis burden are essential to make goals of tuberculosis elimination by 2035 set by global health organizations. 3 the covid-19 pandemic has the potential to substantially impact disease control for a range of diseases. considerable increases in hospitalizations due to covid-19 may overload health systems. for example, in the most heavily impacted regions of italy, the national healthcare service was on the verge of collapse. 4, 5 additionally, health care workers have become sick or are repurposed to control of covid-19 and laboratories are de-prioritized diagnosis of tuberculosis (and other diseases) concentrating on covid-19 diagnosis. there is a concern that, due to health care shortfalls and fear of contracting covid-19, individuals will not seek medical attention when needed. in 2001, the sars epidemic caused a short downfall in tuberculosis case notifications, later regressing to pre-epidemic levels. 6 recent studies have documented substantial decreases in secondary diseases including myocardial infarctions and strokes. 7, 8 in addition, decreases in immunization rates and hiv testing have been reported in some settings. [9] [10] the impact of the covid-19 pandemic on disease control for tuberculosis at a population-level is not well known. [11] [12] [13] to address this knowledge gap, we investigated several markers of tuberculosis control in jiangsu province, china, covering a population of approximately 80 million inhabitants. we compared tuberculosis case notifications, tuberculosis treatment outcomes, and diagnostic screening for multidrug resistance (mdr) among tuberculosis patients from 2015-2020 before and after the covid-19 pandemic began in china. a c c e p t e d m a n u s c r i p t tuberculosis is a reportable disease in china and all persons with tuberculosis are managed through the tuberculosis management information system of jiangsu province. due to this, patient diagnoses can be tracked at a city-and provincial-level over time. we included weekly data from january 2015 to the end of may 2020. we concentrated our analysis on annual markers reported to the world health organization including the absolute number of tuberculosis notifications, notification rate, and tuberculosis treatment success rate. 3 other measures were also analyzed to measure laboratory capacity and screening for multi-drug resistant (mdr) tuberculosis. we calculated the proportion of persons in which treatment previously failed; iii) sputum smear positive tuberculosis patients that were close contacts of mdr-tuberculosis patients; iv) a previous relapse event; or v) new tuberculosis patients who remained smear positive two months into anti-tuberculosis treatment. we compared weekly tuberculosis notifications between 2015-2019 with 2020 levels. we also assessed indicators before and after provincial-wide quarantine (january 24, 2020) and when 90% of businesses were re-opened at the provincial-level (march 27, 2020). we stratified our results at the city-level to investigate whether our results were modified by a certain location in the province. we a c c e p t e d m a n u s c r i p t discussion this is the first data from china reporting the potential indirect impact on tuberculosis control of the covid-19 pandemic. we found a substantial reduction, in excess of 50%, in tuberculosis notifications in 2020 compared to 2015-2019. there is substantial concern that the covid-19 pandemic may impede global tuberculosis elimination goals by 2035 and our results suggest that tuberculosis control may be severely impacted, at least temporarily. tuberculosis treatment outcomes and diagnostic screening for mdr among tuberculosis patients was also reduced in 2020 indicating that health care access may have reduced or health professionals may have been unavailable in dealing with tuberculosis control after being redirected to covid-19 control efforts. vigilance, reprioritization of tuberculosis control practices, and sustained funding and resources for drug resistant screening, diagnoses, and promoting treatment adherence must be immediately conducted in endemic settings to mitigate the damage to tuberculosis control suggested by these results. the decreased number of tuberculosis notifications in 2020 likely suggests lower tuberculosis patient case detection in the province. social distancing interventions, which took place in much of china, may impact mycobacterium tuberculosis transmission as well as health care access. interventions, such as quarantine and social distancing, may have influenced tuberculosis notification numbers by reducing transmission. however, the long incubation period of tuberculosis 14 suggests that massscale interventions would not have shown immediate effects as seen in this study. in addition, reductions in treatment completion and mdr screening during this time period suggest that both deterred health care seeking and community disease management are likely drivers of these trends. a decrease in tuberculosis case notifications in 2020 has also been seen in other recent findings from settings outside of china. 15, 16 in nigeria, there was a 35% and 34% decrease in the number of presumptive and active tuberculosis notifications from january to may 2020 15 while, in south korea, tuberculosis notifications decreased by 24% in 2020 compared to prior years. 16 tuberculosis notifications and diagnoses must continue to be monitored in settings that have seen this reduction. the finding that screening for drug resistance among tuberculosis patients has reduced by 15-17% has important implications to the tuberculosis epidemic in china. this is likely to lead to community transmission of drug resistance at a critical time when sustained tuberculosis control efforts in china has resulted in significant improvement in tuberculosis burden. 1, 2 reductions in mdr screening are likely due to de-prioritization of tuberculosis testing and diagnoses in laboratories which have been a c c e p t e d m a n u s c r i p t re-directed to covid-19 testing. drug resistant screening among tuberculosis patients was altered from 2015-2020 and this may have impacted our results; however, since genexpert became available to all counties in the province in 2020, we would've expected an increase, rather than a decrease, in screening uptake of tuberculosis patients for drug resistance if the covid-19 pandemic had not occurred. monitoring drug resistant tuberculosis trends in late 2020, 2021, and 2022 will be critical to understanding whether this decrease in testing leads to population-wide increases in drugresistance. our analysis suggests that collateral effects of the covid-19 pandemic on tuberculosis control are substantial, with a reduction of 36%-52% in tuberculosis notifications in 2020 compared to 2015-2019. substantial decreases were also seen in patient treatment success and screening for mdr among new and high-risk tuberculosis patients. these findings suggest health care seeking and management are largely responsible for tese trends. urgent attention must be paid to tuberculosis control efforts 13, 17 to mitigate further impact of the covid-19 pandemic on tuberculosis morbidity and mortality. a c c e p t e d m a n u s c r i p t notes acknowledgement: the research was considered part of routine public health surveillance and, due to this, was considered exempt from ethical approval. all data was de-identified prior to use and identifying information was not available. qiao liu, wei lu, limei zhu, and leonardo martinez conceived the study. qiao liu, peng lu, wei lu, and limei zhu collected the data. leonardo martinez analyzed the data and made the first draft of the manuscript. limei zhu, wei lu, and jianming wang participated in the study design. all authors assisted in data interpretation. all authors read and edited the drafted manuscript for important intellectual content. all authors have read and approved the final manuscript. the funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had access to all the data in the study and had final responsibility for the decision to submit for publication. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the effect of tuberculosis control in china tuberculosis prevalence in china, 1990-2010; a longitudinal analysis of national survey data the italian health system and the covid-19 challenge indications for healthcare surge capacity in european countries facing an exponential increase in coronavirus disease (covid-19) cases lowered tuberculosis notifications and deterred health care seeking during the sars epidemic in hong kong collateral effect of covid-19 on stroke evaluation in the united states the covid-19 pandemic and the incidence of acute myocardial infarction impact of covid-19 lockdown on routine immunisation in karachi, pakistan. the lancet global health impact of the covid-19 pandemic on hiv testing and assisted partner notification services impact of covid-19 on tuberculosis control in china anticipating the impact of the covid-19 pandemic on tb patients and tb control programmes tackling two pandemics: a plea on world tuberculosis day. the lancet respiratory medicine lifetime risks, incubation period, and serial interval of tuberculosis impact of covid-19 on tb care: experiences of a treatment centre in nigeria effect of covid-19 on tuberculosis notification, south korea covid-19, tuberculosis, and poverty: preventing a perfect storm a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-331731-c2r0kfaz authors: anugwom, chimaobi m; aby, elizabeth s; debes, jose d title: inverse association between chronic hepatitis b infection and covid-19: immune-exhaustion or coincidence? date: 2020-06-05 journal: clin infect dis doi: 10.1093/cid/ciaa592 sha: doc_id: 331731 cord_uid: c2r0kfaz nan m a n u s c r i p t dear editor, we read with great interest the report by zhao et al, regarding a case of delayed immune response to sars-cov-2 in a patient with hiv and hcv co-infection [1] . the authors stipulate the previous hiv and hcv infection could confer immune dysfunction providing a differential immune response during covid-19 development. this report, as most initial reports, originate in china which has an intermediate-high prevalence of chronic hepatitis b (hbv) infection [2] . we evaluated all peer-reviewed articles, written in the english language, reporting cases of covid-19 infection and specifically defining rates of hbv infection and hospital admission, since december 1 st , 2019 until march 25 th , 2020 and found a surprisingly low prevalence of chronic hbv in covid-19 cases admitted to the hospital. indeed, of the 2054 cases that were reported with this information, only 28 patients (1.36%) were reported positive for hbv. several of these studies reported 0% incidence of hbv among individuals infected with covid-19. we matched the hbv-rates in covid-19 subjects to age-specific rates of hbv reported in the respective geographic areas of origin ( table 1 ). the median age of covid-19 infected individuals in the evaluated studies ranged between 47-51 years, corresponding to hbv rates ranging from 7-11% while the hbv rates of those with covid-19 remained between 0-1.3%. it is unclear whether this is a simple epidemiological "misconnection" or if being chronically infected with hbv impacts the chances of clinically significant infection with sars-cov-2 leading to less hospital admissions, in a similar fashion as that reported by zhao et al to hiv and hcv. in this regard, research has documented that, chronic hbv infection leads to a reduced or absent virus specific t-cell reactivity (although hbv-specific t cells). this phenomenon, a c c e p t e d m a n u s c r i p t known as "immune exhaustion", is manifested by an impaired ability of t-lymphocytes to produce appropriate cytokines secondary to years of continuous, yet inefficient, immune reaction to the virus [3] . immune exhaustion is also frequently observed in chronic hcv infection [4] . in this setting, it is plausible that the exhaustion of t-lymphocytes may affect their ability to respond to other viruses and reduce the degree of "cytokine storm" that has been noticed in covid-19 patients, thus culminating in a less severe disease. similar patterns of immune co-interaction with consequences in clinical presentation and prognosis have been reported in individuals infected with hbv and schistosomiasis [5] . a c c e p t e d m a n u s c r i p t early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv prevalence of hepatitis b virus infection in molecular and transcriptional basis of cd4⁺ t cell dysfunction during chronic infection the path to cancer and back: immune modulation during hepatitis c virus infection, progression to fibrosis and cancer, and unexpected roles of new antivirals helminth-induced immune modulation influences the outcome of acute and chronic hepatitis b virus infection a comparative study on the clinical features of covid-19 pneumonia to other pneumonias clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical progression of patients with covid-19 in epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore we thank drs. lanjuan li (wuhan, china) and barnaby young (singapore, singapore) for providing information from their manuscripts regarding hbv. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-317823-ztawznod authors: yehya, nadir; venkataramani, atheendar; harhay, michael o title: statewide interventions and covid-19 mortality in the united states: an observational study date: 2020-07-08 journal: clin infect dis doi: 10.1093/cid/ciaa923 sha: doc_id: 317823 cord_uid: ztawznod background: social distancing is encouraged to mitigate viral spreading during outbreaks. however, the association between distancing and patient-centered outcomes in covid-19 has not been demonstrated. in the united states social distancing orders are implemented at the state level with variable timing of onset. emergency declarations and school closures were two early statewide interventions. methods: to determine whether later distancing interventions were associated with higher mortality, we performed a state-level analysis in 55,146 covid-19 non-survivors. we tested the association between timing of emergency declarations and school closures with 28-day mortality using multivariable negative binomial regression. day 1 for each state was set to when they recorded ≥ 10 deaths. we performed sensitivity analyses to test model assumptions. results: at time of analysis, 37 of 50 states had ≥ 10 deaths and 28 follow-up days. both later emergency declaration (adjusted mortality rate ratio [amrr] 1.05 per day delay, 95% ci 1.00 to 1.09, p=0.040) and later school closure (amrr 1.05, 95% ci 1.01 to 1.09, p=0.008) were associated with more deaths. when assessing all 50 states and setting day 1 to the day a state recorded its first death, delays in declaring an emergency (amrr 1.05, 95% ci 1.01 to 1.09, p=0.020) or closing schools (amrr 1.06, 95% ci 1.03 to 1.09, p<0.001) were associated with more deaths. results were unchanged when excluding new york and new jersey. conclusions: later statewide emergency declarations and school closure were associated with higher covid-19 mortality. each day of delay increased mortality risk 5 to 6%. nonpharmaceutical interventions, such as social distancing and issuance of emergency public health warnings seeking to modify activity, are recommended to mitigate the spread of viral epidemics and pandemics [1] [2] [3] [4] [5] [6] [7] , including the current sars-cov-2 and its associated covid-19 disease. historical analysis of the 1918-1919 influenza pandemic during the second wave of infections that examined 43 cities in the united states demonstrated an association between earlier school closures and bans on public gatherings with lower mortality [5] . a recent modeling study supported multilayered nonpharmaceutical interventions, including quarantine, school closures, and workplace distancing, for covid-19 [8] . at this stage in the pandemic, the efficacy of social distancing measures on patientcentered outcomes specifically for sars-cov-2 and covid19 have not been demonstrated. in the united states, social distancing measures have been implemented primarily at the local and state levels, with evidence of mistrust for their efficacy or necessity. as these interventions are fundamentally political and decided upon by elected officials, real-time evidence of efficacy would be helpful for informing policy. if social distancing measures were causal for improved outcomes, we reasoned there would be a dose-response, with states implementing distancing measures later experiencing worse outcomes. this rationale is premised on the association retrospectively seen between timing of social distancing measures and mortality during the 1918-1919 influenza pandemic [5] . therefore, we assessed the association between the timing of emergency declarations and school closures, two specific statewide distancing measures, and subsequent covid-19 mortality. we hypothesized that states with delayed emergency declarations and school closures would experience higher covid-19 mortality. a c c e p t e d m a n u s c r i p t this was an ecologic study of publicly available data. the this was a decedent-only analysis of attributed covid-19 deaths in the hopkins coronavirus resource center between january 21, 2020 and april 29, 2020 (n = 55,146 non-survivors). we chose to analyze decedents given the relatively low and variable rates of testing between states [10, 11] , making number of cases unreliable. we reasoned that eventual covid-19 non-survivors were more likely to have experienced severe infection and have undergone testing, thereby making this the most accurate of available metrics to track pandemic spread. our co-primary exposures were the number of days between a state experiencing ≥ 10 covid-19 deaths (standardized across states as day 1) and implementation of a statewide emergency declaration (day of emergency declaration minus day 1), and separately school (kindergarten through grade 12) closure (day of school closure minus day 1). we chose emergency declarations and school closures as a c c e p t e d m a n u s c r i p t the primary exposures because they were unambiguous interventions. other distancing measures, such as bans on public gatherings, closure of non-essential businesses, and shelter-in-place orders were variably implemented between states, using divergent definitions, thresholds for maximum group sizes, and carve-out exemptions. the primary outcome was covid-19 mortality on day 28. we chose the timepoint of 28 days because we reasoned that, if a statewide emergency declarations and school closures impacted mortality, then several weeks would be required to allow for a reduction in transmission, hospitalizations, and mortality. potential state-level confounders considered a priori were 2019 population, population density, percent of the population < 18 years of age, percent ≥ 65 years of age, percent black, percent hispanic, and percent below census-designated poverty threshold. we included the country-level confounder of census-designated division, which divides the country into nine geographic regions. all analyses were performed in stata se/14.2 (statacorp, college station, tx, usa) on april 30, 2020 (ny and mh separately). summary data are presented as mean ± standard deviation (sd) or as proportions, and analyzed for monotonic trends using a non-parametric test of trend across tertiles of states ordered based on timing of emergency declarations or school closings. for our primary analyses, we used multivariable negative binomial regression to test the association between earlier emergency to test the dependence of our results on our assumptions, we performed a secondary analysis where we set day 1 to the day when a state recorded its first covid-19 death. exposures were calculated as before (day of emergency declaration or of school closures minus day 1), while the primary outcome was death on april 29, 2020. the negative binomial model allows for an offset to allow states to have different lengths of follow-up time. these analyses were adjusted for the same confounders as in the primary analyses. second, because deaths per million is a common method to compare localities, we provide an analysis testing the association between deaths per million at the state level after multivariable adjustment relative to timing of emergency declarations or statewide school closures. we set day 1 to equal when a state experienced at least 1 death per million, and followed deaths until april 29, 2020, allowing different follow-up times between states. finally, to account for the potential impact from the excess of deaths in the new york city metropolitan area, all models above were re-run excluding new york and new jersey. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. the corresponding author had full access to the data and had final responsibility for the decision to submit for publication. at the time of analysis on april 29, 2020, 37 of 50 states had experienced ≥ 10 deaths by april 2, 2020, thus ensuring availability of 28-day mortality. these 37 states comprised the cohort for our primary analyses (tables 1 and 2 ). timing of emergency declarations and school closing were highly correlated (r = 0.84, p < 0.001). states declared an emergency at a median of -14 (interquartile range [iqr] -18, -13) days relative to experiencing ≥ 10 covid-19 deaths, with all 37 states declaring an a c c e p t e d m a n u s c r i p t emergency prior to recording at least 10 deaths. states implemented school closures at a median of -9 (iqr -11, -4) days relative to experiencing ≥ 10 covid-19 deaths, with 32 of 37 states (86%) closing schools before experiencing at least 10 deaths. states declaring emergencies and closing schools earlier had a lower population, but were otherwise comparable to states closing schools later. after adjusting for confounders, later emergency declaration was associated with higher mortality ( figure 1a ). assigning the day that a particular state had ≥ 10 covid-19 deaths as day 1, every day a state delayed declaring an emergency increased 28-day mortality by 5% (mrr 1.05, 95% ci 1.00 to 1.09). when assigning day 1 as the day a state experienced its first covid-19 death and using data from all 50 states until april 29, 2020 ( figure 1b) , mortality increased by 5% (mrr 1.05, 95% ci 1.01 to 1.09) for every day of delay. results were consistent when excluding new york and new jersey from both analysis ( figures 1c and 1d) , with later declaration of emergency associated with higher mortality risk. later implementation of a statewide school closure was similarly associated with higher mortality (figure 2a ). assigning the day that a particular state had ≥ 10 covid-19 deaths as day 1, for every day a state delayed implementing a school closure, 28-day mortality risk increased by 5% (mrr 1.05, 95% ci 1.01 to 1.09). when assigning day 1 as the day that a state experienced its first covid-19 death and using all available data from all 50 states until april 29, 2020 ( figure 2b) , for every day a state delayed implementing a school closure, final mortality increased by 6% (mrr 1.06, 95% ci 1.03 to 1.09). results were consistent when excluding new york and new jersey from both analysis ( figures 2c and 2d ), with later implementation of school closures associated with higher mortality risk. a c c e p t e d m a n u s c r i p t when analyzing deaths per million as the primary outcome (supplementary figure 2) , later implementation of emergency declarations was associated with higher mortality (mrr 1.03, 95% ci 1.00 to 1.07), although this did not reach a traditional threshold for statistical significance (p = 0.077). later implementation of statewide school closure was similarly associated with higher mortality (mrr 1.04, 95% ci 1.01 to 1.07), which attained statistical significance (p = 0.014). results were similar when excluding new york and new jersey (supplementary figure 2b and 2d). as different regions of the united states experienced local epidemics, we assessed the cumulative death curves for each census-designated division (supplementary figure 3) . curves grew in all divisions, without evidence for plateauing. census divisions 2 and 3 were among the latest of the nine divisions to implement statewide emergency declarations, whereas divisions 4 and 8, which showed the slowest increase in deaths, were the two earliest. states implementing emergency declarations or school closures later in the course of the pandemic experienced higher covid-19 mortality, with each day of delay increasing mortality risk 5 to 6%. this effect size was attenuated when measured as deaths per million, but still consistent with lower mortality with earlier statewide declarations. to our knowledge, this is the first demonstration of an association between statewide social distancing orders and mortality during covid-19. our results support early social distancing as a nonpharmaceutical intervention for reducing mortality. our study design and results do not directly implicate timing of either emergency declarations or school closings specifically as causal for reduced mortality, although causality is plausible. emergency a c c e p t e d m a n u s c r i p t declarations, for example, have been shown to reduce social contacts. in a time-series analysis conducted by the national bureau of economic research (nber) during the early weeks of covid-19, state-level emergency declarations had the largest reduction in within-state mixing [13] . thus, early state-level social distancing measures, including declaration of an emergency, may have contributed to reducing the spread of covid-19, and by extension lower mortality. the causality of timing of school closures on covid-19 mortality is even less certain. while sarscov-2 appears not to cause as severe a disease in children [14, 15] , children are potential asymptomatic carriers. in an analysis of laboratory-confirmed cases in the united states up to april 2, 2020, 27% of patients < 18 years of age were completely asymptomatic, compared with 7% of patients 18 to 64 years of age [16] . modeling of influenza and a review of nonpharmaceutical interventions for 2003 sars suggested that school closings are effective at reducing transmission between children, but only modestly affected transmission in the larger population, particularly if children were not disproportionately affected by the virus [17, 18] . however, school closings also prompt additional social distancing measures as caretakers reduce their workplace presence and travel, causing indirect social distancing and improving overall population transmission rates [19] [20] [21] [22] [23] . in the aforementioned nber time-series analysis, school closing orders had negligible impact on within-state mixing, but reduced interstate travel by 10% [13] . thus, school closings may directly reduce sars-cov-2 transmission rates by reducing contact among asymptomatic pediatric carriers, and indirectly via changing contact patterns between adults. lastly, as this study is occurring early in covid-19, it is possible that the efficacy of both emergency declarations and school closings are not in reducing total eventual covid-19 mortality, but in reducing peak infection rates and improving hospital surge capacity [24]. alternatively and equally plausibly, both emergency declarations and the timing of school closures may be a proxy for the degree to which a state began to officially and unofficially implement significant social distancing [13] . at the time of analysis, all states had declared a statewide emergency, a c c e p t e d m a n u s c r i p t with 43 of 50 declaring prior to their first recorded covid-19 death. similarly, all had closed schools, and 41 of 50 states had a shelter-in-place order. in all but 2 cases, school closures preceded more restrictive shelter-in-place orders, with these orders occurring simultaneously for 2 states. emergency declarations and school closures were among the first social distancing measures implemented in the united states. thus, our results may reflect how quickly states responded to news about the size and severity of the spreading pandemic, with emergency declarations and school closures being among the first official nonpharmaceutical interventions, rather than protective effects specific to either intervention itself. the majority of states implemented statewide school closures, and all states declared emergencies, prior to experiencing 10 covid-19 deaths. hence, the time to declaring an emergency or implementing school closure relative to how we defined day 1 could have either a positive or a negative value. earlier implementing states were likely responding to the rapid increase in cases being reported in the early hotspot states. this is consistent with data suggesting that early intervention in an exponentially growing pandemic is more efficacious than later interventions [5] . our choice of death as an endpoint was due to concerns about inadequate and imprecise testing, thereby making counts of cases imprecise and highly variable between states. however, death is a lagging indicator, and increased time between these early interventions and eventual non-survival can result in imprecise effect estimates. reassuringly, our conclusions remained unchanged in all analyses performed. states which implemented emergency declarations and school closings later were more populous, and included the early hotspots of washington, california, and new york. it is likely that covid-19 had already attained a foothold in these states, and that subsequent states had the advantage of following their lead after witnessing the exponential increase in cases. this could lead to confounding in our analyses, as there were more deaths in these more populous hotspots. we attempted to control for this in three ways. first, we adjusted for state population, population density, and census-designated geographic division. next, we designated day 1 to start at a fixed number of deaths in order to analyze m a n u s c r i p t states at the same point in the pandemic. finally, given the four-fold higher mortality in the new york city metropolitan area, we performed analyses excluding new york and new jersey. while our analysis using deaths per capita as an outcome, rathe than deaths, provided an attenuated effect size, the direction of the effect was still in favor of earlier nonpharmaceutical intervention orders. our study has limitations. both of our exposures were measured at the state level, while local school districts also closed schools of their own accord prior to state orders. however, this was estimated to only affect ~16% of the population [13] . death rates were based on publicly available data derived from inconsistent testing using assays with imperfect test characteristics and uneven state-level reporting. thus, both exposure and outcome risk being misclassified. when reliable testing and excess mortality data are available, an analysis using those data may yield more precise estimates of the efficacy of early statewide interventions on covid-19 mortality. additionally, we restricted analysis to the early weeks of covid-19 because of concerns regarding accuracy of mortality data after may 1, 2020, as covid-19 and social distancing continued to be politicized in the united states. indeed, multiple states started re-opening in the first weeks of may, and in some cases changed the method and timing of publicly reporting cases and deaths. due to data limitations, we were unable to adjust for potentially important confounders such as outbreaks in long-term care facilities, which may have both accelerated the spread of sars-cov-2 and served as an impetus for physical distancing policies. state-level variation in access to healthcare and availability of hospital and intensive care unit resources were not included, which could also bias the results. our data do not explore the association between duration of school closing orders and outcomes. however, the lesson of the 1918-1919 influenza pandemic is instructive: among 43 cities investigated, no city experienced a second peak of infections while the first set of nonpharmaceutical interventions were in effect, whereas in cities which lifted initial restrictions, death rates increased [5] . finally, ecologic studies of group-level interventions cannot apply to individuals, and we have no metrics of either state-or individual-level adherence to social distancing in this study. a c c e p t e d m a n u s c r i p t however, our study satisfies several criteria for causality between timing of early interventions and mortality, including mechanistic plausibility, prior knowledge, temporal relationship, a dose-dependent effect (earlier versus later orders), and strength and consistency of the association. these results also confirm the utility and necessity of early nonpharmaceutical intervention to reduce mortality in covid-19, and may serve to increase acceptance of social distancing measures by the lay public and by policymakers. we provide evidence of an association between earlier statewide nonpharmaceutical interventions of social distancing and lower mortality in the early weeks of covid-19. specifically, each day of delay in a state declaring an emergency or closing schools increased mortality risk by 5 to 6%. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t figure 2 strategies for containing an emerging influenza pandemic in southeast asia strategies for mitigating an influenza pandemic mitigation strategies for pandemic influenza in the united states targeted social distancing design for pandemic influenza nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic health outcomes and costs of community mitigation strategies for an influenza pandemic in the united states social distancing to slow the u.s. covid-19 epidemic: an interrupted time-series analysis interventions to mitigate early spread of sars-cov-2 in singapore: a modelling study an interactive web-based dashboard to track covid-19 in real time 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n u s c r i p t key: cord-324007-hapzf0fl authors: mcgeer, allison j. title: diagnostic testing or empirical therapy for patients hospitalized with suspected influenza: what to do? date: 2009-01-01 journal: clin infect dis doi: 10.1086/591852 sha: doc_id: 324007 cord_uid: hapzf0fl accumulating evidence supports the use of specific diagnostic tests and antiviral therapies for seriously ill patients with influenza. among available diagnostic tests, reverse-transcriptase polymerase chain reaction is faster than culture and more sensitive than commercial antigen assays. current neuraminidase inhibitors were approved on the basis of their efficacy in ambulatory patients, but seriously ill patients who receive these agents are less likely to die, even when treatment is initiated >48 h after symptom onset. for patients hospitalized with suspected influenza, it is unclear which circumstances warrant diagnostic testing and which warrant the use of empirical therapy. rapid antigen assays may reduce the unnecessary use of other tests and medications but are relatively insensitive, thus eliminating many patients with influenza as candidates for treatment. empirical antiviral therapy ensures that all patients receive treatment promptly, at a cost equivalent to that of diagnostic tests alone, but results in the receipt of treatment by many patients without influenza. for patients hospitalized with suspected influenza, clinicians need to combine these approaches in order to optimize patient care. trials of early therapy (!48 h after symptom onset) with neuraminidase inhibitors (nais) in healthy adults and children. these trials demonstrated that therapy was associated with a significant reduction in the duration and severity of illness and a 40%-60% reduction in the percentage of patients who developed complications or required hospitalization [2] [3] [4] [5] [6] [7] . similar reductions in complications and hospitalization associated with early therapy with oseltamivir have been identified in subsequent observational cohort studies of nursing home residents during influenza outbreaks, in 2 studies in which administrative databases were examined, and in cohort studies of immunocompromised patients [5, [8] [9] [10] . however, there is a difference between the early treatment of otherwisehealthy outpatients and the treatment of patients who require hospital admission. the question becomes, what data do we have on the impact of antiviral therapy for the treatment of influenza in patients requiring hospitalization? there currently are 3 cohort studies that have examined the impact of treatment on patients with community-acquired illness severe enough to warrant hospitalization. in a cohort study by falsey et al. [11] [12] [13] , viral testing was done systematically for patients admitted to rochester general hospital with underlying cardiopulmonary disease and respiratory tract infection, congestive heart failure, exacerbation of chronic lung disease, or acute respiratory viral illness during 4 winter seasons, from 1999 to 2003. of the 193 patients with influenza, 53 received treatment with amantadine or rimantadine, and 15 received treatment with nais. in-hospital mortality among these patients was 6%. the investigators were unable to find an effect of treatment with antiviral drugs. however, it is important to recognize that the study was not powered to detect a clinically significant difference: an analysis based on a cohort of 200 patients has !30% power to detect a 50% reduction in mortality. furthermore, the authors indicated that it appeared that more severely ill patients were more likely to be treated with an antiviral drug, a bias that would decrease the probability of finding a treatment effect. in a retrospective study of adult patients with influenza who were admitted to the prince of wales hospital in hong kong during the 2004-2005 influenza season with fever and respiratory and systemic symptoms, a clinically and statistically significant reduction in length of hospital stay was associated with treatment with oseltamivir [14] . in north america, less severely ill patients would have been screened from this cohort and sent home, but, in hong kong, most patients with fever and respiratory and systemic symptoms were admitted to a hospital regardless of disease severity and were screened for severe acute respiratory syndrome coronavirus and other respiratory viruses. patients who had been symptomatic for р2 days received empirical therapy with oseltamivir; patients who had been symptomatic for у2 days received treatment at the discretion of their physician. in this study, a total of 356 patients were admitted to the hospital, and 257 received treatment with oseltamivir. of those receiving treatment, 161 received treatment within 2 days of symptom onset. patients who received treatment experienced a median reduction in their length of hospital stay of 2 days-an ∼30% reduction-relative to that of patients who did not receive treatment or who received treatment 12 days after symptom onset ( ) [14] . p ! .0001 further support for the use of specific antiviral therapy for hospitalized patients comes from data collected prospectively in a cohort study of patients in toronto, which showed a surprisingly large reduction in mortality even when therapy was started 148 h after symptom onset [15] . this study correlated mortality with specific antiviral therapy over 2 influenza seasons (2004-2005 and 2005-2006) in toronto. of 327 adult patients with laboratory-confirmed influenza who were admitted to a hospital, 106 patients (32%) received treatment with oseltamivir. overall in-hospital mortality in this cohort was 10.7%. although the observation of a treatment effect was not anticipated, owing to the small sample size, patients who received treatment with oseltamivir had a risk of death (or) of 0.21 ( ), corresponding to a point estimate of a 79% reduc-p p .03 tion in mortality, compared with patients who did not receive treatment. given the limitations of this study's methodology, it is not possible to state unequivocally that oseltamivir treatment reduces mortality among patients admitted to a hospital with influenza. on the other hand, because of the established treatment effect in healthy outpatients, the apparent magnitude of the treatment effect in compromised and seriously ill patients, and the established safety of oseltamivir, a placebo-controlled trial to determine the efficacy of antiviral therapy for the treatment of severe influenza may no longer be ethically justifiable. of note, over the 3 seasons of surveillance in the toronto invasive bacterial diseases network (tibdn) study, about onethird of patients were hospitalized within 48 h of symptom onset. in addition, the treatment effect seen in this cohort was not different for patients treated р48 h or 148 h after symptom onset. these data confirm the findings of ison et al. [16] , who demonstrated that hospitalized patients receiving treatment with rimantadine and zanamivir shed influenza virus for several days after hospital admission. both the fact that one-third of patients present early and the fact that treatment may be effective when initiated 148 h after symptom onset in hospitalized patients emphasize how important it is to improve our understanding of severe influenza illness. another interesting finding from the tibdn study is that, during the 2006-2007 influenza season, 2 of 21 patients admitted to an intensive care unit after out-of-hospital cardiac arrest tested positive for influenza [15] . this raises a concern that influenza may trigger ventricular arrhythmias and sudden death and supports the results of a number of cohort studies suggesting that influenza vaccination is protective against sudden death. this accumulating evidence for a treatment effect suggests that it is prudent to establish a policy of antiviral treatment for patients who are seriously ill with influenza. such a policy will have a low risk of adverse events and a low risk of increasing selective pressure for the development of resistant strains of influenza virus. oseltamivir has no proven serious adverse effects [6, 7, 17] . the neurobehavioral adverse events reported primarily in japanese adolescents may have been an effect of either influenza or treatment; only further study will resolve this question. reassuringly, the rate of neurobehavioral adverse events reported to the us food and drug administration by japan is !1 case per 100,000 prescriptions [18] . for patients within the age range typically admitted to an intensive care unit, that rate is probably low enough to be of very limited clinical relevance. with regard to increasing selective pressure for the development of resistant virus strains, it is important to remember that nais are active against only influenza virus. if a patient treated with nais does not have influenza, no selective pressure is being applied, and antiviral resistance will not be increased in the patient population. this differs from the effect of empirical antibiotic therapy: every time an antibiotic is used, selective pressure is applied to the host's normal flora, whether or not the antibacterial is active against the pathogen being targeted. past influenza-management guidelines have not offered much guidance regarding antiviral treatment for patients admitted to a hospital. nonetheless, recent guidelines suggest that antiviral therapy should be offered to patients with severe illness and to those who are most likely to develop complications and/ or to die. for example, the association of medical microbiology and infectious diseases canada/canadian pediatric society recommends antiviral treatment for individuals with severe illness and for those most likely to develop complications from influenza or to die prematurely as a result [19] . the american academy of pediatrics recommends antiviral treatment for high-risk children and for other children with moderate to severe disease [20] . italian guidelines recommend prioritizing therapy for patients at risk of complications, ensuring that patients take the drug as early as possible, and using antiviral therapy only at the seasonal peak of influenza prevalence [21] . a decision to treat with antiviral therapy requires that physicians either rapidly make a reasonably definitive diagnosis of influenza or choose to treat empirically when the probability of influenza is above a certain threshold. the former is obviously preferable, when possible. among adults, acute respiratory illness with fever and early cough has a positive predictive value for influenza of 170% during influenza season [22] . however, many adults, particularly those who are elderly or those who have a significant underlying illness, do not mount an adequate febrile response and may not present with early cough. thus, clinical features alone cannot be used to diagnose influenza. commonly available diagnostic tests for influenza are shown in table 1 [23] [24] [25] [26] . of the available laboratory tests, rt-pcr is preferred for its speed, sensitivity, and specificity but is not currently available to a majority of clinicians. in toronto, most hospital laboratories provide eia testing. on average, eia sensitivity is 50%-70%, compared with that for viral culture, and specificity is ∼95%, depending on the laboratory and the test. however, the use of viral culture as the gold standard for sensitivity may be outdated. table 2 compares the sensitivity of viral culture to that of rt-pcr [26] [27] [28] [29] . the proportion of virus detected by culture in the different studies ranges from 50% to 90%, or ∼70% for purposes of estimation. thus, actual rapid eia sensitivity is not 60% but is closer to 60% of 70%, which is ∼42%. other rapid tests with sensitivities ranging from 24% to 90%, relative to that for viral culture, share the same limitation [24] . the lack of a rapid and sensitive clinical diagnostic test for influenza is problematic. although a number of new and sensitive molecular techniques are being investigated, such tests will not become available for several years [30] . given the limitations of currently available diagnostic tests, empirical therapy is an option worth considering. empirical therapy has the advantage of offering earlier treatment, which is likely to be more effective. it also may be the most costeffective option, because laboratory testing actually may be more expensive than therapy (which costs ∼$60 for a 5-day course). however, empirical therapy has the disadvantage of resulting in many more patients receiving treatment than actually have influenza. the prevalence of influenza in some recent patient cohorts is shown in table 3 [11, 15, 28, 29] . these studies looked at data from different groups of patients, in different years and at different times of year. seasonal and yearto-year variations in the underlying incidence of influenza explain much of the difference observed. overall, these studies suggest that, during most influenza seasons, 10%-15% of adult patients with pneumonia and/or febrile respiratory illness are likely to have influenza virus infection [29] . thus, empirical therapy will result in 5-15 patients without influenza receiving treatment for every patient with influenza. this ratio is similar to that reported in recommendations for the use of empirical therapy for atypical bacterial infection in pneumonia and, thus, is worthy of consideration [31, 32] . on the other hand, the testing of patients provides infor-mation to clinicians that enables more-directed therapy. evidence from both pediatric and adult studies indicates that testing for influenza results in reduced use of antibiotics and possibly reduced use of some other diagnostic tests [12, 33, 34] . such observations suggest that the information is immediately useful to clinicians. however, false-positive test results occur and may mislead clinicians. the benefits and limitations of laboratory diagnostic testing versus empirical antiviral therapy for influenza are summarized in table 4. as a practical matter, each infectious disease specialist must weigh the uncertainties of diagnosis and the effects of treatment to determine the best option for each patient under his or her care. the accumulating evidence suggests that, for patients with acute cardiorespiratory illness requiring hospital admission during influenza season, consideration should be given to either prompt laboratory diagnostic testing and treatment for influenza virus-infected patients or empirical antiviral therapy for influenza. the best choice is made on a case-by-case basis and depends on the severity of illness in the patient being admitted (since earlier therapy for pneumonia is more effective), the probability of influenza virus infection in the individual patient, and the sensitivity of the rapid diagnostic tests available. it is hoped that the introduction of rt-pcr testing into hospital laboratories and the accumulating information from cohort studies and trials of antiviral therapy among severely ill patients with influenza will soon result in a better understanding of effective diagnosis and therapy and in improved outcomes for severely ill patients. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) use of oseltamivir during influenza outbreaks in ontario nursing homes risk of pneumonia and other complications of influenza-like illness in patients treated with oseltamivir effect of oseltamivir on the risk of pneumonia and use of health care services in children with clinically diagnosed influenza impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations symmonds-abrahams m. neuraminidase inhibitors for preventing and treating influenza in children neuraminidase inhibitors for preventing and treating influenza in healthy adults prevention and treatment of influenza in highrisk groups: children, pregnant women, immunocompromised hosts, and nursing home residents safety and tolerability of oseltamivir prophylaxis in hematopoietic stem cell transplant recipients: a retrospective case-control study use of oseltamivir to control influenza complications after bone marrow transplantation respiratory syncytial virus infection in elderly and high-risk adults impact of rapid diagnosis on management of adults hospitalized with influenza pulmonary complications of interpandemic influenza a in hospitalized adults factors associated with early hospital discharge of adult influenza patients antiviral therapy and outcomes of influenza requiring hospitalization in ontario safety and efficacy of nebulized zanamivir in hospitalized patients with serious influenza the safety of oseltamivir in patients with influenza: analysis of healthcare claims data from six influenza seasons post-marketing adverse event reports review of central nervous system/psychiatric disorders associated with the use of tamiflu the use of antiviral drugs for influenza: recommended guidelines for practitioners antiviral therapy and prophylaxis for influenza in children italian guidance on antiviral use for management and prevention of seasonal influenza [abstract p1318 does this patient have influenza? influenza (flu): influenza symptoms and laboratory diagnostic procedures role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics laboratory diagnosis of human seasonal and pandemic influenza virus infection testing of diagnostic methods for detection of influenza virus for optimal performance in the context of an influenza surveillance network diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza incidence and characteristics of viral community-acquired pneumonia in adults surveillance for laboratory confirmed influenza requiring intensive care unit admission in toronto, canada expert consultation on diagnosis of h5n1 avian influenza infections in humans a worldwide perspective of atypical pathogens in community-acquired pneumonia treatment of community-acquired pneumonia-idsa guidelines impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial the impact of rapid antigen testing for influenza on paediatric hospital admissions in a large uk emergency department: a retrospective observation financial support. biocryst pharmaceuticals, inc., provided educational grant support to develop this article and the symposium on which it is based, "antiviral therapy for influenza: challenging the status quo" (san diego), 4 october 2007.supplement sponsorship. this article was published as part of a supplement entitled "antiviral therapy for influenza: challenging the status quo," jointly sponsored by the institute for medical and nursing education and international medical press and supported by an educational grant from biocryst pharmaceuticals, inc.manuscript preparation. margery tamas of international medical press (atlanta) provided assistance in preparing and editing the manuscript.potential conflicts of interest. a.j.m. has served as a consultant to biocryst pharmaceuticals, inc., has received funding for investigator-initiated research under contract with hoffman-laroche and biocryst pharmaceuticals, inc., and is a member of the speakers' bureau for gilead pharmaceuticals. key: cord-324307-2zbm4iwn authors: kam, kai-qian; yung, chee fu; cui, lin; tzer pin lin, raymond; mak, tze minn; maiwald, matthias; li, jiahui; chong, chia yin; nadua, karen; tan, natalie woon hui; thoon, koh cheng title: a well infant with coronavirus disease 2019 with high viral load date: 2020-02-28 journal: clin infect dis doi: 10.1093/cid/ciaa201 sha: doc_id: 324307 cord_uid: 2zbm4iwn a well 6-month-old infant with coronavirus disease 2019 (covid-19) had persistently positive nasopharyngeal swabs up to day 16 of admission. this case highlights the difficulties in establishing the true incidence of covid-19, as asymptomatic individuals can excrete the virus. these patients may play important roles in human-to-human transmission in the community. a well 6-month-old infant with coronavirus disease 2019 (covid-19) had persistently positive nasopharyngeal swabs up to day 16 of admission. this case highlights the difficulties in establishing the true incidence of covid-19, as asymptomatic individuals can excrete the virus. these patients may play important roles in human-to-human transmission in the community. keywords. covid-19; sars-cov-2; infant; 2019 novel coronavirus. in december 2019, health authorities in wuhan, china, were investigating cases of severe viral pneumonia of unknown origin that were epidemiologically linked to the huanan seafood market. sequencing of respiratory tract samples revealed a novel coronavirus, initially termed as 2019 novel coronavirus (2019-ncov). the disease was subsequently named coronavirus disease 2019 (covid-19) and the virus was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1, 2] . to date, the covid-19 outbreak has affected 25 countries and remains a global health concern with an estimated case-fatality rate of 2% [3] . singapore reported its first imported case of covid-19 on 23 january 2020. prior to 3 february 2020, there was no evidence of local humanto-human transmission. on 3 february, a local cluster suggestive of limited community transmission was reported [4] . our patient was the first pediatric case detected in singapore with confirmed covid-19 and he remained largely asymptomatic. our infant was part of a household transmission cluster where both parents and their live-in helper were infected with covid-19. this was the result of an occupational exposure to a large tourist group from china. a case series of 9 infants with confirmed covid-19 showed that the majority of the infants were symptomatic and had strong epidemiological risk factors [5] . understanding the clinical manifestation of covid-19 across all population groups, especially infants and young children, is critical for public health containment measures to be effective. extraction of viral nucleic acid from respiratory specimens was performed using the ez1 virus mini kit version 2.0 (qiagen, hilden, germany). the rna was eluted in 60 μl of buffer and used as template for all assays. two specific real-time reverse-transcription polymerase chain reaction (rrt-pcr) methods, targeting the n and orf1ab genes, were designed to detect the presence of sars-cov-2 in clinical samples. the n gene primer sequences were forward primer 5′ ctc agt cca aga tgg tat ttc t and reverse primer 5′ agc acc ata ggg aag tcc, and the probe sequence was 5′ fam-acc tag gaa ctg gcc cag aag ct-bhq1. thermal cycling was performed at 50°c for 20 minutes for reverse transcription, followed by 95°c for 15 minutes and then 50 cycles of 94°c for 5 seconds and 55°c for 1 minute. the sequences for the orf1ab rrt-pcr were forward primer 5′ tca ttg tta atg cct ata tta acc, reverse primer 5′ cac tta atg taa ggc ttt gtt aag, and probe 5′ fam-aac tgc aga gtc aca tgt tga ca-bhq1. thermal cycling for orf1ab was performed at 50°c for 20 minutes and 95°c for 15 minutes, and 50 cycles of 94°c for 5 seconds, 50°c for 20 seconds, and 72°c for 20 seconds. for both assays, a 20-μl reaction contained 5 μl rna template, 500 nmol each of forward and reverse primer, 150 nmol probe, and 0.2 μl quantitect rt mix (quantitect probe rt-pcr kit, qiagen). all reactions were run on a lightcycler 2.0 instrument (roche, rotkreuz, switzerland). all samples were tested for the endogenous rnase p gene as an internal control. a well 6-month-old boy was referred to kk women's and children's hospital (kkh) on 4 february 2020. his mother and live-in helper were admitted to the singapore general hospital (sgh) isolation unit on 3 february 2020 for pneumonia and were undergoing investigations for covid-19. his mother's occupation involved close contact with tourists from china. she did not have any recent travel history. her symptoms of fever and sore throat started on 29 january 2020. chest radiograph showed left lower zone consolidation, and she was classified as a suspected case of covid-19. her first nasopharyngeal swab on 3 february 2020 was positive for sars-cov-2. the infant's father developed fever and sore throat on 1 february 2020 and was admitted to the national centre for infectious diseases isolation unit on 4 february 2020 for sars-cov-2 testing. as the infant had no remaining well caregivers, he was brought to kkh for clinical assessment and isolation in light of his close contact with confirmed covid-19 cases. the infant was asymptomatic on arrival to the hospital. he was afebrile with no tachypnea. oxygen saturation was 98% on room air and his lungs were clear to auscultation. as his respiratory status was stable, no chest radiograph was performed. a nasopharyngeal specimen taken on admission and tested by rrt-pcr confirmed the diagnosis of covid-19 infection with low cycle threshold (n gene, 15.57; orf1ab gene, 13.73), suggesting high viral load. rapid multiplex respiratory pathogens nucleic acid amplification test (biofire filmarray rp2, biofire diagnostics, salt lake city, utah) was negative for all pathogens, including influenza a and b and 4 human coronaviruses (oc43, 229e, nl63, and hku1). on day 2 of admission, he was found to be viremic with detection of sars-cov-2 in his blood sample via rrt-pcr. however, stool and urine samples from the same day were negative. during this viremic phase, he had 1 temperature record of 38.5°c, which normalized within 1 hour. otherwise, he was afebrile and remained asymptomatic throughout admission. daily nasopharyngeal swabs continued to be positive for sars-cov-2 ( figure 1 ) and eventually became negative on day 17 of admission. biochemical markers showed normal liver function tests on day 2 and day 8 of admission. his full blood count was normal on day 2, but neutropenia (absolute neutrophil count of 0.9 × 10 9 /l [normal range, 1.5-8.5 × 10 9 /l]) occurred on day 8 of admission. repeat testing of his urine on day 9 of admission was negative, but his stool sample became positive for sars-cov-2. he did not have any gastrointestinal symptoms before or throughout this admission. his mother was transferred from sgh to kkh on 6 february 2020 to unite with her child. she became afebrile on 5 february (day 8 of illness) and continued to have dry cough until 11 february (day 14 of illness). her respiratory status was stable without oxygen requirement. her daily nasopharyngeal specimens were positive from 3 february to 6 february at sgh and subsequently fluctuated near the detection limit for sars-cov-2 ( figure 1 ). she had initial leukopenia (2.6 × 10 9 /l [normal range, 4.0-10.0 × 10 9 /l]), neutropenia (1.2 × 10 9 /l [normal range, 2.0-7.5 × 10 9 /l]), and thrombocytopenia (10 6 × 10 9 /l [normal range, 140-440 × 10 9 /l]), which resolved on day 14 of illness. breast milk samples on day 11 of illness were negative for sars-cov-2. her nasopharyngeal swabs returned negative on day 18 of illness. both patients remained well in the kkh isolation unit at the time of writing (day 18 of the infant's admission). we report a confirmed case of covid-19 in a 6-month-old infant who was well even though there was evidence of viremia. apart from a single transient temperature of 38.5°c, the infant had no clinical signs or symptoms. we detected a high viral load from the nasopharynx of our infant from the day of admission. samples from the nasopharynx continued to remain positive up to day 16 of admission. our infant likely acquired the sars-cov-2 virus from a household member, but it was difficult to ascertain the day of infection as there were no reported symptoms. similar to reports of adult covid-19, we confirm the detection of sars-cov-2 rna in the stool of our infant. the frequency and duration of viral secretion in infant stool remains uncertain. our infant's stool sample was negative on day 2 of admission but positive on day 9 of admission. during the global sars-cov epidemic, the majority of children had a benign course of illness with mild respiratory symptoms and there were no deaths reported in the pediatric population [6, 7] . the reason for young children < 12 years of age being less affected by the infection was unknown. one of the hypotheses proposed was a blunted immune dysregulation response toward the sars-cov infection in children [8] . as covid-19 originates from the β-coronavirus genus group which sars-cov belongs to, we may observe similar illness trajectories in both pediatric infections. following the outbreak of covid-19 that began in wuhan and export of cases to countries worldwide, there was an urgent need to contain the spread of the novel virus. quick and accurate identification of cases for isolation or quarantine remains a key pillar of public health efforts to contain an emerging epidemic. this strategy is dependent on accurate case definitions to identify patients for further management, including testing if available. most of the current case definitions used globally are developed based on clinical signs and symptoms collated from covid-19 in adults, which include either acute respiratory symptoms or more severe signs of lower respiratory tract infection [9] . while most adults with covid-19 infection have fever, respiratory symptoms, and/ or chest radiograph changes, current data suggest that the majority of the infected children have mild or no symptoms [10] [11] [12] . our case highlights a likelihood of poor performance of current case definitions used to contain covid-19, especially in infants. there is an urgent need to better understand the full clinical spectrum of covid-19 in the pediatric population in order to refine public health containment strategies. world health organization. who director-general's remarks at the media briefing on severe acute respiratory syndromerelated coronavirus-the species and its viruses, a statement of the coronavirus study group novel coronavirus (2019-ncov) situation report-21 confirmed cases of local transmission of novel coronavirus infection in singapore novel coronavirus infection in hospitalized infants under 1 year of age in china clinical presentations and outcome of severe acute respiratory syndrome in children other members of the hospital for sick children sars investigation team. children hospitalized with severe acute respiratory syndrome-related illness in toronto severe acute respiratory syndrome (sars) in neonates and children centers for disease control and prevention. flowchart to identify and assess 2019 novel coronavirus clinical features of patients infected with 2019 novel coronavirus in wuhan diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster key: cord-335038-q32ghvsv authors: huang, jiao; xie, nianhua; hu, xuejiao; yan, han; ding, jie; liu, pulin; ma, hongfei; ruan, lianguo; li, gang; he, na; wei, sheng; wang, xia title: epidemiological, virological and serological features of covid-19 cases in people living with hiv in wuhan city: a population-based cohort study date: 2020-08-17 journal: clin infect dis doi: 10.1093/cid/ciaa1186 sha: doc_id: 335038 cord_uid: q32ghvsv background: we aimed to describe the epidemiological, virological and serological features of coronavirus disease 2019 (covid-19) cases in people living with hiv (plwh). methods: this population-based cohort study identified all covid-19 cases among the whole plwh in wuhan city, china, by april 16, 2020. the epidemiological, virological and serological features were analyzed based on the demographic data, temporal profile of nucleic acid test for sars-cov-2 during the disease, and sars-cov-2-specific igm and igg after recovery. results: from january 1 to april 16, 2020, 35 of 6001 plwh have experienced covid-19, with the cumulative incidence of covid-19 to be 0.58% (95%ci: 0.42%-0.81%). among the covid-19 cases, 15 (42.86%) had severe illness, with 2 deaths. the incidence, case-severity and case-fatality of covid-19 in plwh were comparable to that in the entire population in wuhan. 197 persons had cart discontinuation, of whom 4 persons experienced covid-19. risk factors for covid-19 were age ≥50 years old and cart discontinuation. the median duration of sars-cov-2 viral shedding among confirmed covid-19 cases in plwh was 30 (iqr: 20-46) days. cases with high hiv viral load (≥20 copies/ml) had lower igm and igg levels than those with low hiv viral load (<20 copies/ml) (median s/co for igm, 0.03 vs. 0.11, p<0.001; median s/co for igg, 10.16 vs. 17.04, p=0.069). conclusions: efforts need to maintain the persistent supply of antiretroviral treatment to elderly plwh aged 50 years or above during the covid-19 epidemic. the coinfection of hiv and sars-cov-2 might change the progression and prognosis of covid-19 patients in plwh. coronavirus disease 2019 (covid-19) is an emerging infectious disease, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which was first reported in the latterly december 2019 in wuhan, china [1] . according to the who status report, more than 16 million covid-19 cases have been reported worldwide until july 30 [2] . has emerged as a great challenge to global public health. since no vaccine and effective drugs are available against this infectious disease, non-pharmaceutical interventions were implemented to slow the spread and flatten the epidemic curve of covid-19 [3] . although governments and communities have been dedicated to maintaining hiv service provision for people living with hiv (plwh), antiretroviral treatment for plwh may be hindered by the covid-19 epidemic [4] . considering the large number of plwh, it is urgent to evaluate whether the incidence of covid-19 in plwh differs from that in the general population. cases with coinfection of sars-cov-2 and hiv have been reported in several countries [5] [6] [7] . but only one study calculated the covid-19 incidence in plwh based on hiv patients in a single hospital [8] . the population-based incidence of covid-19 in plwh needs to assess this population at risk of sars-cov-2 infection comprehensively. previous studies have found that plwh with low cd4 cell count, high hiv viral load and not taking antiretroviral treatment have an increased risk of other respiratory infections [9] . however, limited data were available for the risk of sars-cov-2 infection in plwh. several studies have demonstrated the antibody response to sars-cov-2 infection in the general population [10, 11] , which may inform vaccine intervention in the future. nevertheless, there is no information currently on the antibody against sars-cov-2 infection in plwh. guidance has been proposed by us department of health and human services and eacs european aids clinical society to m a n u s c r i p t 5 provide information about the proper response to covid-19 in plwh [12, 13] . but guidance like these needs more evidence to refine their recommendation. in the present study, we performed a population-based cohort study to calculate the cumulative incidence of covid-19 in plwh from january 1 to in wuhan city, china, and compared it with the entire population of wuhan. furthermore, we described the virological and serological features of covid-19 cases in plwh. all individuals in wuhan who were tested positive for hiv have been reported to wuhan center for disease control (cdc) through the china national hiv/aids comprehensive response information management system (crims) [14] . according to crims requirement, a standardized form was used to collect hiv-positive persons' information, including basic demographic characteristics (gender, date of birth, education level), mode of hiv acquisition and baseline cd4 cell count and hiv viral load. the local cdc or designated hospital staff followed up them every year for their cd4 cell count and hiv viral load at least once [15] . hiv-positive persons who met the chinese national treatment criteria were referred to the china national free antiretroviral treatment programme (nfatp) to receive combination antiretroviral therapy (cart) [16] . on december 31, 2019, 6001 plwh resided in wuhan city had been included in crims. covid-19 has been listed as a class b infectious disease on january 20, 2020 in china [17] . all covid-19 cases must be reported to the national notifiable infectious disease report system (nnidrs) within 2 hours after diagnosed [18] . all covid-19 cases a c c e p t e d m a n u s c r i p t 6 reported to nnidrs in wuhan have been rechecked and verified on april 16, 2020[19] . therefore, the covid-19 cases in plwh were identified by linking the individual information from these two systems in wuhan using the unique id number on april 17, 2020 . according to the national guideline, covid-19 cases were categorized into confirmed cases, clinically diagnosed cases, suspected cases, and asymptomatic cases. and the severity status of covid-19 cases was categorized as mild, moderate, severe, or critical. the details could be found elsewhere [3, 20] . in this study, covid-19 cases with severe or critical illness were classified into severe cases group. otherwise, they were classified into non-severe cases group. among the 6001 plwh, 474 (7.90%) persons were not on cart before dec demographic characteristics included gender, date of birth, education level, duration of hiv infection, mode of hiv acquisition, cart regimens and treatment status, cd4 cell count and hiv viral load at last routine medical visit within the previous 12 months were obtained from crims. for covid-19 cases, information including date of onset, date of diagnosis, date of death (if applicable) and case type and clinical severity were extracted from nnidrs. we also collected temporal profiles of rt-pcr results for testing sars-cov-2 in each confirmed case. in addition, we obtained the covid-19 incidence among the general population by the street where the plwh's living address located to indicate their chance of infection by sars-cov-2. if the covid-19 incidence among the general population for the street was ≤0.66% (median level of the covid-19 incidence among all the streets in wuhan), plwh located in this street were classified as having low chance, otherwise, they were deemed to have high chance. serum samples were taken from each alive covid-19 cases in plwh on may 18, 2020. sars-cov-2-specific igm and igg were detected by magnetic chemiluminescence enzyme immunoassay using commercial kits following the manufacturer's instructions, which have been described elsewhere [10, 22] . the antibody level was expressed as the chemiluminescence signal value divided by the cutoff value (s/co). igm or igg was defined as positive if the s/co value was higher than 1.0; otherwise, it was regarded to be negative. a c c e p t e d m a n u s c r i p t 8 the covid-19 incidence was estimated assuming a poisson distribution and described for the entire plwh population and subgroups. we used poisson regression to estimate incidence rate ratios (irrs) to compare the covid-19 incidence in subgroups of plwh. univariate and multivariate modified poisson regressions with robust variance were used to evaluate the relationship between characteristics of plwh with the covid-19 occurrence [23] . besides, we imputed the missing data on the basis of multivariable imputation and performed sensitivity analyses retaining all plwh to explore the risk factors of covid-19 among plwh. we used the direct method to calculate the covid-19 incidence standardized by age and gender for plwh and to compare the difference in covid-19 occurrence between plwh and the general population in wuhan. we derived standardization weight from the age and gender distribution of the general population of wuhan in 2018. a similar method was used to calculate case-severity and case-fatality standardized by age and gender for plwh, with the number of covid-19 cases in different age groups obtained from the previous study as a standard [3] . all analyses were conducted using sas statistical software version 9.4 (sas institute inc) and r project version 3.6.1 (http://cran.r-project.org). a two-sided p value of <0.05 was considered statistically significant. this study was reviewed and approved by the institutional review board of the wuhan a c c e p t e d m a n u s c r i p t from january 1 to april 16, 2020, 35 (0.58%) plwh had experienced covid-19, including 22 (62.86%) confirmed cases, 11 (31.43%) clinical cases and 2 (5.71%) asymptomatic cases. the median age of plwh (52, iqr: 36-57 years) for covid-19 cases was higher than that for non-covid-19 cases (37, iqr: 29-52 years, p=0.004). most table 1 ). the cumulative incidence of covid-19 was 0.58% (95%ci: 0.42%-0.81%). there is no significant difference between the covid-19 incidence for males (0.61%, 95%ci: 0.43%-0.86%) and females (0.35%, 95%ci: 0.09%-1.39%, p=0.444). covid-19 incidence among plwh aged ≥50 years was 1.18%, which was higher than that of plwh below 50 years table 2 ). the standardized rates of case-severity and case-fatality of covid-19 in plwh were also similar to that in the entire population in wuhan (table 2) . we included the 5004 (83.39%) persons with available data for hiv viral load to analyze the risk factors for covid-19 in plwh, with 984 (16.49%) and 3 (8.57%) plwh exclude from non-covid-19 and covid-19 cases groups (supplementary figure 1) . the multivariate poisson regression analysis showed positive associations between covid-19 occurrence and older age and cart discontinuation after adjusting for other variables (table 3 ). compared to plwh aged below 50 years with cart continuation, plwh aged 50 years or above with cart discontinuation were at sharply increased risk for covid-19 (adjusted irr=16.86, 95%ci: 4.71-60.26) ( table 4 ). the sensitivity analyses results using multivariable imputation of missing data were consistent with the above analyses retaining only patients with complete data (supplementary table 3 a c c e p t e d m a n u s c r i p t the temporal profile of rt-pcr results in 22 confirmed covid-19 cases in plwh was shown in figure 2 . table 7 ). in this cohort study, we reported the covid-19 incidence among plwh in wuhan during the covid-19 epidemic. the incidence, case-severity and case-fatality of covid-19 in plwh were comparable to that in the entire population in wuhan. the covid-19 incidence among plwh aged 50 years or above was twice higher, compared to plwh below 50 years. the median duration of viral shedding was 30 days for confirmed covid-19 cases in plwh. higher antibody levels were observed in cases with high hiv viral load than those with low hiv viral load. although several studies recently reported the covid-19 cases in plwh in hospitals, there are few population-based studies having focused on covid-19 of plwh. our study showed that plwh have similar risk of covid-19 compared to that in the general population during covid-19 epidemic. the incidence of covid-19 among plwh (0.58%) in wuhan was lower than that in hiv-infected individuals (1.8%) in madrid [8] . it may be one of the reasons that plwh in wuhan (mean age 40.7 years) were much younger than hiv-infected individuals in madrid (mean age 53.5 years), since old age was found to be positively associated with covid-19 in plwh [24] . in spite of the different incidence, the age and case type were similar in the 35 covid-19 cases (wuhan, median age 52 years old, 63% confirmed cases) and 51 covid-19 cases (madrid, mean age 53.3 years old, 68% confirmed cases) from these two studies. the case-fatality of covid-19 in plwh varied 4%-28.6% in different studies [25] [26] [27] [28] , further systematic studies are needed to clarify the disparity. a c c e p t e d m a n u s c r i p t 13 maintaining antiretroviral therapy during the covid-19 epidemic is urgent for the health of plwh, especially in elderly person. out study firstly suggested that plwh aged over 50 years with cart discontinuation had over ten times risk of sars-cov-2 infection than young plwh with art continuation. similar findings from studies on the coinfection of hiv and tuberculosis demonstrated that antiretroviral therapy for hiv-positive adults could lower the incidence and mortality of people having coinfection of hiv and tuberculosis [29, 30] . our findings provide the evidence to support the suggestion from nhh interim guidance for covid-19 and persons with hiv that the elderly persons with hiv are at the highest risk of covid-19 [12] . although the supply of antiretroviral drugs against hiv had been disrupted during the "lockdown period" in wuhan, most of plwh in wuhan had antiretroviral drugs supply with the help of the cdc staffs and volunteers from the community based organizations (cbos) during the covid-19 epidemic. however, designed hospitals for hiv care services have been closed, which means plwh may have not proper antiretroviral therapy without any timely examination. the challenge would have been the toughest if the covid-19 epidemic duration lasts over three months. therefore, it is critical to make a response policy and strategy to provide a timely art treatment for plwh during the covid-19 epidemic. our findings suggested that coinfection of hiv and sars-cov-2 may change the development and prognosis of covid-19 in plwh. the median interval from symptom onset to viral clearance of confirmed covid-19 cases in plwh was 30 days in this study, which was longer than that of covid-19 cases without hiv infection (20 days) [31] . this indicates that covid-19 cases in plwh may have delayed viral clearance for sars-cov-2 because of immunosuppression, although clinical improvement of covid-19 in plwh was not worse than that of individuals without hiv infection as described in the present study and other published studies [25, 32] . furthermore, our study found that the level of hiv viral load we thank all staff members at municipal and district center for disease control and prevention and designated hospital for data collection. we acknowledge all medical staff members and community volunteers who are working on the frontline of caring for patients and collecting the data. the views expressed in this study are those of the authors and do not represent the official position of wuhan center for disease control and prevention. this work is funded by the fundamental research funds for the central universities (2020kfyxgyj066). all authors declare that they have no conflicts of interest. a c c e p t e d m a n u s c r i p t m a n u s c r i p t m a n u s c r i p t m a n u s c r i p t 25 m a n u s c r i p t 27 the boxplots indicate medians (middle line) and third and first quartiles (boxes), while the whiskers indicate 1.5× the interquartile range (iqr) above and below the box. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 31 figure 3 a novel coronavirus from patients with pneumonia in china coronavirus disease 2019 (covid-19) situation report -192. 2020 *cited association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan maintaining hiv care during the covid-19 pandemic co-infection of sars-cov-2 and hiv in a patient in wuhan city covid-19 in patients with hiv: clinical case series hiv/sars-cov-2 co-infected patients in istanbul, turkey description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort cd4 cell count and the risk of aids or death in hiv-infected adults on combination antiretroviral therapy with a suppressed viral load: a longitudinal cohort study from cohere antibody responses to sars-cov-2 in patients with covid-19 sars-cov-2 antibody responses do not predict covid-19 disease severity interim guidance for covid-19 and persons with hiv. 2020 *cited development of a unified web-based national hiv/aids information system in china antiretroviral therapy to prevent hiv transmission in serodiscordant couples in china (2003-11): a national observational cohort study effect of earlier initiation of antiretroviral treatment and increased treatment coverage on hiv-related mortality in china: a national observational cohort study announcement on the pneumonia infected with noval coroavirus. 2020 *cited province hcoh. statement on the revision of the covid-19 epidemic in hubei province on predictors of first-line antiretroviral therapy discontinuation due to drug-related adverse events in hiv-infected patients: a retrospective cohort study clinical and immunological assessment of asymptomatic sars-cov-2 infections easy sas calculations for risk or prevalence ratios and differences incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study clinical characteristics and outcomes in people living with hiv hospitalized for covid-19 outcomes among hiv-positive patients hospitalized with covid-19 sars-cov-2 infection in persons living with hiv: a single center prospective cohort clinical outcomes and immunologic characteristics of covid-19 in people with hiv optimal timing of antiretroviral therapy initiation for hiv-infected adults with newly diagnosed pulmonary tuberculosis: a systematic review and metaanalysis opportunistic diseases diminish the clinical benefit of immediate antiretroviral therapy in hiv-tuberculosis co-infected adults with low cd4+ cell counts clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study covid-19 and people with hiv infection: outcomes for hospitalized patients plwh: people living with human immunodeficiency virus; msm: men who have sex with men; iqr: inter quartile range; cart: combination antiretroviral therapy. nrtis: nucleoside reverse transcriptase inhibitors nnrtis: non-nucleoside reverse transcriptase inhibitors; pis: protease inhibitors; in this study, only lopinavir/ritonavir (lpv/r) was in this category of antiretroviral plwh: people living with human immunodeficiency virus *according to the report from wuhan municipal health commission on estimated based with the number of covid-19 cases in different age and gender groups obtained from the published study as a standard 3 a c c e p t e d m a n u s c r i p t key: cord-327862-zcg3baym authors: luo, yiqi ruben; chakraborty, indrani; yun, cassandra; wu, alan h b; lynch, kara l title: kinetics of sars-cov-2 antibody avidity maturation and association with disease severity date: 2020-09-14 journal: clin infect dis doi: 10.1093/cid/ciaa1389 sha: doc_id: 327862 cord_uid: zcg3baym the kinetics of igg avidity maturation during sars-cov-2 infection was studied. the igg avidity assay used a novel label-free immunoassay technology. it was found that there was a strong correlation between igg avidity and days since symptom onset, and peak readings were significantly higher in severe than mild disease cases. a c c e p t e d m a n u s c r i p t 3 in the race to identify correlates of covid-19 immunity, the focus has been on characterizing the production of sars-cov-2 antibodies. most immunocompetent individuals with symptomatic infections develop detectable sars-cov-2 antibodies within 2 weeks of symptom onset. 1 the sars-cov-2 igg antibody response is more robust in severe cases of covid-19 at all time-points after seroconversion. 1 preliminary findings suggest that the magnitude of the antibody response correlates with virus neutralizing power, but some hypothesize that the antibodies could promote disease progression. 2 influential factors in the distinction between protective antibodies and those that promote pathology include antibody isotype, concentration, specificity, post-translational modifications, and avidity for the virus. most sars-cov-2 antibody studies to date have focused on the quantity of specific isotypes rather than the quality of the antibodies. antibody avidity, or functional affinity, is a measure of the maturation of the humoral immune response after both infection and vaccination and typically increases over time. for some viruses, the measurement of antibody avidity is used to differentiate acute infection from past exposure or vaccination (e.g. toxoplasmosis, sars-cov infection) and to detect re-infection in the presence of prolonged virus-shedding. 3, 4 here we report the development of a method to characterize sars-cov-2 igg avidity maturation in covid-19 patients from initial diagnosis through convalescence. the igg avidity assay was established on a novel label-free immunoassay platform gator analyzer (gator bio, palo alto, ca) to measure sars-cov-2 igg avidity to the virus spike protein receptor-binding domain (rbd). the platform utilizes thin-film interferometry and can measure the entire time course of formation of an immune complex on a sensing probe without attaching a reporter. 5 the sensing probe in use was pre-coated with recombinant rbd (his-a c c e p t e d m a n u s c r i p t 4 tagged, sino biological, wayne, pa). serum samples were diluted 10-fold in running buffer (pbs with 0.02% tween 20, 0.2% bsa, and 0.05% nan 3 ) for analysis. the steps of the igg avidity assay included 1) dipping the sensing probe in running buffer for a baseline measurement, 2) formation of rbd-igg immune complex on the sensing probe, 3) dissociation of loosely bound igg using either running buffer or 3 m urea in running buffer, and 4) formation of rbd-igg-anti-igg immune complex using 10 μg/ml anti-igg in running buffer (goat antihuman igg antibody, jackson immunoresearch, west grove, pa). the sensing probe was washed in running buffer for 1 min between the steps, and regenerated in glycine ph 2.0. the signal increase in the final step, which is proportional to the quantity of rbd-igg-anti-igg immune complex on the sensing probe, was measured. as other isotypes of antibodies might bind to rbd in the second step, the measurement of the rbd-igg-anti-igg immune complex enhanced the assay specificity. the igg avidity index was calculated as the ratio of the readout with the dissociation agent (urea) to the reference (running buffer), presented as a percentage. the performance of the igg avidity assay was verified for precision and specificity. precision was evaluated using a spiked serum sample prepared by adding a standard anti-rbd igg (absolute antibody, oxford, uk) in a negative serum sample. the spiked serum sample was measured simultaneously by a row of 7 sensing probes, and the assay was repeated 4 times with regeneration of the sensing probes. the cv among the sensing probes was 4.5%, and the cv among regeneration cycles was 10.4%. the spiked serum sample was also measured in 4 consecutive days and the cv was 13.7%. specificity was evaluated using a set of 28 serum samples from individuals tested rt-pcr negative for sars-cov-2 and positive for other respiratory viruses (4 coronavirus hkv1, 1 coronavirus 229e, 2 coronavirus oc43, 11 human rhinovirus/enterovirus, 4 human metapneumovirus, 3 respiratory syncytial virus, 2 parainfluenza a c c e p t e d m a n u s c r i p t 5 type 1 virus, 1 adenovirus). no formation of rbd-igg-anti-igg immune complex was observed for these samples, providing negative results in the igg avidity assay. the study was approved by the institutional review board of the university of california. all testing was performed on remnant serum samples collected for routine clinical testing and stored at -20°c prior to testing. individual and serial serum samples (n = 168) from patients with pcr-confirmed covid-19 (n = 90, 51% male, median age 49, 24% admitted to the icu) were analyzed. based on least squares linear regression analysis, there was a strong correlation between igg avidity and days since symptom onset (p < 0.0001) ( figure 1a ). sars-cov-2 igg avidity did not correlate with igg concentration, as measured previously using a quantitative immunoassay (1) . the kinetics of igg avidity maturation in 13 patients for whom serial samples (≥ 3) were available is shown in figure 1b . in all patients, igg avidity increased over time. peak measurements, which included the last measurement per 7-day interval per patient, were used to compare igg avidity by disease severity. peak readings were significantly higher for specimens from icu than non-icu patients for the first month after symptom onset (1-4 weeks) and thereafter, using the t-statistic (p < 0.0001) ( figure 1c ). aw reports scientific advisory board fees from et healthcare, outside the submitted work. no conflict for any other authors. a c c e p t e d m a n u s c r i p t 7 magnitude and kinetics of anti-sars-cov-2 antibody responses and their relationship to disease severity temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study clinical utility of avidity assays use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection development of label-free immunoassays as novel solutions for the measurement of monoclonal antibody drugs and antidrug antibodies antibody avidity maturation during severe acute respiratory syndrome-associated coronavirus infection key: cord-303240-tv1ta3z5 authors: althoff, keri n; coburn, sally b; nash, denis title: contact tracing: essential to the public health response and our understanding of the epidemiology of covid-19 date: 2020-06-11 journal: clin infect dis doi: 10.1093/cid/ciaa757 sha: doc_id: 303240 cord_uid: tv1ta3z5 nan m a n u s c r i p t as the pandemic in the us continues to evolve, data from contact tracing can continue to inform our understanding about sars-cov-2 transmission and guide the public health response. 1 a key finding of rosenberg et al. was the statewide positivity rate (proportion of molecular tests positive for sars-cov-2) of 33% in march, increasing from a daily minimum of 11% to 48% as the outbreak surged. 1 although the first covid-19 case was confirmed in new york on march 1, these positivity rates signaled wide-spread community transmission occurring in march. the second key finding was the very high proportion of household contacts also infected with sars-cov-2, which suggests a high likelihood of transmission within households, corroborating findings from wuhan, china. 1,2 although cdc has guidance for how to care for ill household members with covid-19, the guidance would be challenging to follow if: 1) the person ill is the only adult in a household with school-aged children or others who need care; or 2) there is not a separate bedroom and bathroom for isolation (or a source to improve air circulation if separate space is not available); or 3) the caretaker does not have access to gloves, disinfectants, or a washing machine. 3 these limitations are likely to be more common among people of lower socioeconomic status, who are also experiencing a disproportionate burden of covid-19 illness and impact. 4 contact tracers support individuals in isolation and equipping them with the ability to send gloves, disinfectant, and fans may also help reduce household transmission. a c c e p t e d m a n u s c r i p t contact tracing can be extremely labor intensive in part due to missing and incorrect information available to contract tracers. 5 the study by rosenberg et al., prompts the question: can technology, electronic medical records, and other innovative strategies (like at-home testing or selfcollection of specimens) be leveraged alongside existing public health infrastructure to improve the efficiency, reach, and impact of larger-scale test and trace initiatives? rosenberg et al. described that contact tracing and testing associated with their investigation included "covid-19 home testing teams and alternative specimen collection sites" which "enhanced community testing, improving individuals' access to testing, and decreasing healthcare system burden." 1 this raises important considerations for jurisdictions that are trying to rapidly expand testing as part of contact tracing efforts. leaving home to get tested may increase the risk of sars-cov-2 acquisition among those seeking a test who are not infected and increase the risk of onward community transmission among those who are infected. contact tracing that includes home-based molecular and/or serologic specimen collection to trigger isolation and quarantine may be especially critical when both testing and physical distancing are necessary to achieve the public health goals of epidemic control and mitigation of community transmission. as of this writing, there are a handful of attempts to deploy at-home testing and/or self-collection and mailing of specimens for sars-cov-2 testing as part of public health efforts and research, however the u.s. food and drug administration has recently halted a program in seattle from returning results to those wishing to be tested. [6] [7] [8] despite recent regulatory challenges, research to inform regulations permitting novel and effective public health approaches is urgently needed. and given the unprecedented nature of this pandemic and the response, the importance of generating evidence and learning from public health implementation activities such as those reported by rosenberg et al. cannot be overstated and must continue. as the covid-19 epidemic in the us evolves, the "re-opening" plans for many geographic regions include test and trace as essential elements to keeping transmission low. contact tracing and testing a c c e p t e d m a n u s c r i p t of household members of those newly diagnosed with sars-cov-2 was possible with existing contact tracing resources in new york state when the number of new diagnoses was low. however, it becomes infeasible when widespread community transmission takes hold; the control strategies must rapidly shift to non-pharmaceutical interventions, especially physical distancing. once these interventions succeed in reducing community transmission, physical distancing restrictions will be lessened (i.e. "re-opening"), and test and trace strategies will again be deployed to facilitate epidemic control and interruption of sars-cov-2 transmission via isolation and quarantine. during this phase of the epidemic, utilizing technology to aid contact tracing may reduce the number of days a potentially infectious person is not quarantined or in isolation. the north dakota department of health created the care19 app that uses gps to track location via mobile phone, assigns users a random id, and requires consent from covid-19 cases to use their information for contact tracing and forecasting. 9 utah rolled out the healthy together app to use bluetooth and gps data connected with individual identifiers. 10 if consent is provided, the contact tracer gains access to information about the contact's movement and uses that information to reduce gaps in recollection, effectively reducing the time spent and maybe even improving the accuracy of the information. apple and google are reportedly developing technology that will not track users' locations (to protect privacy), but will use bluetooth technology to determine if someone has been in contact with a covid-19 case. 11 the cdc has provided helpful guidance to understand the value of digital contact tracing and evaluate these tools regarding both case management and proximity tracking. 12 the demand for contact tracers is high as jurisdictions attempt to preserve the progress made in reducing r 0 through physical distancing interventions. the johns hopkins bloomberg school of public health recently released the "covid-19 contract tracing" coursera course and has waived the fees for a certificate. on may 16, 2020, the first day the course was offered, there were >130,000 learners enrolled world-wide and 7 million page views. a c c e p t e d m a n u s c r i p t decisions must be made now to protect human health with the data available. as demonstrated by rosenberg et al., data collected via contact tracing in the early phase of the us outbreak is important to understanding the epidemiology of covid-19. test and trace initiatives will yield equally important information in the current phase of the epidemic as communities begins to "re-open." the contact tracers who will continue to log endless hours to protect the health of the population against covid-19, and subsequently the stability of our healthcare systems, should be supported and commended. covid-19 testing, epidemic features, hospital outcomes, and household prevalence epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study us centers for disease control and prevention. caring for someone sick at home or other non-heatlhcare settings covid-19 and racial/ethnic disparities identification and monitoring of international travelers during the initial phase of an outbreak of covid-19 -california us approves at-home, saliva-based coronavirus test seattle coronavirus assessment network (scan). scan project update a national prospective cohort study of sars/cov2 pandemic outcomes in the office of govenor gary r herberg. state of utah releases "healthy together apple and google partner on covid-19 contract tracing technology preliminary criteria for the evaluation of digital contact tracing tools for covid-19 a c c e p t e d m a n u s c r i p t notes funding: kn althoff was supported by funding from the us national institutes of health, including the na-accord of iedea (u01 ai069918) and r01 ag053100. sb coburn was supported by a predoctoral training award from the us national cancer institute (f31ca247610). d nash was supported by funding from the us national institutes of health, including together 5000 (ug3ai133675) and central africa iedea (u01ai096299). drs. keri althoff, denis nash, and mrs. sally coburn report no conflicts of interest.a c c e p t e d m a n u s c r i p t key: cord-335907-93gwmo0v authors: de man, peter; paltansing, sunita; ong, david s y; vaessen, norbert; van nielen, gerard; koeleman, johannes g m title: outbreak of covid-19 in a nursing home associated with aerosol transmission as a result of inadequate ventilation date: 2020-08-28 journal: clin infect dis doi: 10.1093/cid/ciaa1270 sha: doc_id: 335907 cord_uid: 93gwmo0v nan m a n u s c r i p t dear editor, with great interest we read the appeal to address airborne transmission of covid-19 by morawska and milton in your journal [1] . recently, we were involved in an outbreak in a dutch nursing home that was likely to be the result of aerosol transmission in a setting of inadequate ventilation. this outbreak therefore illustrates the risks for which the authors are warning us. in total, 17 (81%) residents from one of the seven wards in a nursing home with psychogeriatric residents were diagnosed with covid-19 as confirmed by rt-pcr ( figure 1 ) [2] . subsequently, 17 (50%) healthcare workers (hcws) of the same ward were also tested positive. in contrast, all tests of the 106 hcws or 95 residents in the 6 other wards were negative. in the week of this outbreak a low prevalence of covid-19 was reported, only 493 (0.77%) positive cases were detected in the netherlands, whereas the weekly rate was 8391 (21.5%) during the national peak in april 2020 [3]. to prevent and control covid-19 infections all hcws in this nursing home had been assigned to specific wards and did wear surgical masks during patient contacts since april 26, 2020 [4] . hcws did not wear masks during not patient related activities and breaks. residents were housed in individual rooms and spend part of the day in shared living rooms, some residents were mobile. therefore, contact and thus direct transmission between residents cannot be excluded. because of the remarkable increase of covid-19 infections in a very short time period despite the use of surgical masks, the ventilation system of the outbreak ward was investigated in addition to routine source and contact tracing. this ward was renovated, including the installation of a co 2 controlled energy-efficient ventilation system. in this a c c e p t e d m a n u s c r i p t system, the indoor air was only refreshed with outside air based on real time co 2 concentration measurements. if the co 2 concentration did not exceed 1000 ppm, the ventilation cabinets recirculated indoor air back into the ward without filtration. in this situation, low co2 production by inactive nursing home patients might have limited the ventilation with outside air. moreover, this ward was additionally cooled by two air conditioning units, which recirculated air through a 1 mm mesh dust filter. in contrast, the other six wards were ventilated with outside air. a c c e p t e d m a n u s c r i p t none to declare m a n u s c r i p t it is time to address airborne transmission of covid-19 detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr current information about the novel coronavirus (covid-19) bilthoven: rivm allowing visitors back in the nursing home during the covid-19 crisis: a dutch national study into first experiences and impact on well-being m a n u s c r i p t key: cord-305394-wwabxlgr authors: venter, w d francois; nel, jeremy title: covid-19: first data from africa date: 2020-08-31 journal: clin infect dis doi: 10.1093/cid/ciaa1293 sha: doc_id: 305394 cord_uid: wwabxlgr nan m a n u s c r i p t africa is no stranger to infectious disease pandemics prior to sars-cov-2, with recent major outbreaks spanning ebola to listeria and measles, and ongoing high background incidence of malaria, hiv and tb. most countries on the continent have severe health resource constraints, and the focus of many far richer countries hit by the sars-cov-2 epidemic -pcr-guided contact tracing to prevent spread, reliance on intensive-care and ventilation to deal with those who became severely ill -struck fear into african public health practitioners, health workers and the public, where sophisticated laboratory infrastructure and high-tech intensive care is often severely rationed (1) . there was speculation that the continent may be relatively protected from the worst of covid by its younger population age structure (and fewer associated chronic comorbidities linked to covid-19 mortality), limited public transport, possibly greater circulating seasonal coronaviruses, and even antiretroviral therapy programs with anti-coronavirus activity. however, the possibility of hiv and tb being risk factors for covid severe events, both relatively unusual in the richer countries, could mean worse consequences in these populations (2) . dr mary-anne davies presents important data from the western cape province, the epicenter for south africa's initial wave of infections, in this edition of cid (3) . south africa as a major business and tourism hub was always likely to be one of the earliest african countries struck. initial severe lockdown measures were announced in march as the first internal cases in international travelers were reported. community seeding, especially in poorer and more densely populated communities, was delayed, but infections rapidly accelerated, and at the time of writing (late august 2020), the country has the fifth largest epidemic in the world (4). this data is from an ongoing surveillance cohort that has previously generated rich data on disease patterns in the western cape, and currently continues to provide near real-time updates on the impact of pcr-confirmed sars-cov-2 on factors ranging from death to oxygen consumption within hospitals. data from electronic clinical information systems is synthesized with laboratory, pharmacy and administrative data, providing a powerful population level dataset. reported data from other national surveillance systems from across the country confirms much of davies' findings, although her dataset is remarkable in its detail. key strengths of the paper include a dataset covering over 3 million healthcare users in the western cape province, and the use of both hospitalized and nonhospitalized cases and deaths davies' data shows similar mortality risk factors, including age, sex, diabetes (especially uncontrolled diabetes), hypertension and renal disease to other cohorts from richer countries. the data do provide vital information on south africa's other two continuing pandemics, with hiv and past or current tb all giving a roughly two-fold increased risk of death. earlier reports from europe and north america had not found a clear association with mortality, but they were limited by small sample sizes and selection biases. davies' cohort had about 4000 hiv positive patients with covid in it -about 40 times more than all the other published case series combined to date (5) . the dataset shows an association between hiv infection and increased mortality, with a hazard ratio after adjusting for age, sex and other comorbidities of 2.14 (95% ci 1.7-2.7). the possibility of residual confounding exists, but davies makes a plausible case for hiv being an independent risk factor for covid-19-related death. interestingly, davies' dataset does suggest a higher mortality in hiv patients with virological failure and/or pre-covid cd4 counts under 200 cells/µl compared with those who were virally suppressed, though there is substantial overlap in the confidence intervals, m a n u s c r i p t and may be confounded by social factors associated with non-adherence, as well as covid-19 mortality. whether antiretroviral therapy somehow mitigates covid outcomes has been widely speculated in the hiv scientific literature, with in-vitro data suggesting tenofovir has activity against coronaviruses (6) . intriguingly, in the multivariate analysis davies found that patients receiving tenofovir disoproxil fumarate as part of their antiretroviral therapy saw a statistically significant reduction in mortality (ahr 0.41, p=0.007). this supports observational data from a spanish cohort that similarly suggested better outcomes for those on tenofovir-based antiretroviral therapy (5) . however, extreme care should be taken interpreting the tenofovir data, as the association is at high risk of confounding and channeling bias. patients not on tenofovir in south africa are very likely to have underlying renal dysfunction, or be on second-line therapy, which is often again associated with coexisting social issues that may make them more vulnerable to covid-19 consequences. davies' data again makes a substantial addition to the covid-19 literature with respect to tuberculosis, a previously unreported topic. both active tuberculosis and past tuberculosis were associated with an elevated risk of mortality in the study, the latter presumably on the basis of residual lung damage and consequent poor respiratory reserve. what does this data mean for the rest of africa? we should be wary of extrapolating too far -africa has enormous diversity, and western cape demographics differ markedly even from the rest of south africa. however, the data is broadly similar to other regions in the world, and so the immediate priority for african health systems remains to rapidly and practically design systems to protect the elderly and those with chronic diseases or tb. for south africa, a sigh of relief at a relatively small increase in mortality in hiv and tb should be quickly tempered; diabetes was the second commonest cause of death in the country pre-covid-19, and most patients in the country have poor glucose control, a major risk factor from davies' data (7) . in addition, obesity is an independent risk factor for covid-19 mortality in other cohorts, and while this was not collected in this dataset (weight and height was not available for this cohort), the country's obesity epidemic, especially among women, is well documented. among hiv patients, again more among women, the large-scale introduction of dolutegravir in late 2019 may markedly aggravate obesity and covid mortality in the near-6 million south african patients on antiretrovirals (8) . sadly, south africa has not learnt from other african countries and their epidemics (2) . when ebola struck in west africa, the consequence of locking down the society and suspending vaccine programs extracted a mortality from measles alone estimated to be similar to that from ebola (9) . initially lauded by the who for the speed and severity of covid-19 lockdown measures, the south african response became increasingly militarised (over 200 000 people charged or incarcerated since the start), characterized by bizarre decisions on commerce, an incoherent pcr-based testing and tracing program, poor food support programs, corruption around procurement of personal protective equipment, and chaotic re-opening of schools, with a temporary decimation of vaccination and hiv and tb programs (10) . the country will face years of continuing infectious and other diseases well beyond this sars-cov-2 era, that may well lead to a far greater mortality than covid-19. low-and-middle income countries will need to ponder future pandemic responses that pit any current epidemic against hard-won public health interventions. m a n u s c r i p t finally, the value of having integrated surveillance systems such as the western cape's that can rapidly inform urgent public health responses in real time are demonstrated in this paper. african governments and donors should invest more in these systems, as a major adjunct to public health programs. fv reports research grants from bill and melinda gates foundation, unitaid, usaid, samrc, and viiv healthcare; drug donations from gilead sciences and viiv healthcare; and honoraria from gilead, viiv, mylan, merk, adcock-ingram, aspen, abbott, roche, and j&j, all outside the submitted work. j.n. has no potential conflicts to disclose. covid-19: shining the light on africa tackling covid-19: can the african continent play the long game? j glob health risk factors for covid-19 death in a population cohort study from the western cape province who coronavirus disease (covid-19) dashboard coinfection: case reports, retrospective cohorts and outcomes triphosphates of the two components in descovy and truvada are inhibitors of the sars-cov-2 polymerase (preprint) mortality and causes of death in south africa: findings from death notification weight gain and integrase inhibitors reduced vaccination and the risk of measles and other childhood infections post-ebola covid-19 lockdowns in low-and middle-income countries: success against covid-19 at the price of greater costs a c c e p t e d m a n u s c r i p t key: cord-323125-qtlevnbt authors: al hosani, farida ismail; kim, lindsay; khudhair, ahmed; pham, huong; al mulla, mariam; al bandar, zyad; pradeep, krishna; elkheir, kheir abou; weber, stefan; khoury, mary; donnelly, george; younis, naima; el saleh, feda; abdalla, muna; imambaccus, hala; haynes, lia m; thornburg, natalie j; harcourt, jennifer l; miao, congrong; tamin, azaibi; hall, aron j; russell, elizabeth s; harris, aaron m; kiebler, craig; mir, roger a; pringle, kimberly; alami, negar n; abedi, glen r; gerber, susan i title: serologic follow-up of middle east respiratory syndrome coronavirus cases and contacts—abu dhabi, united arab emirates date: 2019-02-01 journal: clin infect dis doi: 10.1093/cid/ciy503 sha: doc_id: 323125 cord_uid: qtlevnbt background: although there is evidence of person-to-person transmission of middle east respiratory syndrome coronavirus (mers-cov) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. methods: a seroepidemiological investigation was conducted among mers-cov case patients (cases) and their household contacts to investigate transmission risk in abu dhabi, united arab emirates. cases diagnosed between 1 january 2013 and 9 may 2014 and their household contacts were approached for enrollment. demographic, clinical, and exposure history data were collected. sera were screened by mers-cov nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. results: thirty-one of 34 (91%) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. mers-cov antibodies were detected in 13 of 24 (54%) case patients with available sera, including 1 severely symptomatic, 9 mildly symptomatic, and 3 asymptomatic case patients. no serologic evidence of mers-cov transmission was found among 105 household contacts with available sera. conclusions: transmission of mers-cov was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. these results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission. although there is evidence of person-to-person transmission in household and healthcare settings [10] [11] [12] [13] [14] , more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. these data would be of importance to the public health response given that approximately 25% of confirmed mers-cov cases reported to the world health organization have been described as mildly symptomatic or asymptomatic [15] . during 1 january 2013-9 may 2014, the department of health-abu dhabi (doh) investigated 65 laboratory-confirmed cases and conducted extensive contact investigations in both household and healthcare settings [5] . through these investigations, 72% of the laboratory-confirmed cases reported no symptoms or mild illness [5] . contacts of case patients were tested by diagnostic pcr assays; however, results could include false negatives due to the 14-day incubation period. in this investigation, we use serological detection of mers-cov antibodies to evaluate if asymptomatic or mildly ill case patients had detectable mers-cov antibodies, estimate transmission rates from known cases to their household contacts, and identify potential risk factors. this investigation occurred in the emirate of abu dhabi, which occupies >80% of the uae's total area [16] and is comprised of 3 regions: abu dhabi (capital city), al ain region, and al dhafra. the emirate of abu dhabi has a population of 2.8 million (2015 estimate) [17] . the al ain region borders oman and saudi arabia and houses the second largest city in the emirate, al ain city. while al ain city is an oasis, the rest of the region primarily consists of desert and mountains. the al dhafra region is mainly desert and rural with approximately 285 000 residents and a population density of 8 residents/km 2 [18] . all laboratory-confirmed mers-cov cases (n = 65) in the emirate of abu dhabi diagnosed between 1 january 2013 and 9 may 2014 and their household contacts (n = 452) were eligible for the investigation. these cases were a convenience sample during the ongoing mers-cov outbreak. two of the 431 (0.5%) household contacts tested for mers-cov during initial contact investigations were pcr positive and eligible to be enrolled as cases for our investigation (figure 1 ). the enrolled case was a healthcare worker who might have been exposed by another coworker, who also lived in the case's household; therefore, the enrolled case was a result of either household or healthcare transmission prior to this investigation's initiation. the case not enrolled in this investigation was exposed in the household. household contacts were defined as any person who stayed at least 1 night at the same location as the case patient during the 14 days prior to the case patient's symptom onset or the date of first positive specimen if the case patient was asymptomatic. excluded cases included palace workers and other high-level officials; their associated household contacts were also excluded. for each mers-cov case identified in the investigation, clinical information, including symptoms, was collected using the international severe acute respiratory and emerging infection consortium form, which was filled out in real time by healthcare providers and subsequently verified by retrospective chart review. in abu dhabi during this time period, all individuals who tested positive for mers-cov were admitted to a healthcare facility for observation and infection control regardless of symptom status. the same definitions for case severity were used as in al hosani et al [5] including the following: asymptomatic cases reported no symptoms at the time of a positive test as recorded by a healthcare provider in the medical chart; mildly symptomatic cases reported symptoms, such as pharyngitis, rhinorrhea, or cough, and did not require oxygen during their hospitalization; and severely symptomatic cases required supplemental oxygenation during their hospitalization, ranging from nasal cannula to mechanical ventilation. using data collected from doh's surveillance of mers-cov cases, households with mers-cov case patients were approached. household contacts who were eligible for the investigation included those that had been identified through contact investigations associated with the case patient performed by doh officials within 24 hours' notification. three attempts were made to contact each household. if no response was received after 3 attempts, the household was not enrolled. households that agreed to be enrolled were given an appointment at the local disease prevention and screening center for questionnaire administration and serum collection. questionnaires were administered in english, arabic, or, if an interpreter was available, the participant's native language. data collected included demographics; residence/household description; exposure history to other mers-cov cases, healthcare settings, and animals; travel history; and medical history, including any long-term effects reported by case patients. for deceased case patients, a proxy completed the case patient questionnaire using recall. the real-time reverse-transcription pcr (rrt-pcr) results were obtained from the doh surveillance data. upper (ie, nasopharyngeal, oropharyngeal) and lower respiratory tract specimens (ie, sputum, bronchoalveolar lavage fluid, tracheal aspirates) were analyzed using rrt-pcr in the sheikh khalifa medical center laboratory. additional laboratory result verifications were performed in a random sample of 23 specimens using nucleocapsid-based rrt-pcr [5] . serum samples were inactivated using 2 × 10 6 rads gamma irradiation and stored at ≤ -70°c until use. screening of serum specimens by mers-cov nucleocapsid enzyme-linked immunosorbent assay (elisa) was performed at the sheik khalifa medical city in abu dhabi, uae and the centers for disease control and prevention (cdc), atlanta, georgia. titers of ≥1:400 were reported as positive. recombinant full length mers-cov nucleocapsid protein indirect elisa was used to screen serum specimens as described by al-abdallat et al [19] . serum samples were tested for the presence of neutralizing antibodies to mers-cov using a microneutralization assay (mnt) [19] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell lysis in at least 1 of the 3 triplicate wells. positive and negative controls were included for each mnt performed and included back-titration and mock-infected cells. titers of ≥1:20 were reported as positive. all work with live mers-cov was done in biosafety level 3 containment at the cdc. immunofluorescence assays (ifas) were performed by screening sera at a dilution of 1:50 and 1:100 on paraformaldehyde-fixed, acetone-methanol permeabilized mers-cov (strain mers-cov hu/england-n1/2012) infected or uninfected control vero cells. antihuman immunoglobulin g, m, and a fluorescein isothiocyanate conjugate was used to detect anti-mers-cov antibodies in human serum, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole to allow identification of individual mers-cov-infected cells. fluorescence was detected using a zeiss axioimager fluorescence microscope. the positive control for the assay is a serum sample from a patient infected with mers-cov hu/ england-n1/2012. a positive result was scored when these 3 conditions were met: cells were evenly stained (instead of punctate staining); fluorescence intensity was higher than that of the negative controls; and signal intensity declined with serial dilution. a minimum of 2 negative controls were included with each ifa. approximately 10% of specimens negative by nucleocapsid elisa were screened by both ifa and mnt to confirm the negative result. mers-cov antibody positivity was defined as one of the following: (1) 2 of 3 tests (ie, mers-cov nucleocapsid elisa, mers-cov mnt, and ifa) were positive; or (2) mers co-v mnt was the only positive test. household survey data were entered into electronic forms in epi info 7 version 7.1 (cdc). quality control and assurance were performed through epi info 7 intelligent codes programmed into the forms. household survey data were merged with the laboratory results, and descriptive analysis was completed. differences in proportions were compared using the mantel-haenszel χ 2 test, while differences in continuous variables were compared using the student t test. p < .05 was considered statistically significant. data analysis of the merged dataset was conducted with sas version 9.3 software (sas institute, cary, north carolina). following local customs, informed consent was obtained from the head of the household, who provided consent for all members of a household; however, each individual was still able to decline participation. this investigation was determined by doh and cdc to be part of a public health response, not research, and therefore not subject to institutional review board review. thirty-four case patients' households were included (supplementary table 1 ). household residences ranged in size from 7 m 2 to 1100 m 2 (interquartile range [iqr], 70-200 m 2 ). a median of 4 individuals (range, 1-30) lived in the households 14 days prior to the diagnosis of a mers-cov household case patient. more than half of mers-cov case patients shared a bathroom with others in the household. all households reported having air conditioning. thirty-four cases of 65 (52%) and 124 household contacts of 452 (27%) participated (table 1) . females comprised a higher proportion of case patients compared with household contacts (70.6% vs 53.2%), and case patients were older compared with household contacts (median, 42 years vs 31 years). most case patients and contacts were from the al ain region of the abu dhabi emirate. seventy-one percent (n = 24) of case patients reported working in a healthcare setting 14 days prior to diagnosis, with nurses being most represented (24%, n = 8) ( table 1) ; only 24% (n = 30) of household contacts worked in a healthcare setting 14 days prior to a case patient's diagnosis. compared with household contacts, case patients less frequently reported visiting or owning a farm (12% vs 14%), but reported camel exposure more frequently (12% vs 7%). household contacts reported the following frequent exposures to mers-cov case patients: hugging (54%, n = 67), using the same bathroom (51%, n = 63), sharing meals (49%, n = 61), and kissing or nose-kissing (ie, rubbing tips of noses against one another) (48%, n = 60). case patients reported a higher proportion of underlying medical conditions than household contacts, including diabetes (18% vs 5%, p = .01), hypertension (27% vs 7%, p < .001), kidney failure (6% vs 2%, p = .03), and heart failure (6% vs 0%, p < .01) ( table 1 ). case patients also reported taking medications for any illness more frequently than contacts (38% vs 17%). three case patients (9%) reported limitation to activities due to mers-cov with a median duration of 5 days (iqr, 4-24 days) ( table 1) . normal activities were resumed at a median of 5 days (iqr, 3-7 days). of 34 case patients, 17 (50%) reported being asymptomatic, 14 (41%) reported being mildly symptomatic, and 3 (9%) were severely symptomatic. age and proportion having underlying medical conditions increased with symptom severity ( table 2) . symptom duration did not have any noticeable trend with symptom severity (data not shown). all severe case patients were treated in the intensive care unit, as well as 1 asymptomatic case, who had underlying diabetes, hypertension, and kidney disease. the median days hospitalized increased with symptom severity ( table 2) . sera were obtained from 24 of 34 (71%) case patients and 105 of 124 (85%) household contacts. among the 24 case patients with available sera (table 3) among the 13 case patients with detectable antibodies against mers-cov, all of them were aged <60 years, with a median age of 43 years, compared to a median of 32 years for case patients without detectable antibodies (table 4 , p = .04). number of days of pcr positivity was notably higher among those who had detectable antibodies compared to those who did not (median, 15 days vs 2 days, p = .01). we describe the results of follow-up of 34 mers-cov case patients and 124 of their household contacts from the emirate of abu dhabi during 2013-2014. notably, serologic testing did not find any evidence of mers-cov transmission in the households of mers-cov case patients in our investigation, suggesting that viral transmission from asymptomatic or mildly symptomatic individuals to household contacts does not readily occur. sera were tested with a combination of 3 different laboratory assays (nucleocapsid elisa, ifa, and mnt); we feel confident that individuals identified as "negative" did not seroconvert. although there was clear evidence of household transmission in 1 household not enrolled in this investigation, our investigation's results did not show evidence of additional household transmission. overall, our findings support current recommendations that home isolation may be appropriate for asymptomatic cases and close contacts who are ill and do not require hospitalization in consultation with local public health departments [20, 21] . because this investigation occurred during may-june 2014, many case patients were recruited from the april 2014 healthcare-associated outbreak at an al ain region hospital [22] . a kingdom of saudi arabia study found that while healthcare personnel were at high risk for infection, most illness was relatively mild and could be unrecognized, highlighting potential undetected transmission of the virus to others [23] . in our investigation, case patients tended to be younger (30-59 years) , and most reported working in a healthcare setting 14 days prior to their diagnosis where they were exposed to a similar to previous studies, case patients with severe disease had higher frequency of comorbid conditions and required intensive care, including intubation [24] [25] [26] . in a recent investigation from south korea, patients with a higher host infectivity, which included evaluation of pcr cycle threshold values, along with higher numbers of contacts, were more likely to transmit mers-cov [27] . it is likely that most of the primary case patients in this investigation had lower host infectivity. while our investigation found that some asymptomatic or mildly symptomatic case patients had detectable antibodies, we did not find any detectable antibodies in 11 asymptomatic and mildly symptomatic case patients. other studies also did not find detectable antibodies in some asymptomatic and mildly ill cases [6, 28] . if seroconversion is to occur in case patients, studies have demonstrated that this usually occurs within the first month of illness [28] [29] [30] . for the majority of case patients with detectable antibodies, we found persistence of antibody response for several months after the initial diagnosis, even close to a year. additionally, these case patients had a longer duration of mers-cov pcr positivity than those who did not have detectable antibodies, indicating a potential relationship between longer viral shedding and seroconversion. previous studies have demonstrated that asymptomatic and mildly symptomatic case patients can test pcr positive >2 weeks from lower respiratory tract specimens [5, 31] . our investigation's serology results do not provide additional evidence of transmission to household contacts, though there is evidence from other settings to suggest limited household transmission [11] . also, very low rates of household transmission have been reported during hospital-based outbreaks [19, 32] . more robust transmission studies involving larger numbers of case patients representing a range of clinical and demographic characteristics and their contacts are needed to further investigate risk exposures. there are several limitations to this investigation. first, serum samples were collected at varying intervals after illness onset for each case patient, potentially affecting serology results. the duration of antibody response is unknown. second, recall bias might have led to the misclassification of symptom severity among household contacts; however, for case patients, to minimize this bias, we relied upon retrospective medical chart review, though this also might not be as complete since it depended on the initial healthcare provider's history and physical. third, these case patients were immediately isolated in hospitals after pcrpositive results were discovered. the removal from the household setting might have reduced exposure to household contacts although the case patients were residing with household contacts at the time of the contact investigations. last, because our investigation did not detect household transmission, we cannot comment on any behaviors or exposures that would increase risk among household contacts of case patients. in summary, we did not document additional household transmission in this investigation that included a preponderance of asymptomatic and mildly symptomatic confirmed mers-cov case patients. our investigation findings support the recommendation to consider home isolation for asymptomatic and mildly ill cases that do not require hospitalization while using proper precautions, including face masks, frequent hand washing, and minimizing exposure to the case patient in the household [20, 21, 33] . while no vaccines or antivirals against mers-cov are currently available, reducing transmission through effective infection control management remains a major priority. understanding transmission risk for different mers-cov-infected patients who live in different settings will be important data that must be factored into prevention strategies. further studies on human-to-human transmission in different settings should be conducted to inform mers-cov prevention and control guidelines. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): current situation 3 years after the virus was first identified world health organization. who mers-cov global summary and risk assessment risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia response to emergence of middle east respiratory 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and clinical description management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance implementing home care and isolation or quarantine of people not requiring hospitalization for mers-cov transmission of middle east respiratory syndrome coronavirus infections in healthcare settings risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea serologic responses of 42 mers-coronavirusinfected patients according to the disease severity viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker health care worker contact with mers patient, saudi arabia interim guidance for healthcare professionals acknowledgments. the authors gratefully thank the participants; the teams from the disease prevention and screening center-al ain, the disease prevention and screening center-abu dhabi, ghayathi hospital, and silla hospital that supported this investigation; and suvang trivedi and seyhan boyoglu-barnum at the centers for disease control and prevention (cdc) for technical support.disclaimer. the conclusions, findings, and opinions expressed herein are those of the authors and do not necessarily reflect the official position of the us department of health and human services, the us public health service, the cdc, or the authors' affiliated institutions.financial support. this work was supported by the cdc. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-327069-vjlisnui authors: driscoll, amanda j.; karron, ruth a.; morpeth, susan c.; bhat, niranjan; levine, orin s.; baggett, henry c.; brooks, w. abdullah; feikin, daniel r.; hammitt, laura l.; howie, stephen r. c.; knoll, maria deloria; kotloff, karen l.; madhi, shabir a.; scott, j. anthony g.; thea, donald m.; adrian, peter v.; ahmed, dilruba; alam, muntasir; anderson, trevor p.; antonio, martin; baillie, vicky l.; dione, michel; endtz, hubert p.; gitahi, caroline; karani, angela; kwenda, geoffrey; maiga, abdoul aziz; mcclellan, jessica; mitchell, joanne l.; morailane, palesa; mugo, daisy; mwaba, john; mwansa, james; mwarumba, salim; nyongesa, sammy; panchalingam, sandra; rahman, mustafizur; sawatwong, pongpun; tamboura, boubou; toure, aliou; whistler, toni; o’brien, katherine l.; murdoch, david r. title: standardization of laboratory methods for the perch study date: 2017-06-15 journal: clin infect dis doi: 10.1093/cid/cix081 sha: doc_id: 327069 cord_uid: vjlisnui the pneumonia etiology research for child health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. blood, respiratory specimens, and urine were collected from children aged 1–59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. a standardized testing algorithm and standard operating procedures were applied across all study sites. site laboratories received uniform training, equipment, and reagents for core testing methods. standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory. the primary aim of the pneumonia etiology research for child health (perch) study was to provide a contemporary picture of the microbial etiology of severe pneumonia in young children from developing countries [1] . one of the defining characteristics of the study was the use of a standard case definition and a rigorous training program to achieve standardization of case assessments and specimen collection [2] . the standardization of laboratory methods in the perch study was equally important to ensure comparability across study sites and for accurate and meaningful interpretation of pneumonia etiology results. we have previously described the process leading to the perch diagnostic testing strategy [3] [4] [5] [6] . here we describe the laboratory methods used in perch and the procedures to ensure standardization and quality. sites. to build capacity at the sites, and in alignment with the priorities of the bill & melinda gates foundation, all perch testing was done locally, with the exception of quality assurance testing and a select subset of specialized assays, which were performed at the study reference laboratory (canterbury health laboratories, christchurch, new zealand), which also served as the study specimen and isolate biorepository. the perch site laboratories were located in kilifi, kenya; basse and banjul, the gambia; bamako, mali; lusaka, zambia; soweto, south africa; nakhon phanom, sa kaeo, and nonthaburi, thailand; and matlab and dhaka, bangladesh. study sites were selected through an open, global site solicitation and selection process. as there was little support in the budget to expand existing infrastructure, preference was given to sites with well-established research laboratories and with experience in pediatric pneumonia studies. blood, nasopharyngeal and oropharyngeal (np/op) swabs, induced sputum (is), and urine were collected from all perch cases at enrollment. cases who were intubated had endotracheal aspirate collected in lieu of induced sputum. gastric aspirate and pleural fluid specimens were collected when clinically indicated. at select sites, lung aspirates (bangladesh, the gambia, mali, south africa) and postmortem specimens (thailand, south africa) were also collected. among perch controls, blood, np/op swabs, and urine were collected at enrollment at all sites. standardized recommendations for specimen storage prior to laboratory evaluation were provided in standard operating procedures (sops) ( table 1 ). all specimens were collected within walking distance or within a 2-hour drive of the study laboratory, with the exception of matlab, bangladesh, where specimens were transported once or twice a day from the field hospital to the laboratory in dhaka, and basse, the gambia, where specimens were transported 1 to 2 times weekly to the fajara laboratory for molecular testing. the transportation of all specimens was done under controlled temperature conditions, as stipulated in the study sop. standardized testing algorithms were developed for each body fluid type among cases and controls ( figure 1 ). following testing, residual volumes of body fluid specimens were stored at -80°c to facilitate future research, with particular attention to bioethical considerations [7] . bacterial isolates cultured in pure growth were stored at -80°c unless classified as contaminants. barcode labels were used on specimen collection containers, data collection forms, and laboratory requisition forms [8] . in the laboratory, specimen aliquots and isolates were archived in 2-ml sterile cryovials with silicone o-rings, labeled with thermostable labels, and inventoried using freezer management software [8] . laboratory data were entered into an electronic data capture system to allow for real-time study monitoring [8] . to standardize molecular testing, an automated nucleic acid extraction platform and standardized polymerase chain reaction (pcr) assays, described below, were deployed across all study sites. the perch laboratory director reviewed all pcr results files prior to their inclusion in the study database. for pcr evaluation of respiratory specimens, we used the fasttrack diagnostics respiratory pathogens 33 multiplex pcr kit (ftd resp-33 kit) (fast-track diagnostics, sliema, malta). np/ op specimens were collected in viral transport medium (universal transport medium [utm], copan diagnostics, bresica, italy) and refrigerated at 2°c-8°c for a maximum of 8 hours, or frozen at -80°c prior to nucleic acid extraction. induced sputum, pleural fluid, and lung aspirate specimens were collected in saline in universal containers and either refrigerated at 2°c-8°c for a maximum of 24 hours, or frozen at -80°c prior to nucleic acid extraction. total nucleic acid extraction was performed on respiratory specimens using the nuclisens easymag platform (biomérieux, marcy l'etoile, france). four hundred microliters of each respiratory specimen (np specimen in utm, induced sputum aliquot in normal saline, pleural fluid aliquot, or lung aspirate aliquot) was eluted to a final volume of 60-110 μl nucleic acid. prior to extraction, induced sputum specimens were digested with 1:1 dithiothreitol and incubated at ambient temperature until any mucus was broken down. the ftd resp-33 kit is a real-time pcr arranged in 8 multiplex groups for the detection of the following 33 viruses, bacteria, and fungi: influenza a, b, and c; parainfluenza viruses 1, 2, 3, and 4; coronaviruses nl63, 229e and haemophilus influenzae species. the k. pneumoniae target was not used in any of the final analyses because of difficulties with assay specificity, as has been found elsewhere [9] . positive, negative, and internal extraction controls were included in each run. quantitative pcr (qpcr) data were generated through the creation of standard curves using 10-fold serial dilutions of plasmid standards provided by ftd on an approximately quarterly basis at each study site, with calculation of pathogen density (copies/milliliter) from the sample cycle threshold (ct) values. because the results for the known standards were highly consistent across laboratories, standard curve data from all sites were pooled to create "standardized" standard curves for each pathogen target; data points beyond 2 standard deviations of the mean were excluded. quantitative pcr was performed at each site using an applied biosystems 7500 (abi-7500) platform (applied biosystems, foster city, california). cycling conditions were 50°c for 15 minutes, 95°c for 10 minutes, and 40 cycles of 95°c for 8 seconds followed by 60°c for 34 seconds. whole blood samples were collected into a dedicated edta (ethylenediaminetetraacetic acid) tube and either refrigerated at 2°c-8°c for a maximum of 3 days, or frozen at -80°c prior to nucleic acid extraction. total nucleic acid extraction was performed in batches with 200 μl of whole blood extracted and eluted to a final volume of 100 μl nucleic acid using the nuclisens "specific b" protocol. extracted dna was frozen at -80°c until undergoing pcr for detection of the autolysin (lyta) gene. streptococcus pneumoniae nucleic acid was detected in whole blood using a qpcr assay based on a method from the us centers for disease control and prevention [10] . mastermix containing 12.5 μl of gene expression mastermix (applied biosystems, life technologies, california), 0.5 μl of each of the 10 μm forward and reverse primers and probe, 1 μl of molecular-grade water, and 10 μl of template dna was used per reaction. quantification standards consisting of lyta plasmids (fast-track diagnostics, sliema, malta) diluted 1:10 from 10 7 copies/ml to 10 2 copies/ml were run in triplicate on every plate. a no-template control, consisting of molecular-grade water, was likewise run in triplicate. cycling conditions of 95°c for 10 minutes followed by 40 cycles of 95°c for 15 seconds and 60°c for 1 minute were applied on an abi-7500 instrument. exponential amplification curves with a ct value of <40 cycles were considered positive and quantified using the standard curve. all samples positive for bordetella pertussis were tested for bordetella holmesii [11] . a high proportion of samples positive for h. influenzae type b, especially from countries with hib vaccine, called into question the specificity of that pathogen target. consequently, we retested these positive samples with an established hib assay [12] and used the results from this second assay in our analyses. due to intermittent contamination of the nucleic acid extraction lysis buffer with legionella species, samples positive for legionella were retested at the reference laboratory [13] . because few whole blood samples from thailand and bangladesh were positive for lyta pcr, we retested a random sample of 100 documented pneumococcal carriers (np/op specimens positive for s. pneumoniae) from both sites using the perch lyta pcr assay at the reference laboratory. no false negative blood results were found. np/op swabs from all 33 cases with clinical signs or history of measles were tested for measles virus by pcr using the nucleoprotein gene target [14] at the reference laboratory. suspected measles was defined as a history of measles in the past 3 months, measles rash at admission, or measles diagnosis at admission or discharge. organism identification was done according to standard microbiological methods that were documented in sops and clarified at each site at the outset; antimicrobial susceptibility testing followed the clinical and laboratory standards institute (clsi) guidelines [15] . antimicrobial susceptibility was tested using overnight growth of pure isolates, using the disk diffusion methodology when possible. streptococcus pneumoniae isolates that had reduced susceptibility to penicillin by the oxacillin screen were tested by etest (biomérieux, low-dose strips) or a commercially available broth mic method (trek diagnostic systems) to measure minimum inhibitory concentrations (mic) to penicillin. enterobacteriaceae were screened for extended spectrum β-lactamase production using a cefotaxime (30 µg) disk and a ceftazidime (30 µg) disk. zone sizes ≤27 mm for cefotaxime or ≤22 mm for ceftazidime were confirmed by the double disk diffusion test, following the clsi guidelines. organism identification was confirmed for each specimen type and is described below. antibiotic susceptibility testing results were confirmed in a sample of 10% of isolates from all sites with 100% concordance of results. blood cultures were incubated per manufacturer instructions using automated systems (bactec [becton dickinson, sparks, maryland] in kenya, south africa, the gambia, mali, and zambia; bact/alert [biomérieux, marcy l'etoile, france] in bangladesh and thailand). culture bottles were incubated within 24 hours of specimen collection. alarm-positive culture specimens were plated on to 5% sheep or horse blood, chocolate, and macconkey agar. specimens were incubated for 5 days and then discarded. specimens that were alarm-positive but subculture negative were tested using the binaxnow streptococcus pneumoniae antigen card (alere, scarborough, maine) if the gram stain was either negative or revealed gram-positive cocci. organisms were identified and antimicrobial susceptibility testing was performed according to clsi methods. any isolate classified as a contaminant was not stored; all other organisms were identified then stored at -80°c. organisms were defined, a priori, as contaminants (table 2) . of 195 stored blood culture isolates, 146 (72%) were shipped for confirmatory identification at the reference laboratory; of these, 121 (83%) were concordant with the original result. in the absence of strong evidence otherwise, organisms meeting the a priori contaminant definition were considered as contaminants. candida species was also considered a blood culture contaminant if the patient recovered without antifungal treatment. np swabs in skim milk tryptone-glucose-glycerin (stgg) medium were frozen at -80˚c overnight, then thawed and processed using a broth-enrichment step to enhance pneumococcal carriage recovery [16, 17] . streptococcus pneumoniae was identified by colony morphology, susceptibility to optochin, and bile solubility testing. samples were inoculated onto 5% sheep or horse blood agar with 5 µg of gentamicin per milliliter and incubated at 35°c ± 2°c for 18-24 hours. following subculture, each morphologically distinct pneumococcal colony was isolated and stored, with up to a maximum of 4 isolates per plate. efforts were made to process sputum specimens within 2, and no more than 24, hours following collection. gram-stained smears were made from the most purulent portion of each induced sputum specimen. the number of epithelial cells and neutrophils per low-powered microscopic field were counted and recorded for the purpose of assessing specimen quality [18] . microorganisms seen in the smear were described according to classic gram stain morphotypes, with the number of bacterial morphotypes seen per high-powered field recorded to assist in interpretation of culture results. the most purulent portion of each specimen was inoculated onto sheep or horse blood, chocolate, and macconkey agars, streaked out using the 4-quadrant streaking method, and incubated at 35°c for 48 hours. cultures were examined at 24 hours and 48 hours, and predominant organisms were identified and quantified according to the furthest quadrant with visible colonies (first quadrant = scanty; second quadrant = 1+; third quadrant = 2+; fourth quadrant = 3+). background mixed oropharyngeal flora, including α-hemolytic streptococci, commensal neisseria, coagulase-negative staphylococci, yeasts (except cryptococcus), diphtheroids, and capnocytophaga were quantified as a group but not identified further. induced sputum specimens were also cultured for mycobacteria by standard liquid culture methods. gram stains were performed on all pleural fluid and lung aspirate specimens, and the number of leukocytes per low-powered field and bacterial morphotypes per high-powered field was recorded. each specimen was cultured by plating onto chocolate and macconkey agar and also inoculated in appropriate broth (blood culture bottles, tryptone soy broth, brain heart infusion, and brucella broth) and overnight incubation at 35°c-37°c. all plated and broth growth was examined at 24 hours and identified according to standard microbiological methods. pleural fluid supernatant was assayed for protein and glucose and tested using the binaxnow streptococcus pneumoniae antigen card. pleural fluid and lung aspirate specimens were cultured for the presence of mycobacteria in liquid culture. of 20 available pleural fluid and lung aspirate isolates, 12 (60%) were shipped to the reference laboratory for confirmation and all had their original organism identification confirmed. pneumococcal capsular serotyping was performed by the following methods: quellung reaction (zambia, south africa), pcr deduction of pneumococcal serotypes [19] followed by quellung reaction if there were mixed or ambiguous results by pcr (thailand, mali, the gambia, bangladesh), or latex agglutination at pool level with quellung reaction for final typing and pcr confirmation of a subset of isolates as a quality control procedure (kenya). mixed or ambiguous results that could not be resolved at the study sites were serotyped by quellung reaction at a reference laboratory (national institute for communicable diseases, johannesburg, south africa or the institute of environmental science and research [esr], porirua, new zealand). serotyping for all pneumococcal isolates isolated from sterile sites as well as a sample of 50-75 pneumococcal isolates from the np swab culture was verified by quellung at the esr laboratory. haemophilus influenzae were identified at all sites using standard microbiological methods; serotype b was identified by slide agglutination. additional (non type b) serotyping was performed in south africa and the gambia by slide agglutination. for all other sites, serotyping beyond type b was done by pcr at the reference laboratory (canterbury health laboratories, christchurch, new zealand) [20] . a bioassay was performed on enrollment serum samples, from all cases and controls, to detect antibiotic activity. a 6-mm filter paper disc was inoculated with 20 µl of serum and placed on a mueller-hinton plate seeded with a 0.5 mcfarland suspension of a fully sensitive staphylococcus aureus strain (atcc 25923). any zone of inhibited bacterial growth around the disc after 18-24 hours' incubation was recorded as evidence of serum antibiotic activity. serum from all cases was assayed for c-reactive protein (crp). samples from south africa were assayed in country using crp gen3 immunoturbidometric assay (roche diagnostics, milan, italy). all other samples were assayed at the reference laboratory in christchurch, new zealand, using crp vario immunoturbidometric assay (roche diagnostics). a subset of 682 control samples was assayed for crp as part of an analysis to evaluate its diagnostic utility [21] . a complete blood count was performed on all cases. hemoglobin testing for controls was also carried out in the gambia, mali, and south africa. thalassemia testing for cases and controls was done in thailand. culture of induced sputum and gastric aspirate specimens for mycobacteria was performed using liquid media in established tuberculosis testing laboratories at all sites. antimicrobial susceptibility testing was performed on all mycobacterium tuberculosis isolates. isolates of nontuberculous mycobacteria were identified at the reference laboratory by 16s ribosomal rna and rpob sequencing [22] [23] [24] . malaria testing was performed by rapid antigen test or microscopy for all cases at sites with endemic malaria (kenya, the gambia, mali, zambia) and in south africa when clinically indicated. human immunodeficiency virus (hiv) testing was done on all cases at all sites apart from bangladesh, and for controls at all africa sites with the exception of the gambia (which has an hiv infection prevalence of <2%). testing was done by serum antibody assay, followed by pcr confirmatory antigen testing for cases and controls <18 months of age. in zambia and south africa, cd4 assessments were performed or collected from referral facilities for all hiv-infected cases and controls. all respiratory specimens were tested for p. jirovecii nucleic acid by fast-track pcr. additionally, induced sputum, endotracheal aspirate, pleural fluid, and lung aspirate specimens were tested for p. jirovecii by immunofluorescence (south africa) and toluidine blue staining (zambia). laboratory sops were developed in collaboration with site investigators for all core laboratory procedures. all sites underwent a period of training and pilot testing prior to study initiation. following demonstration of successful performance, a site activation letter allowed formal study enrollment to commence [8] . the perch laboratory director, in conjunction with other team members, visited and evaluated each study laboratory prior to study piloting and provided advice on areas for additional improvement of facilities or training of staff. major equipment, including the nucleic extraction platform and pcr thermocyclers, was procured centrally and installed at each site laboratory. in addition, maintenance contracts were provided for the period of the study. training on the nucleic acid extraction system was provided by biomérieux at installation. fast-track pcr training was provided in-person by fast-track diagnostics over a period of 3 days at each site. trained staff received a certificate of completion and the site laboratory was required to pass an external quality assurance assessment before beginning molecular diagnostic testing on study samples. sites were trained on induced sputum slide reading at an initial training and again at a midstudy refresher training. a subset of approximately 10% of slides from each site were stored following reading and later sent to the reference laboratory where they were audited. sites that were unable to send slides had a random sample checked by the laboratory director during study oversight visits. key sops were reviewed with laboratory scientists from all sites at an investigator meeting prior to study initiation, training on the data capture system and freezer inventory software was provided remotely via a webinar prior to the start of the study. a working group including the laboratory director and representatives from all site laboratories was convened throughout the study to harmonize practices and troubleshoot problems at periodic investigator meetings and through regular teleconferences. a midstudy in-person refresher training on core laboratory procedures was conducted for all sites in august 2012. the laboratory director visited each study site at least twice over the course of the study to provide on-site monitoring. laboratory quality indicators were monitored using the real-time data entry system and were used to identify areas for improvement over the duration the study [8] . electronic laboratory data reports were generated from the database and reviewed at regular intervals by the laboratory director. in addition, digital pcr results files were rechecked at the reference laboratory to confirm accurate interpretation of pcr quantification curves. discordant interpretations of results were discussed with the laboratories and corrected in the database. an external quality assessment (eqa) program was set up by fast-track diagnostics to monitor performance of the fasttrack respiratory pcr and whole blood lyta pcr assays at each site. for the fast-track respiratory eqa, laboratories were supplied with a series of 12 samples containing mixtures of plasmids at various concentrations at 3-to 4-month intervals. each laboratory was required to test the samples using its routine ftd resp-33 assay and standard procedures. panels for the whole blood lyta pcr eqa were dispatched from ftd at the same frequency and included blinded plasmid samples containing the lyta target in a range of concentrations. for each round of eqa, an individual performance report was provided along with details of overall performance for all sites. reports and practical feedback allowed participants to identify and resolve potential problems whilst monitoring the effectiveness of their laboratory quality assurance processes. site-to-site variation was also assessed using these reports. all sites were enrolled in an eqa program for the microbiological assessment of respiratory specimens, organized by the royal college of pathologists of australasia quality assurance programme. eqa panels were dispatched on a quarterly basis and consisted of simulated clinical specimens for the isolation of pathogens, bacterial identification and antimicrobial susceptibility testing. most module shipments consisted of 2 specimens containing either a pure culture or a mixture simulating a clinical specimen with normal body flora. following each dispatch, results were reviewed by the laboratory director and discussed with the site laboratories. we faced challenges in applying such a high level of standardization to a complex study across diverse research sites. sites varied considerably in their prior level of experience with the perch laboratory methods and therefore required varying levels of assistance and oversight. assuring that each laboratory had standard equipment in place and was comprehensively trained meant that the initiation of study enrollment and full specimen testing was delayed by weeks to months in some instances. maintaining a high level of involvement and in-person oversight required regular travel by the laboratory director in addition to frequent communication by phone and email. additionally, local approvals to ship specimens and isolates for confirmatory testing resulted in long delays in the reference laboratory receiving samples from some sites. despite these challenges, we were able to achieve the highest methodological standards across a variety of circumstances, and demonstrated the ability to set up very complex molecular diagnostics in challenging environments. achieving standards was a very positive motivator among laboratory staff, especially in laboratories that had not used international standards before. the value of regular feedback to the staff was evident in our study and we observed laboratory capacity and technical skills improve rapidly over a short period of time, with some of the laboratories without prior similar experience becoming the highest performers. applying a high level of standardization required considerable effort in the study planning stage and throughout the enrollment and testing period, but in the end this effort was outweighed by our confidence that results were accurate and comparable across sites. perch was one of the largest pneumonia etiology studies ever undertaken, with a complex testing algorithm applied to >9500 individuals distributed over 9 enrollment centers in 7 different countries. considerable efforts were made to perform as much of the laboratory testing at the study sites as possible, and to ensure cross-site standardization of testing methods. as well as providing confidence in the perch analyses, our experiences provide evidence that multisite studies involving extensive laboratory assessments and including complex molecular diagnostics can be undertaken at research sites in a variety of settings and circumstances, including those with limited prior experience. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. author contributions. a. j. d. and d. r. m. drafted the manuscript. d. r. m. supervised all laboratory activities for the study. all authors were involved in the development of standard operating procedures, study conduct, data collection, and/or data management. all authors reviewed and approved the manuscript. a. j. d. and d. r. m. had final responsibility for the decision to submit for publication. pneumonia etiology research for child health. introduction standardization of clinical assessment and sample collection across all perch study sites bhat n; pneumonia methods working group; perch core team. laboratory methods for determining pneumonia etiology in children use and evaluation of molecular diagnostics for pneumonia etiology studies disk diffusion bioassays for the detection of antibiotic activity in body fluids: applications for the pneumonia etiology research for child health project evaluation of fast-track diagnostics and taqman array card real-time pcr assays for the detection of respiratory pathogens bioethical considerations in developing a biorepository for the pneumonia etiology research for child health project data management and data quality in perch, a large international case-control study of severe childhood pneumonia aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested case-control study of the drakenstein child health study evaluation and improvement of real-time pcr assays targeting lyta, ply, and psaa genes for detection of pneumococcal dna pneumonia etiology research for child health (perch) study group. pertussis-associated pneumonia in infants and children from low-and middle-income countries participating in the perch study detection of haemophilus influenzae type b by real-time pcr direct detection and differentiation of legionella spp. and legionella pneumophila in clinical specimens by dual-color real-time pcr and melting curve analysis development of quantitative gene-specific real-time rt-pcr assays for the detection of measles virus in clinical specimens performance standards for antimicrobial susceptibility testing; twenty-first informational supplement revisiting pneumococcal carriage by use of broth enrichment and pcr techniques for enhanced detection of carriage and serotypes evaluation of a medium (stgg) for transport and optimal recovery of streptococcus pneumoniae from nasopharyngeal secretions collected during field studies microscopic analysis and quality assessment of induced sputum from children with pneumonia in the perch study molecular detection methods and serotyping performed directly on clinical samples improve diagnostic sensitivity and reveal increased incidence of invasive disease by streptococcus pneumoniae in italian children real-time pcr for determining capsular serotypes of haemophilus influenzae association of c-reactive protein with bacterial and respiratory syncytial virus-associated pneumonia among children aged <5 years in the perch study detection and identification of mycobacteria by amplification of rrna genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory rpob-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria acknowledgments. we offer sincere thanks to the patients and families who participated in this study. we acknowledge the significant contributions of the perch study group and all perch investigators. we offer our gratitude to the members of the pneumonia methods working group and perch expert group for their time and lending expertise to assist the perch study group. see supplementary materials for a full list of names. key: cord-337972-otfloo64 authors: kao, shang jyh; yang, fwu lin; hsu, yung hsiang; chen, hsing i title: mechanism of fulminant pulmonary edema caused by enterovirus 71 date: 2004-06-15 journal: clin infect dis doi: 10.1086/421021 sha: doc_id: 337972 cord_uid: otfloo64 pulmonary edema (pe) may occur with enterovirus 71 (ev71) infection. we monitored arterial pressure (ap) and heart rate (hr) in patients with ev71 infection and analyzed the variability of ap and hr. sympathetic activity, ap, and hr increased with respiratory stress. thereafter, parasympathetic activity increased with decreases in ap and hr. the lungs showed edema with inducible nitric oxide synthase (inos) expression. destruction of the medial, ventral, and caudal medulla may lead to sympathetic overactivation, causing blood to shift to the lungs. the pathogenesis of pe may also involve inos and nitric oxide. pulmonary edema (pe) may occur with enterovirus 71 (ev71) infection. we monitored arterial pressure (ap) and heart rate (hr) in patients with ev71 infection and analyzed the variability of ap and hr. sympathetic activity, ap, and hr increased with respiratory stress. thereafter, parasympathetic activity increased with decreases in ap and hr. the lungs showed edema with inducible nitric oxide synthase (inos) expression. destruction of the medial, ventral, and caudal medulla may lead to sympathetic overactivation, causing blood to shift to the lungs. the pathogenesis of pe may also involve inos and nitric oxide. epidemic or sporadic infection with enterovirus 71 (ev71) usually occurs during the summer months. children infected with ev71 develop rashes on the hand, foot, and mouth. most patients recover without complications. some patients manifest signs such as headache, profuse sweating, lethargy, paraparesis, and weakness of the limbs. several patients infected with ev71 have died of fulminant pulmonary edema (pe) soon after infection [1, 2] . the pathogenesis of pe is still unknown. chang et al. [2] postulated that viral destruction of the cns may be the cause of death. central sympathetic activation resulting in systemic vasoconstriction and the shift of blood volume to the pulmonary region of the circulatory system, as demonstrated in earlier findings from our laboratory, was used to explain centrogenic pe [2, 3] . however, studies by chang et al. [1, 2] did not provide pathological evidence of cns lesions, nor did they assess autonomic activity. in the present study, we report cns lesions in patients associated with pe due to enterovirus infection. spectral analysis of arterial pressure (ap) and heart rate (hr) variability were employed as a measure of autonomic activities [4] [5] [6] [7] . expression of nitric oxide synthase (nos) mrna in the lungs was performed using rt-pcr. the purpose was to elucidate the possible involvement of nitric oxide in the pathogenesis of acute pe associated with enterovirus infection. subjects and methods. during a 2-year period, we obtained data from a total of 48 patients (29 male and 19 female subjects), aged 6-18 years, who were treated during the summer months (may-august 2001 , 2002 in hospitals in taipei and hualien. all subjects had developed rashes on the hand, foot, and mouth. for each of these patients, enterovirus virus infection with hand, foot, and mouth disease was suspected. each subject underwent chest radiography and a routine physical examination at admission. blood cell counts, blood gas levels, and biochemical factors, such as glucose level, creatinine level, urea nitrogen level, and hemoglobin level, were assessed. csf samples were obtained and used to determine protein concentration and wbc count. swab samples from the throat, rectum, and vesicles were obtained. neurological and respiratory signs were observed. during episodes of acute respiratory distress syndrome (ards), ap and hr were continuously monitored with use of a polygraph and a tape recorder, allowing a retrospective analysis of ap and hr variability [4, 7] . consent for autopsy of patients who died of acute pe was obtained from the patients themselves (in 4 cases) or from their relatives. postmortem specimens obtained from the brain and lungs were examined for pathological lesions. rt-pcr was used for detection of inos mrna expression in the lungs. rt-pcr was performed according to the manufacture's instructions (alpha-innotech). consent for autopsy of patients who died of acute pulmonary edema was obtained from the patients themselves or from their relatives. the study was approved by the institutional board for human studies of taipei medical university. results. isolates from swabs samples obtained from the rectum, throat, and vesicles were identified as ev71 by immunofluorescence with monoclonal antibody (chemical international) [ figure 1a ). signs such as profuse sweating, insomnia, increased startle reflex, tachycardia, dyspnea, vomiting, cold skin, and elevated oral temperature developed after admission. after receipt of intensive care and respiratory therapy over a period of 8 days, the symptoms gradually subsided in 27 of the patients (15 male and 12 female patients). twenty-one patients (14 male and 7 female patients) developed severe dyspnea, hyperglycemia, leukocytosis, and decreased blood oxygen partial pressure. ap and hr fluctuation ensued. in these 21 patients, frothy pink secretions (and even fresh blood) was expelled from the endotracheal tube. each of these 21 patients died within 4 h after the development of ards. in each, the protein concentration and wbc count in csf samples were greatly elevated. chest radiography revealed marked lung infiltration ( figure 1b) . autopsy was performed with prior consent for 4 of the 21 patients who died of acute pe. figure 2 shows an immunohistochemical stain of brain lesions using an antienterovirus monoclonal antibody [8, 9] . the brain lesions were characterized by the formation of nodules in axonal and dendritic processes ( figure 2a) . distribution of the viral lesions was pre-dominantly in the medial and caudal medulla (figure 2b). a horizontal section (figure 2c) revealed the destruction of the ventral medulla. these regions are considered to be the central depressor areas [10] . accordingly, the sympathetic drive was increased following the destruction of the depressor areas. this contention was confirmed by spectral analysis of the ap and hr variabilities that occurred during the episode of ards. figures 3 and 4 show changes in ap and hr in a representative patient at the onset of illness (a), 1 h after onset (b), and 30 min before death (c). before the onset of ards, the patient's ap and hr were within normal limits. later, the ap and hr (the reverse of the r-r interval) increased and fluctuated. before death, there was a dramatic decrease in ap and hr. the patient's blood glucose level ‫ע(‬ se) was mmol/l at admission. hypergly-7.6 ‫ע‬ 0.4 cemia (blood glucose level ‫ע‬ se, mmol/l) devel-26.7 ‫ע‬ 2.5 oped. at admission, the patient's wbc count ‫ע‬ se was 15.6 ‫ע‬ 3.2 ϫ 10 9 cells/l. an elevated wbc count (wbc count ‫ע‬ se, 48.6 ‫ע‬ 9.4 ϫ 10 9 cells/l) was noted at the onset of ards. in samples of the csf, wbc count and protein concentration were cells/l and mg/dl, respectively. discussion. our laboratory has performed a series of studies on the mechanisms of acute pe induced by cerebral compression or increased intracranial pressure [3, [11] [12] [13] . the pe associated with ards is usually fatal. in brief, central sympathetic overactivation produces systemic hypertension and vasoconstriction. these changes cause a shift of the blood to the pulmonary circulatory system [3] . we extended the study from animals to human subjects. acute pe can be induced in rare cases of japanese b encephalitis, fat embolism, breast cancer with lymphangitis, and rupture of intracranial aneurysm [14] . the lesions in the cns following ev71 infection that were observed in the current study matched those that we have previously observed in patients who died of japanese b encephalitis [14] . in a recent review article [13] , we described and discussed early and recent studies of ards in animals and humans and recommended further studies on the mechanism, prevention, and treatment of ards in the clinical practice of intensive care. the present study also supports the contention that hyperglycemia, leukocytosis, and elevated protein levels in csf are risk factors for the development of ards due to enterovirus infection [1, 2] . the relationship between hyperglycemia and ards remains an interesting topic for future study. our research demonstrated enhanced inos mrna expression in the hemorrhagic lung. the increase in inos expression does not definitely indicate that nitric oxide formation is detrimental to the lung. our previous studies [15, 16] found that exogenous and endogenous nitric oxide were toxic to the lung in cases of sepsis and of ischemia and reperfusion. therefore, nitric oxide production may be responsible for the pathogenesis of ards due to ev71 infection, and selective inos blockers may be therapeutic agents for ards induced by ev71 or by other viral infections. in the present report, we did not measure the pulmonary ap, which was demonstrated to be high during the development of pe [3, 11, 14, 16] . it is well known that nitric oxide exerts vasodilatory effects in various vascular beds. the production of nitric oxide through inos could reduce pulmonary hypertension. on the other hand, it may increase the oxygen free radicals and recruit more capillaries to increase pulmonary microvascular permeability. in this connection, we found that endogenous and exogenous nitric oxide reduced pulmonary hypertension but increased the capillary filtration coefficient and the extent of injury in an isolated rat's lung that was subjected to ischemia and reperfusion [16] . viral lesions in the brain were found in the cortex, pons, medulla, cerebellum, and spinal cord. the brain stem lesions were predominantly in the ventral, medial, and caudal medulla. these areas have been considered to the central depressor or sympathetic inhibitory mechanisms in the vasomotor center [10] . destruction of these areas causes an increase in the sympathetic drive. in a recent report from our laboratory [14] , we also found that viral lesions caused by japanese b encephalitis were similar to those caused by ev71 infection. however, whether the same brain stem lesions are caused by other neurotropic viruses (such as coronavirus, rabies, and others) remains to be investigated. in conclusion, the present study suggests that death due to ev71 infection likely results from brain stem lesions. the production of nitric oxide through inos may also be involved in the pathological changes in the lung. fulminant neurogenic pulmonary edema with hand, foot and mouth disease clinical feature and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease centrogenic pulmonary edema induced by cerebral compression in rats preeclamptic pregnancy is associated with increased sympathetic and decreased parasympathetic control of hr spectral analysis of systemic arterial pressure and heart rate signals as a prognostic tool for the prediction of patient outcome in intensive care unit brain death assessment using instant spectral analysis of heart rate variability diminished vasomotor component of systemic arterial pressure signals and baroreflex in brain death use of avidin-biotin-peroxidase complex (abc) in immunoperoxidase technique: a comparison between abc and unlabeled antibody (pap) procedures japanese encephalitis: immunocytochemical studies of viral antigen and inflammatory cells in fatal case localization of central cardiovascular control mechanism in lower brain stem of the cat pulmonary edema and hemorrhage resulting from cerebral compression participation of regional sympathetic outflows in the centrogenic pulmonary pathology acute respiratory distress syndrome acute pulmonary oedema: rare causes and possible mechanisms the lung is the major site that produces acute pulmonary oedema in endotoxin shock nitric oxide mediates lung injury induced by ischemia-reperfusion in rats we are grateful to dr. c. t. hu (skanhex technology) for the spectral analysis of ap and hr variabilities and to ms. c. c. chang for the preparation of this manuscript.financial support. national science council (grants nsc 90-2320-b-320-04 and nsc 91-2320-b-320-008), shin-kong wu-ho su memorial foundation, and outstanding scholarship development foundation (1998)(1999)(2000)(2001)(2002)(2003). key: cord-325455-e464idc0 authors: atchison, christina; pristerà, philippa; cooper, emily; papageorgiou, vasiliki; redd, rozlyn; piggin, maria; flower, barnaby; fontana, gianluca; satkunarajah, sutha; ashrafian, hutan; lawrence-jones, anna; naar, lenny; chigwende, jennifer; gibbard, steve; riley, steven; darzi, ara; elliott, paul; ashby, deborah; barclay, wendy; cooke, graham s; ward, helen title: usability and acceptability of home-based self-testing for sars-cov-2 antibodies for population surveillance date: 2020-08-12 journal: clin infect dis doi: 10.1093/cid/ciaa1178 sha: doc_id: 325455 cord_uid: e464idc0 background: this study assesses acceptability and usability of home-based self-testing for sars-cov-2 antibodies using lateral flow immunoassays (lfia). methods: we carried out public involvement and pilot testing in 315 volunteers to improve usability. feedback was obtained through online discussions, questionnaires, observations and interviews of people who tried the test at home. this informed the design of a nationally representative survey of adults in england using two lfias (lfia1 and lfia2) which were sent to 10,600 and 3,800 participants, respectively, who provided further feedback. results: public involvement and pilot testing showed high levels of acceptability, but limitations with the usability of kits. most people reported completing the test; however, they identified difficulties with practical aspects of the kit, particularly the lancet and pipette, a need for clearer instructions and more guidance on interpretation of results. in the national study, 99.3% (8,693/8,754) of lfia1 and 98.4% (2,911/2,957) of lfia2 respondents attempted the test and 97.5% and 97.8% of respondents completed it, respectively. most found the instructions easy to understand, but some reported difficulties using the pipette (lfia1: 17.7%) and applying the blood drop to the cassette (lfia2: 31.3%). most respondents obtained a valid result (lfia1: 91.5%; lfia2: 94.4%). overall there was substantial concordance between participant and clinician interpreted results (kappa: lfia1 0.72; lfia2 0.89). conclusion: impactful public involvement is feasible in a rapid response setting. home self-testing with lfias can be used with a high degree of acceptability and usability by adults, making them a good option for use in seroprevalence surveys. lateral flow immunoassays (lfia) offer a rapid point-of-care (poc) approach to novel coronavirus (sars-cov-2) antibody testing. while lfias may not currently be accurate enough for individual-level clinical decisions (1, 2) , they are valuable as a public health tool. on a population level, by conducting seroprevalence surveys through widespread random sampling of the general public, and by adjusting for the sensitivity and specificity characteristics of the lfia used, it is possible to estimate the levels of past infection with sars-cov-2 in the community (3) . however, testing hundreds of thousands of people would be impractical if it required a blood sample to be drawn followed by processing in a laboratory. one solution is to use self-sampling and selftesting in the home with participants reporting results to the researchers. however, there is limited understanding of public acceptability and usability of these lfias in the home setting, as most are currently designed as poc tests performed by healthcare professionals. self-sampling and self-testing are widely used in healthcare for monitoring, for example in diabetes management (4) , and for diagnostics, for example for hiv (5, 6) . there are many advantages in terms of uptake, cost, patient activation and scale (4, 6) , but also potential disadvantages in relation to validity, usability and practicality which should be explored (6, 7) . usability research on hiv selftesting has generally found good acceptability, the devices easy to use and high validity in interpretation of self-reported test results (7) (8) (9) . however, these hiv test kits were designed for selfsampling and self-testing and went through several iterations before designs were appropriate for home use and therefore the same levels of acceptability and usability for home-based self-testing for sars-cov-2 antibody using lfias cannot be assumed. as part of the real-time assessment of community transmission (react) programme (10), we evaluated the acceptability and usability of lfias for use in large seroprevalence surveys of sars-cov-2 antibody in the community. a c c e p t e d m a n u s c r i p t 5 we evaluated two lfias with different usability characteristics from five lfias being validated in parallel in our laboratory-based study (11). both lfias required a blood sample from a finger-prick and produced a self-read test result after 10 or 15 minutes. lfia1 (guangzhou wondfo biotech co ltd) was a cassette-based system containing a "control" indicator line and a "test" indicator line (for detection of combined igm and igg antibodies). lfia2 (fortress orient gene biotech co ltd) was a cassette-based system containing a "control" indicator line and separate indicator lines for igm and igg ( figure 1 ). in early may 2020 we carried out rapid, iterative public involvement and a pilot usability study including an online forum with four discussion groups (n=37), a study of lfia1 test use with volunteers (n=44) and a broader public sample (n=234), and a nested observation and interview study (n=25). further details on the methods, including how we recruited participants from our existing involvement networks, are available online (supplement, s1). the test kits dispatched in the pilot study included one test cassette, one button-activated 28g lancet and a 2ml plastic pipette, alongside an instruction booklet also containing a weblink to an instructional video. based on findings from the pilot study, for the larger population-based usability study, the lancet and pipette were replaced with two pressure-activated 23g (larger) lancets and a smaller 1ml plastic pipette, respectively. the design and language in the instructional booklet and video were changed and an alcohol wipe was also included in the kit. a c c e p t e d m a n u s c r i p t 6 in late may 2020 we carried out a larger population-based usability study of a representative sample of the adult population (aged 18 years and over) in england. we used addresses from the postal address file to draw a random sample of 30,000 households in england to which study invitation letters were sent. we allowed up to four adults aged 18 and over in the household to register for the study. self-testing lfia kits were then posted to each registered individual. on completion of the test, participants recorded their interpretation of the result as part of an online survey, with the option of uploading a photograph of the test result. reminder letters were sent to participants who had not completed the online survey or uploaded a photograph within 10 days of test kits being dispatched. metrics to evaluate usability and acceptability were based on the hiv self-testing literature (5, 6, 8) and were measured as the percentage of participants responding to specific closed questions in the online survey. the questionnaire used is available as an online supplement (supplement, s2). the main outcome was usability of the lfia kits. this was defined as a participant's ability to complete the antibody test, and how easy or difficult it was to understand the instructions and complete each step in the process. acceptability was measured in terms of people consenting to and using the provided self-test, and the proportion who reported they would be willing to repeat a self-administered finger-prick antibody test in the future. analyses were conducted in stata (version 15.0, statacorp, texas, usa). data obtained from the questionnaires on acceptability and usability were summarised by counts and descriptive statistics, and comparisons were made between lfia1 and lfia2 using pearson's chisquared test. multivariate regression was used to identify sociodemographic factors independently associated with the proportion of participants conducting the test that achieved a valid result. variables that appeared to be associated (p<0.05) in the unadjusted analyses were considered in the adjusted analyses. adjusted odds ratios (aor) and 95% confidence intervals (ci) were estimated. associations with a p-value <0.05 in the adjusted analyses were considered statistically significant. agreement between participant-interpreted and clinician-interpreted result for test outcomes (negative, igg positive, invalid, unable to read) was assessed using the fleiss kappa statistic. a c c e p t e d m a n u s c r i p t 8 (11). therefore, we used the same operational definition of positivity for lfia2. participants were informed in the instructions to consider igm results as negative. the study obtained research ethics approval from the south central-berkshire b research ethics committee (iras id: 283787). overall, 315 members of the public contributed feedback during the involvement and pilot. this led to changes in the design and language used in the instructional video and booklet, the type and number of lancets and the size of pipette in the lfia kits (further details available online, supplement s1). for the national study, 25,000 household invitation letters were sent, and 17,411 individuals registered for the study from 8,508 households. due to the maximum number of kits we had available for the study, 14,400 participants were selected at random from those who registered. thus, 10,600 lfia1 kits were distributed, with 8,754 individual user surveys completed (82.6% response rate), and 3,800 lfia2 kits were distributed, with 2,957 individual user surveys completed (77.8% response rate). most commonly, two adults participated per household (table 1 ). baseline characteristics of study participants are shown in table 1 . the median age of participants across lfia1 and lfia2 was 51.0 years (range 18 to 95). there were some differences between lfia1 and lfia2 participants by ethnicity, region and household size. acceptability of self-testing was high ( a c c e p t e d m a n u s c r i p t 9 as in the pilot, most respondents were willing to perform another finger-prick antibody test in the future (lfia1: 98.3%; lfia2: 97.0%). only a minority preferred to do the antibody test in a clinical care or community setting than at home. as with the pilot study, respondents with children showed a high willingness to perform the antibody test on them, and this proportion increased with the age of the children (table 2) . in the pilot study most people (86.5%, 225/260) who attempted the test managed to complete it. however, significant usability issues were identified, including challenges with the lancet to obtain a blood drop and the pipette to transfer the blood to the sample well. the problems with the lancet led to some participants using alternative objects to draw blood, including pins and sewing needles, while others opened the lancet casing to access the blade. some people reported minor problems putting buffer into the buffer well. this led to the inclusion of two lancets and changes to the instructions for the national study. in the national study, almost all participants who attempted the antibody test reported completing it (97.5% for lfia1 and 97.8% for lfia2) ( table 2) . reasons for not completing the test are shown in the table. of lfia1 participants who reported damaging the test, the majority reported either accidentally removing the entire lid off the buffer bottle and spilling the solution all over the test cassette or putting the blood and buffer in the wrong well. for lfia2, few participants damaged the test and they all reported putting the blood and buffer in the wrong well. about one in four participants asked someone to help them to administer the test. most found the instructions easy to understand (figures 3), but as in the pilot, participants reported some difficulties in performing the test. for lfia1, difficulties with using the pipette were reported by 17.7% (1,512/8,521) of participants. in addition, 10.6% (908/8,556) had difficulties applying the blood to a c c e p t e d m a n u s c r i p t 10 the sample well ( figure 3 ). therefore, for lfia2 the instructions were changed to omit the use of the pipette and instead directly transfer blood from the finger-prick site to the sample well. however, participants still found creating a blood drop from the finger-prick site (23.2%; 664/2,862) and then applying the blood to the well (31.3%; 894/2,858) difficult. lfia2 was deployed after lfia1 because there was a delay in arrival of lfia2 from the supplier. this differential timing in dispatch of the kits had the unexpected benefit of allowing us to make iterative changes to the instructions. overall, 7.4% of lfia1 and 4.8% of lfia2 participants reported an invalid result (table 3 ). there was some variation in the proportion of participant-reported invalid results using lfia1 by age and gender. the higher the number of participants registered for the study in the same household, the lower the odds of the participant reporting an invalid result. no sociodemographic factors were associated with a participant-reported invalid result using lfia2 (table 4 ). after adjusting for sociodemographic differences between lfia1 and lfia2 participants, there was no difference between lfia1 and lfia2 in terms of being able to read the result (1.1% vs. 0.81%; aor 0.76 (95% ci: 0.48-1.2); p 0.24). but a lower percentage of invalid test results were reported by lfia2 participants (7.9% vs. 4.8%; aor 0.64 (95% ci: 0.53-0.77); p <0.001). table 5 shows concordance between participant and clinician interpreted results in the national study. for lfia1, there was substantial agreement overall (kappa 0.72 (95% ci: 0.71-0.73); p<0.001), however there were important differences: while there was 100.0% agreement for results reported as negative, and 98.5% agreement for invalid results, a clinician confirmed only 62.8% of participantreported positives. visible reasons (from the photograph) were insufficient blood volume to cover the bottom of the sample well, or insufficient movement of the blood and buffer solution across the result window. in addition, the clinician was able to interpret the results of all but four out of 66 a c c e p t e d m a n u s c r i p t 11 (6.1%) tests from the photographs of results participants reported as "unable to read". the four results unreadable by the clinician were because blood had leaked out of the sample well and obscured the result window. of participant-reported unable to read results, 78.8% were clinicianinterpreted as invalid (for all these tests, the control and "test" lines were both absent). table 5 ). the clinician could not interpret the results from 10 photographs reported as readable results by participants, reasons included blurred photographs and shadowing obscuring the indicator lines. overall, we found that self-testing with the two lfia kit designs used in this study was highly acceptable among adults living in england. high acceptability of in home self-testing is in keeping with self-sampling and self-testing studies in diabetes management (4), and hiv diagnostics (5, 6) . the majority of participants who attempted the test successfully completed it despite some continued difficulties with using the pipette (lfia1), creating a drop of blood from the finger-prick site (lfia2) and applying the blood to the sample well (lfia1 and lfia2). based on these findings, we proportion of invalid tests for lfia2 could reflect the better designed buffer bottle (which was a significant issue for lfia1), better performance characteristics of lfia2 over lfia1 (e.g. easier movement of the blood and buffer solution across the result window), or improved ability to get sufficient blood in the sample well using the direct blood transfer technique over using a pipette. participants' ability to obtain a valid test result using lfia1 varied marginally by age and gender and increased with the number of participants registered for the study in the same household. the latter observation is not surprising as observing another household member performing the test is likely to improve the performance of others in the same household. no sociodemographic differences in obtaining a valid result were found for lfia2. of note, about one in four participants reported that they had help administering the test, irrespective of lfia used. this could put individuals living alone at a disadvantage in terms of usability. however, comparing those that had help to those that did not, we found no difference in ability to complete the test (97.7% vs. 98.2%; p 0.08) or reported invalid results (6.1% vs. 7.0%; p 0.06). overall, there was good agreement between self-reported results and those reported by a clinician. therefore, our findings broadly support self-reporting of home-based test results using lfias. but the public and individual health impact of misinterpreting a test result that is negative but read as positive is a concern as an individual could falsely conclude that they have antibodies for sars-cov-2 and may change their behaviour as a result. to mitigate against this, and given the scientific our study is original because focusing on the acceptability and usability of lfias for self-testing for sars-cov-2 antibody in a home-based setting has not been done at such scale in the general population. it provides an attractive solution for conducting large seroprevalence surveys. the study has, however, some limitations. study participants may not be representative of the general adult population of england. however, we had data on the england population profile (2011 census data (13)), as well as the study registration profile and survey completion profile of the study participants which gave us an indication of response bias. our sample was broadly similar to the england population profile. in addition, the usability study was conducted in parallel with our laboratorybased study of performance characteristics of lfias. as such, we did not know the accuracy of the lfias chosen for the usability study at the time, or whether either would perform well enough in the laboratory to be considered for the large national seroprevalence study planned as part of the react programme (10). however, given that the majority of commercially available lfias have a similar cassette-based design and test result read out to lfia1 or lfia2 we were confident that our results would be generalisable and applicable to whichever lfia was finally selected. findings from our laboratory-based study, including the performance characteristics of lfia1 and lfia2 are forthcoming (11). overall, our study has demonstrated that home-based self-testing lfias for use in large communitybased seroprevalence surveys of sars-cov-2 antibody are both acceptable and feasible. although this study identified a few usability issues, these have now been addressed. lfia2, fortress orient gene, has been selected for a large national seroprevalence study as part of the react programme. this decision was based on criteria including the usability and acceptability determined in this study, a c c e p t e d m a n u s c r i p t 21 years where asked whether they would carry out the test on children of that age living in their households. denominator is th e number of participants who reported having children of that age living in their household. m a n u s c r i p t 23 antibody testing for covid-19: a report from the national covid scientific advisory panel evaluation of nine commercial sars-cov-2 immunoassays prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study self-monitoring of blood glucose in type 2 diabetes: recent studies acceptability, feasibility, and individual preferences of blood-based hiv self-testing in a population-based sample of adolescents in kisangani, democratic republic of the congo reliability of hiv rapid diagnostic tests for self-testing compared with testing by health-care workers: a systematic review and meta-analysis interferences and limitations in blood glucose self-testing: an overview of the current knowledge usability assessment of seven hiv self-test devices conducted with lay-users in we thank key collaborators on this work --ipsos mori: stephen finlay, john kennedy, duncan key: cord-335691-lsuwsm43 authors: jackson, michael l.; neuzil, kathleen m.; thompson, william w.; shay, david k.; yu, onchee; hanson, christi a.; jackson, lisa a. title: the burden of community-acquired pneumonia in seniors: results of a population-based study date: 2004-12-01 journal: clin infect dis doi: 10.1086/425615 sha: doc_id: 335691 cord_uid: lsuwsm43 background. pneumonia is recognized as a leading cause of morbidity in seniors. however, the overall burden of this disease—and, in particular, the contribution of ambulatory cases to that burden—is not well defined. to estimate rates of community-acquired pneumonia and to identify risk factors for this disease, we conducted a large, population-based cohort study of persons aged ⩾65 years that included both hospitalizations and outpatient visits for pneumonia. methods. the study population consisted of 46,237 seniors enrolled at group health cooperative who were observed over a 3-year period. pneumonia episodes presumptively identified by international classification of diseases, ninth revision, clinical modification codes assigned to medical encounters were validated by medical record review. characteristics of participants were defined by administrative data sources. results. the overall rate of community-acquired pneumonia ranged from 18.2 cases per 1000 person-years among persons aged 65–69 years to 52.3 cases per 1000 person-years among those aged ⩾85 years. in this population, 59.3% of all pneumonia episodes were treated on an outpatient basis. in multivariate analysis, risk factors for community-acquired pneumonia included age, male sex, chronic obstructive pulmonary disease, asthma, diabetes mellitus, congestive heart failure, and smoking. conclusions. on the basis of these data, we estimate that roughly 915,900 cases of community-acquired pneumonia occur annually among seniors in the united states and that ∼1 of every 20 persons aged ⩾85 years will have a new episode of community-acquired pneumonia each year. the risk of pneumonia increases markedly with age, and pneumonia is a leading cause of death among seniors [1, 2] . in the united states, an estimated 600,000 hospitalizations for community-acquired pneumonia (cap) [3] and 59,000 deaths attributed to pneumonia and influenza occur among seniors every year [4] . despite the importance of cap in this population, information on the epidemiology of cap among seniors in the united states is limited. in particular, because many prior studies of pneumonia have included only hospitalized patients, the increasingly important contribution of outpatient disease is not well defined. to better assess the burden of cap in seniors and to iden-bases that record immunizations, medication prescriptions, radiographic test reports, and international classification of diseases, ninth revision, clinical modification (icd-9-cm)-coded diagnoses associated with outpatient visits and hospitalizations. each member also has a paper medical record that included copies of hospital admission and discharge summaries and outpatient and emergency department visit notes. hospitalizations for pneumonia among the study population had been identified and validated as part of a prior study of pneumococcal vaccine effectiveness [5] . in that study, hospitalizations assigned a discharge diagnosis of pneumonia (icd-9-cm codes 480-487.0) or streptococcal or pneumococcal bacteremia (icd-9-cm codes 038.0, 038.2, 041.0, 041.2, and 320.1) were selected for chart review. cases of nosocomial pneumonia, defined as cases in which pneumonia symptoms developed after hospitalization or in which the patient had been hospitalized in the previous 7 days, were excluded. a confirmed episode of cap involving hospitalization was defined by an indication that, at the conclusion of their clinical evaluation, the treating physicians considered pneumonia to be the etiology of the presenting illness. episodes of pneumonia treated on an outpatient basis were presumptively defined by an outpatient or emergency department visit with a pneumonia icd-9-cm code that was associated with both prescription of antibiotics and obtainment of a chest radiograph within 14 days after the visit. an outpatient episode of cap was defined as confirmed if the chart review indicated that pneumonia was the most likely diagnosis attributed to the illness by the treating physician and if the patient had not been hospitalized in the prior 7 days. definition of baseline covariates. to assess potential risk factors for pneumonia among the study population, a series of baseline covariates was defined from ghc administrative data sources and the regional cancer registry. these variables include ischemic cardiac disease, congestive heart failure, chronic obstructive pulmonary disease (copd), asthma without copd, renal disease, stroke, dementia, lung cancer, serious nonlung cancer, other cancer, diabetes mellitus, receipt of prednisone, receipt of other immunosuppressive medications, pneumonia hospitalization in the year prior to cohort entry, use of home oxygen, and receipt of home health care. the full definitions of these variables are provided in table a1 in appendix a. smoking status was defined on the basis of data routinely collected at ghc during outpatient visits [5] . information on smoking status was missing for 12% of study subjects. these subjects differed in several characteristics from persons with known smoking status. alternate analyses were conducted that excluded persons who were missing smoking information, but because the results did not vary substantially from the results of analyses of the entire study population, the results presented include persons with and persons without smoking data. rates of pneumonia. crude event rates were calculated by dividing the number of cases by the cumulative person-time in each age-and sex-specific stratum. crude event rates were based on all events occurring during the study period and could include multiple events per person. to examine seasonal variation in pneumonia rates, biweekly crude rates were calculated by dividing the number of cases occurring in 2-week intervals by the cumulative person-time in each interval. we plotted these rates over time and compared the pattern of variation during and outside of influenza seasons with the pattern of proportionate mortality from pneumonia and influenza in the united states, as defined by national vital statistics data [6] . influenza seasons were defined on the basis of local and national surveillance data [7] [8] [9] [10] . statistical analysis. differences in rates were assessed using the x 2 test. multivariate cox proportional hazards models [11] were used to evaluate the association of baseline covariates with the time to first of each outcome event during the study period. additional models were fit to test for the presence of an interaction between age or sex and other covariates with the risk of pneumonia. the attributable risk, attributable risk percentage, population-attributable risk, and population-attributable risk percentage of cap cases associated with smoking were all calculated by comparing the crude rates of cap in current smokers with the rates of cap in former smokers and those who have never smoked using standard formulas [12] . the cohort consisted of 46,237 persons who contributed 122,605 person-years of observation during the study period. overall, 42% of the subjects were male, 53% were aged 65-74 years at baseline, and 38% were aged 75-84 years at baseline (table 1). hospitalizations for pneumonia. a total of 2486 hospitalizations associated with a pneumonia icd-9-cm code among 2161 persons were identified. information on 2371 (95%) of these events was available for review. of these events, 45 were readmissions for pneumonia and 119 were episodes of nosocomially acquired pneumonia. of the 2207 remaining hospitalizations, a clinical diagnosis of cap was confirmed for 1411 (64%); 1264 cohort members accounted for these cases. an additional 2 confirmed hospitalizations for pneumonia were identified by chart review of hospitalizations associated with a bacteremia icd-9-cm code. thus, 1413 confirmed capassociated hospitalizations among 1266 persons were identified during the study period. a total of 3153 episodes of presumptive outpatient pneumonia among 2736 persons were identified on the basis of administrative data, and records of 3124 (99%) of these events were available for review. of these events, 86 episodes were excluded because a record of care for a respiratory illness was not verified, and 129 were excluded because the respiratory illness was associated with a hospitalization. of the remaining 2909 events, a diagnosis of cap was confirmed for 2063 (71%), which occurred among 1881 cohort members. rates of cap and the percentage of cases resulting in hospitalization increased with age (table 2). the rate of pneumonia was consistently higher among men than among women, across all age groups and for both hospitalized patients and outpatients with pneumonia, although the difference was not always statistically significant. overall, 59.3% of cap episodes were treated on an outpatient basis. among persons aged у85 years, the rate of cap was 52.3 events per 1000 person-years, which is roughly 1 episode of pneumonia for every 20 persons per year. among persons hospitalized for cap, 12.5% died within 30 days after hospital admission. among outpatients with pneumonia, 0.4% died within 30 days after the first diagnosis. overall, 3.6% of all the deaths in the cohort during the study period occurred within 30 days after a cap diagnosis. we found that the peak rates of all cap (both hospitalizations and outpatient visits for pneumonia) coincided with periods of influenza viral circulation (figure 1). in addition, the seasonal pattern of cap in our study population closely mirrored the temporal pattern of the percentage of deaths attributed to pneumonia and influenza nationally. similar seasonal patterns were seen in analyses restricted to either outpatients or hospitalized patients, and the proportion of pneumonia episodes requiring hospitalization did not vary between influenza and noninfluenza periods, either for the cohort overall or by age group (data not shown). the attributable risk for smoking among the study cohort was 12.3 cap events per 1000 person-years, and the attributable risk percentage for smoking was 30.8%. that is, assuming there is a causal relationship between smoking and cap, we estimate that ∼30% of episodes of pneumonia among smokers were due to smoking. the population-attributable risk for smoking (among the population of persons with smoking data) was 1.3 cap events per 1000 person-years, and the population-attributable risk percentage for smoking was 4.4%. that is, among the population of persons with smoking data, ∼4% of pneumonia events were due to smoking. there was no significant difference in the attributable risks of smoking across age groups or between men and women. in multivariate analysis, age, male sex, current smoking, diabetes mellitus, congestive heart failure, lung cancer, serious nonlung cancer, copd, asthma without copd, dementia, stroke, receipt of prednisone, use of home oxygen services, greater number of outpatient visits, and hospitalization for pneumonia in the year prior to the study start date were independently associated with risk of all cap (table 3) . in general, indicators of chronic illness at baseline were more strongly [7] [8] [9] [10] . for comparison, the biweekly proportion of all deaths in the united states attributed to pneumonia and influenza (p&i), as reported by national vital statistics data, is also presented [6] . associated with risk of hospitalization for pneumonia than of outpatient visits for pneumonia. the variable most strongly associated with all cap was copd. there was no significant interaction between age or sex and other covariates on risk of pneumonia. to our knowledge, this is the first contemporary study to assess rates of both hospitalizations and outpatient visits for pneumonia among community-dwelling seniors in the united states, and it provides, to our knowledge, the only populationbased assessment of risk factors for cap among seniors in the united states. our study builds on previous population-based studies of hospitalizations and outpatient visits for cap conducted among communities in finland [13, 14] and among the ghc population in the 1970s and early 1980s [15] , each of which had substantially fewer seniors in their study populations. extrapolating our age-specific rates to the population of seniors in the united states who were not in nursing homes, as reported in 2000 census data [16] , we estimate a disease burden of 551,600 cases of cap treated on an outpatient basis and 364,300 cases of cap resulting in hospitalization, for a total of 915,900 cases of cap. these rates also indicate that ∼1 of every 20 persons aged у85 years will have a new episode of cap each year. our rates of hospitalizations for cap are consistent with those reported by marston et al. [1] in a centers for disease control and prevention-sponsored prospective study of radi-ographically confirmed hospitalization for cap patients in 2 counties in ohio in 1991. after adjusting for differences in the age distribution of persons aged у65 years in the 2 study populations, our age-adjusted hospitalization rate of 10.4 cases per 1000 person-years was nearly identical to the rate of 10.1 cases per 1000 person-years among community-dwelling seniors reported by marston et al. [1] . the ohio study did not, however, include an assessment of outpatient visits for cap, which is particularly important, given the trend towards outpatient treatment that has occurred over the past 2 decades. we found that, even among persons aged у80 years, nearly one-half (49.3%) of cap events were treated on an outpatient basis. inclusion of outpatient episodes allows a more comprehensive estimate of the burden of disease and a more accurate assessment of risk factors for cap. persons with chronic conditions who develop cap are more likely to be hospitalized for treatment than are persons without chronic conditions. this means that assessments restricted to hospitalizations for cap will likely overestimate the association of those conditions with the risk of cap. the availability of administrative data on the ghc population allowed us to define the presence of underlying conditions consistently across the study population. because of this, we were able to assess the independent association of those characteristics with risk of pneumonia in multivariate analyses. as previously reported among adults, we found that copd, immunosuppression, smoking, and congestive heart failure were all independently associated with disease risk among seniors [17] [18] [19] [20] [21] . in addition, we also found that diabetes, lung cancer, serious nonlung cancer, and previous hospitalizations for pneumonia were also risk factors for cap among elderly persons. in this cohort, rates of cap were higher in men than in women, and male sex was a risk factor for cap even after adjusting for age, smoking, and the presence of chronic medical conditions. a survey of hospitalizations associated with a pneumonia icd-9-cm code reported in medicare claims [3] found a significantly higher risk among men than among women, as did a follow-up assessment of the risk of a hospitalization associated with a pneumonia diagnosis among participants of the first national health and nutrition examination survey (nha-nes i) [17] . neither of these studies assessed whether male sex was associated with pneumonia after adjusting for other risk factors, so it was not clear in these studies whether male sex was simply a marker for a higher prevalence of other risk factors. a population-based study involving 4175 elderly persons in finland found that crude cap rates did not differ between men and women and that male sex was not significantly associated with cap after accounting for age and certain chronic medical conditions and risk factors, including asthma, receipt of immunosuppressive therapy, and heart disease [21] . because our study population was much larger than that of the finnish study, we have more power to detect an independent risk of cap associated with male sex if such a risk truly exists. however, it is possible that the difference in risk we observed could be the result of confounding by factors unmeasured in our population. we found that current smokers were at an increased risk of hospitalization for cap, which is consistent with previous reports. previous case-control studies have shown that smoking is associated with an increased risk of pneumonia [19, 22] . one study has reported that, among adults who quit smoking, the excess risk of cap appears to have decreased 5 years after quitting [22] . we estimate that, among elderly smokers, nearly one-third of pneumonia episodes can be attributed to smoking, suggesting that smoking cessation could potentially reduce the risk of cap among smokers by an important degree. among our study population, the seasonal pattern of pneumonia closely followed the pattern of pneumonia and influenza mortality nationally. correlations between rates of hospitalization for acute respiratory disease and patterns of influenza activity have been reported in adult populations in other geographic areas [23] [24] [25] . the 1999-2000 influenza season is recognized as having been more severe than the several preceding seasons [10] , and timing of the peak rate of cap for the elderly ghc population in this study period closely matched the peak proportional pneumonia and influenza mortality rates in the united states, occurring in late december 1999 and early january 2000. this correlation suggests that data from health management organizations (hmos) on pneumonia-related hospitalizations and outpatient visits could be an additional method for identifying periods of influenza virus circulation or quantifying the severity of influenza seasons. rates of cap in this population were high, especially among some subgroups of subjects, such as those aged у85 years, and there was close correlation with influenza virus circulation. this is despite the fact that vaccination rates for both influenza and pneumococcal polysaccharide vaccines were high in this population. annual influenza vaccination rates among the study population were 170%, and 175% of subjects received pneumococcal vaccine either prior to or during the study period, emphasizing the need for other measures for preventing pneumonia in seniors. the burden of illness could potentially be reduced by the availability of other vaccines, such as pneumococcal conjugate vaccines or vaccines effective against human metapneumovirus or respiratory syncytial virus infection. alternatively, increased emphasis on other strategies to reduce the risk of influenza infection, such as vaccination of caregivers and household contacts, could potentially impact disease risk. one limitation of this study is the possible underascertainment of pneumonia events, because our case finding was primarily restricted to medical encounters assigned a pneumonia icd-9-cm code, and so would not capture pneumonia episodes associated exclusively with other diagnosis codes. for outpatient pneumonia, we would also have missed episodes in which a chest radiograph was not ordered. we cannot estimate the extent to which underascertainment may have influenced our results. however, this problem is likely to be more significant for pneumonia treated on an outpatient basis (in which a health care provider may only see a patient early in the course of the illness and assign a diagnosis before the evaluation has been completed) than in cases in the hospital setting (in which the discharge diagnoses are assigned at the end of the hospital stay and should represent a more complete assessment of the illness episode). a second limitation is that the study population includes enrollees of a single health maintenance organization in a single geographic area, and the findings may not be generalizable to the us population as a whole. as noted above, however, our age-standardized rate of cap-related hospitalizations was essentially identical to that observed in the study of cap in ohio [1] . this similarity, along with the close correspondence between the observed rates over time in our study and the national proportional rates of mortality due to pneumonia and influenza, suggest that our study population is not dissimilar from the elderly population of the united states as a whole. our method for detection of both hospitalizations and outpatient visits for cap among a defined population of hmo enrollees has several advantages for conducting pneumonia surveillance. this type of surveillance can identify high-risk subgroups, can track trends in pneumonia incidence over time, and is suitable for both prospective and retrospective studies. surveillance of this type would be particularly useful for studying the effect of new vaccines or the introduction of new pathogens, such as severe acute respiratory syndrome coronavirus, on rates of pneumonia. incidence of communityacquired pneumonia requiring hospitalization. results of a populationbased active surveillance study in ohio. the community-based pneumonia incidence study group deaths: final data for 1999 hospitalized community-acquired pneumonia in the elderly: age-and sex-related patterns of care and outcome in the united states deaths: preliminary data for effectiveness of pneumococcal polysaccharide vaccine in older adults vital statistics mortality data, multiple cause detail update: influenza activity-united states and worldwide, 2000-01 season, and composition of the 2001-02 influenza vaccine update: influenza activity-united states and worldwide, 1997-98 season, and composition of the 1998-99 influenza vaccine update: influenza activity-united states and worldwide, 1998-99 season, and composition of the 1999-2000 influenza vaccine update: influenza activity-united states and worldwide, 1999-2000 season, and composition of the 2000-01 influenza vaccine time-dependent covariates in the cox proportionalhazards regression model epidemiologic methods: studying the occurrence of illness incidence of communityacquired pneumonia in the population of four municipalities in eastern finland clinical efficacy of pneumococcal vaccine in the elderly: a randomized, single-blind populationbased trial rates of pneumonia during influenza epidemics in seattle census 2000 briefs: the 65 years of age and older population prospective study of pneumonia hospitalizations and mortality of us older people: the role of chronic conditions, health behaviors, and nutritional status risk factors for community-acquired pneumonia in adults: a population-based case-control study risk factors for community-acquired pneumonia diagnosed upon hospital admission. british thoracic society pneumonia study group risk factors for community-acquired pneumonia diagnosed by general practitioners in the community risk factors for pneumonia in the elderly proportion of community-acquired pneumonia cases attributable to tobacco smoking serious morbidity and mortality associated with influenza epidemics acute respiratory disease hospitalizations as a measure of impact of epidemic influenza influenza-associated morbidity and mortality in young and middle-aged women financial support. vaccine safety datalink contract with the america's health insurance plans, funded by the centers for disease control and prevention.potential conflicts of interest. k.m.n. has received grant support from aventis-pasteur. l.a.j. has received grant support from aventis-pasteur and has served as a consultant to chiron vaccines. all other authors: no conflicts. key: cord-335767-omm04fg5 authors: raabe, vanessa n; lighter, jennifer; caplan, arthur l; ratner, adam j title: importance of pediatric inclusion in covid-19 therapeutic trials date: 2020-05-27 journal: clin infect dis doi: 10.1093/cid/ciaa656 sha: doc_id: 335767 cord_uid: omm04fg5 pediatric patients are excluded from most coronavirus disease 2019 (covid-19) therapeutic trials. we outline a rationale for the inclusion of children in covid-19 therapeutic trials, which enabled us to include children of all ages in a therapeutic covid-19 trial at our institution. over 4.8 million people worldwide have been diagnosed with coronavirus disease 2019 (covid-19), a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), including over 1.5 million cases and over 90 000 attributable deaths in the united states [1] . supportive care remains the mainstay of covid-19 therapy, and the infectious diseases society of america guidelines for covid-19 treatment recommend use of antiviral and immunomodulatory medications suspected to have activity against covid-19 only in the context of a clinical trial [2] . unfortunately, children are not being given the opportunity to participate in most us-based covid-19 clinical therapeutic trials. as of 6 may 2020, options to participate in antiviral clinical trials targeting sars-cov-2, aside from convalescent plasma trials, are limited to adolescents ages 12 or older (nct04292730, nct04292899, nct04335552) with the exception of a hydroxychloroquine treatment study at our institution (nct04369742) open to children of all ages. we advocate that pediatric patients should be given equal opportunities to enroll in covid-19 therapeutic clinical trials and outline our rationale for inclusion of children in our hydroxychloroquine treatment trial. children are not simply small adults. they have inherent biological differences that evolve with age and can manifest as alterations in immunity, disease pathophysiology, drug metabolism, therapeutic effects, and drug-associated toxicities compared to adults. infection with sars-cov-2 clearly demonstrates significant differences in clinical presentation and severity related to age [3] . given these differences in covid-19 manifestations, therapeutic data abstracted from adult treatment trials may not be reflective of the risks and benefits of therapeutic agents in children. although most children infected with covid-19 do not develop immediate severe illness, covid-19 infection leading to hospitalization, intensive care admission, and death have been reported among children in the united states [4] . as a basic principle of justice, children significantly affected by covid-19 should be given equal opportunities to receive potentially active therapeutic agents against covid-19 in the safest manner possible: via structured clinical trials. well-designed therapeutic clinical trials incorporate routine, structured safety monitoring that maximizes the likelihood of detecting toxicities associated with therapeutic agents of unproven benefit beyond that offered in an off-label clinical context. close monitoring for adverse events, integrating opportunities to monitor drug levels into the trial design if pediatric pharmacokinetics/pharmacodynamics of the investigational agent have not been established, and routine incorporation of criteria for halting the therapeutic agent in clinical trials make this the safest approach for use of an investigational or off-label therapeutic agent in children, particularly when the degree of direct benefit that the agent provides to the child in uncertain. for newly identified diseases, such as the covid-19 pandemic, that immediately pose a direct threat to a large number of children worldwide, delays in initiation of pediatric clinical trials while awaiting adult data pose a potentially preventable safety issue for children and conflicts with the bioethical principle of beneficence. not only do children and the medical community lose out on opportunities to gain the highest level of clinical evidence for efficacy in a vulnerable population, but due to the lack of availability of pediatric trials, many children hospitalized for covid-19 are receiving off-label use of therapeutic agents with unproven benefit against covid-19. off-label prescribing is common practice among children hospitalized in the united states [5] ; however, this approach presents safety risks by lacking the structured safety monitoring provided by clinical trials, does not further advance knowledge to provide benefit to future children with the same condition, and fails to encourage long-term follow-up to detect potential harms. in addition to offering enhanced safety monitoring, enrollment of children in clinical trials ensures that parents and older children are fully informed of the potential risks and benefits associated with use of the therapeutic agent, a conversation often omitted when medications are prescribed off-label. this preserves the autonomy of the parent(s)/guardian(s) and older children capable of providing assent to make informed decisions on behalf of their child and themselves in a setting in which risks and potential benefits need to be closely weighed because therapeutic efficacy is not yet proven. as covid-19 was only recognized in the last few months, adequately powered data using the gold standard of biomedical evidence for therapeutic efficacy, randomized, double-blind, placebo control trials, is lacking for all leading covid-19 treatment candidates. current evidence suggesting agents have a potential therapeutic benefit against covid-19 mainly relies on case series, in vitro studies, and in vivo animal studies against sars-cov-2 or related coronaviruses. the extent of evidence for potential benefit of each of these agents needs to be taken into account when considering either off-label use or clinical trial use for pediatric patients to ensure the potential direct benefit to the child outweighs the commensurate risks associated with the potential therapeutic agent. only agents with scientific evidence supporting potential direct benefit to the child's condition with low potential toxicities should be considered for pediatric use until additional efficacy data are available in adults. as the therapeutic benefit of these agents in humans is unclear, enrollment of children in randomized, double-blind, placebo control trials remains ethical. if a therapeutic agent is proven to be efficacious for covid-19 therapy in adults, it may no longer be ethical to enroll acutely infected pediatric patients in randomized, double-blind, placebo control trials as it would deprive children in a placebo arm from receiving a known efficacious therapy. therefore, including children in covid-19 therapeutic trials immediately during the pandemic period may provide the only ethical window of opportunity for obtaining the gold-standard level of efficacy data for covid-19 therapeutics in children. moreover, depriving children worldwide the opportunity to have therapeutic recommendations based on the same high-quality level data as adults would be an injustice to this vulnerable population. pediatric covid-19 therapeutics trials will require multicenter collaboration to achieve appropriately powered data, and existing clinical trial networks should be mobilized to facilitate this approach. this approach of using only therapeutic agents with the maximum likelihood of providing direct benefit to pediatric trial participants, enhanced safety monitoring beyond that used in routine clinical care for off-label medication use, and obtaining both written assent from capable children and informed consent from parent(s)/guardian(s) is consistent with the special protections afforded to children involved in research studies by the us department of health and human services under 45 cfr part 46. we advocate that covid-19 therapeutic clinical trials should open enrollment to pediatric patients during these early months of the covid-19 pandemic according to the bioethical principles of autonomy, beneficence, and justice. inclusion of children in these clinical trials is feasible under current regulations, provides direct benefit to pediatric trial participants from a safety perspective compared to off-label prescribing, and provides systematic collection of the highest quality of evidence for covid-19 therapeutics in a vulnerable population where sars-cov-2 infection behaves differently from adults. even if sufficient power cannot be obtained for children, stratified age-based analysis of pediatric cohorts embedded in large-scale clinical trials may at least provide preliminary data on whether a therapeutic response difference exists between children and adults to assist with the design of future therapeutic trials and a first look at whether adult data may be generalizable to the pediatric population. excluding children from covid-19 therapeutic trials based on age alone risks causing undue harm and perpetuates medical inequalities among a vulnerable population who deserve equal opportunities to receive investigational therapeutic agents in a safe, structured, systematic manner. financial support. this work was not supported by external funding. v. r. receives salary support from the national institutes of health vaccine and treatment evaluation units and the national emerging special pathogens training and education center. potential conflicts of interest. a. r. receives personal fees from pfizer not related to this work. all other authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. available at infectious diseases society of america guidelines on the treatment and management of patients with covid-19: infectious diseases society of america severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states cdc covid-19 response team. coronavirus disease 2019 in children: united states off-label drug use in hospitalized children key: cord-318615-uhh3owcx authors: xiang, fei; wang, xiaorong; he, xinliang; peng, zhenghong; yang, bohan; zhang, jianchu; zhou, qiong; ye, hong; ma, yanling; li, hui; wei, xiaoshan; cai, pengcheng; ma, wan-li title: antibody detection and dynamic characteristics in patients with covid-19 date: 2020-04-19 journal: clin infect dis doi: 10.1093/cid/ciaa461 sha: doc_id: 318615 cord_uid: uhh3owcx background: the corona virus disease 2019 (covid-19) caused by the corona virus 2 (sars-cov-2) has been rapidly spreading nationwide and abroad. a serologic test to identify antibody dynamics and response to sars-cov-2 was developed. methods: the antibodies against sars-cov-2 were detected by an enzyme-linked immunosorbent assay (elisa) based on the recombinant nucleocapsid protein of sars-cov-2 in patients with confirmed or suspected covid-19 at 3-40 days after symptom onset. the gold standard for covid-19 diagnosis was nucleic acid testing for sars-cov-2 by rt-pcr. the serodiagnostic power of the specific igm and igg antibodies against sars-cov-2 was investigated in terms of sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and consistency rate. results: the seroconversion of specific igm and igg antibodies were observed as early as the 4(th) day after symptom onset. in the confirmed patients with covid-19, sensitivity, specificity, ppv, npv, and consistency rate of igm were 77.3% (51/66), 100%, 100%, 80.0%, and 88.1%, and those of igg were 83.3.3% (55/66), 95.0%, 94.8%, 83.8%, and 88.9 %. in patients with suspected covid-19, sensitivity, specificity, ppv, npv, and consistency rate of igm were 87.5% (21/24), 100%, 100%, 95.2%, and 96.4%, and those of igg were 70.8% (17/24), 96.6%, 85.0%, 89.1%, and 88.1%. both antibodies performed well in serodiagnosis for covid-19 rely on great specificity. conclusions: the antibodies against sars-cov-2 can be detected in the middle and later stage of the illness. antibody detection may play an important role in the diagnosis of covid-19 as complement approach for viral nucleid acid assays. a novel corona virus (sars-cov-2) disease (covid-19) was first identified and outbroke in wuhan city, hubei province, china. a total of 93, 090 patients with covid-19 had occurred globally by march 5, 2020 [1] . accurate and fast diagnosis of the causative sars-cov-2 is important to isolate the patients with covid-19 timely and stop the epidemics, as well as save people's lives. viral nucleic acid detection using real-time polymerase chain reaction (rt-pcr) assay, which has been developed and used for detection of sars-cov-2 rapidly, remains the standard diagnosis of covid-19 [2] . while a large number of the "suspected" cases with typical clinical covid-19 features and/or identical specific computed tomography (ct) scan were not diagnosed [3, 4] . moreover, rt-pcr assay which was time consuming and laborious needed special equipment resulted to limit its usage especially in remote areas. the human antibody response which is crucial for the clearance of the initial virus infection has been widely used to help diagnosis virus infection. compared to rt-pcr assays, the detection of antibody assays are often faster, less expensive, easy-to-use and accessible to staff without laboratory training. here, we detected dynamics characteristics and magnitude of antibody response in patients with covid-19, and evaluated serodiagnostic value of elisa-based igm, igg tests for covid-19 pneumonia. the sensitivity and specificity of antibody tests for detection of igm and igg were presented and the clinical application of these antibody assays for serodiagnosis of covid-19 was discussed. patients at union hospital, tongji medical college, huazhong university of science and technology were evaluated from january 19, 2020 to march 2, 2020. during this period, igm and igg antibody responses to sars-cov-2 virus infection were analyzed. the gold standard for diagnosis is nucleic acid testing for sars-cov-2 by rt-pcr tests of nasopharyngeal and/or oropharyngeal swabs sample. rt-pcr tests were performed for patients who presented with a history of 1) travel or residential history in wuhan or local endemic areas; or 2) the epidemioloical history of contact with patients who have been confirmed with covid-19 pneumonia or individuals present fever or respiratory symptoms from these areas within 14 days; or 3) a clustering outbreak, combined with clinical manifestation of 1) fever and /or respiratory symptoms, or 2) positive findings similar to covid-19 pneumonia on chest computerized tomography (ct) scan, or 3) laboratory tests showing reduced lymphocytes and white blood cell counts in the early stage. in the case of an initial negative rt-pcr test, repeated testing was performed at intervals of one day or more. the diagnosis of laboratory confirmed covid-19 defined as positive nucleic acid tests for sars-cov-2 by rt-pcr assays. the diagnosis of suspected covid-19 was based on one of the epidemiological history and two of the clinical manifestations but the rt-pcr of sars-cov-2 was negative. in confirmed cases, the patient showing fever and respiratory symptoms with radiological findings of pneumonia was defined as normal case, while the case meeting any of the following criteria was defined as severe case: 1) respiratory distress (≧30 breaths/ min); 2) oxygen saturation≤93% at rest; 3) arterial partial pressure of oxygen (pao2)/fraction of inspired oxygen (fio2) ≤ 300mmhg (l mmhg=0.133kpa); 4) cases with chest imaging that shows obvious lesion progression within 24-48 hours >50% shall be 6 managed as severe cases. in the control group, samples from healthy blood donors or from patients with other disease hospitalized in the same hospital. the detailed diagnosis standards were presented in online table s1 . total rnas were extracted from nasopharyngeal and/or oropharyngeal swabs samples of patients suspected of having sars-cov-2 infection within 2 hours using the respiratory sample rna isolation kit. in brief, 40 μl of cell lysates were transferred into a collection tube followed by vortex for 10 seconds. after standing at room temperature for 10 minutes, the collection tube was centrifuged at 1000 rpm/min for 5 minutes. the suspension was used the serum sars-cov-2 antibodies (igm and igg) of the subjects were detected using a sandwich enzyme linked immunosorbent assay (elisa kits, livzon inc, zhuhai, p.r.china, lot number of igm: 20200308, igg: 20200308). for detection of igm, 100 µl diluted serum (1:100) was added into the 96-well microplate (coated with n protein) and then incubated for 1 h at 37℃. after washing, 100 µl secondary antibody (against human igm) labeled with conjugate was added into the wells and then incubated for 30 min under 37℃. following the second wash cycle, 100 µl substrate was added into the wells and incubated for 15 min under 37℃. at last, stop solution was added into the wells to terminate the reaction. the optical density of each well was determined by a microplate reader set to 450 nm within 30 min. the ratio of optical density to the cut off value (optical density of the blank well + 0.1) was reported as the antibody concentration. for detection of igg, the dilution factor was changed (1:20) and the cut off value was modified (optical density of the blank well + 0.13). all statistical analyses were performed using spss 20.0. the diagnostic value of elisabased igm and igg antibody test for covid-19 was based on sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy. log-normal distribution was used to fit the time distribution of igm and igg antibody seropositve rate, as well as calculative seroconversion rate of the two antiobdies. 8 ethics approval was exempted from institutional review board of the hospital since we collected and analyzed all data from the patients according to the policy for public health outbreak investigation of emerging infectious diseases issued by the national health commission of the people's republic of china. 85 patients with confirmed diagnosis and 24 patients with suspected diagnosis were recruited in this study. the demographic and clinical characteristics were showed in tabel 1. comorbidities in patients were recorded including hypertension, diabetes, surgical operation, malignancies, chronic lung disease, and chronic renal diseases. healthy donors who were doctors and nurses working in the hospital, and patients with other lung disorders including bacterial pneumonia (n=5), acute exacerbation of chronic obstructive pulmonary disease (aecopd, n=2), lung cancer (n=3), empyema (n=1), interstitial lung disease (n=2) and pleuritis (n=1) were included as control group. detection of igm and igg antibody on different periods (table 2.) we evaluated specificity of igm and igg antibodies based on elisa from 216 serum sample of 85 confirmed covid-19 pneumonia patients. serum obtained in different periods after 9 symptom onset. the igm and igg antibodies were detected positive as early as on the 4 th day after onset (table 2) . some patients were observed positive for igm (7 patients) and igg (6 patients) within 7 days after illness onset, respectively. the seropositive rate of igm increased gradually and notably. igg was increased sharply on the 12 th day after onset. we used a log-normal distribution to fit the time distribution of igm and igg antibody seropositive rate (figure 1 ). the results from the 60 samples in the control group showed that 3 cases were positive for igg, while all cases were negative for igm. to monitor the kinetics of serological antibodies within covid-19 patients, serological antibodies were tested consecutively once the initiative appearance time of igm and/or igg antibody were detected in 29 confirmed patients. a log-distribution was used to fit the seroconversion time of the two antibodies ( figure 2 ). igm cumulative seroconversion increased quickly from the 9 th day as well as igg increased from the 11 th day after symptom onset. in our investigation, both antibodies were seropositive in nearly all the patients within the illness course for more than 30 days. to evaluate the diagnostic potential of serological igm and igg antibodies detection, 66 patients with confirmed covid-19 pneumonia were evaluated and compare to standard rt-pcr assays. serum sample were obtained from patients with disease course being or more than 13 days and less than 29days. the results of serological test for igm and igg were showed in table 3 . compared with rt-pcr, the sensitivity, specificity, ppv, npv, and as shown in table 4 , 24 patients had covid-19 pneumonia manifestations while being negative at least twice for respiratory tract nucleic acid tests. patients were evaluated independently by two experienced physicians. the serological tests for igm and igg showed in table 5 during the immune responses against pathogens infection, igm are usually produced earlier than igg antibody. however, both igm and igg antibodies against sars-cov-2 were detected as early as the 4 th day after symptom onset, and the appearance of igm and igg antibodies seems earlier than sars, another severe coronavirus pneumonia [9] . in our study, the seropositive rate of igg was observed decreased around the 28 th days after illness onset. the seropositive rate should not decrease on this time point, we thought this may be due to the small sample size, there were only 7 serum samples collected from 7 patients. diagnostic value of serological test was evaluated in patients with confirmed and suspected covid-19 pneumonia. depending on patients whose disease course (>=13 days from the disease onset), specificity and positive predictive value of igm antibody were very high up to 100%, which indicated that the igm can be used as a good mark for diagnosis of covid-19. however, the sensitivity, negative predictive value and consistency rate of igm were 73.2%, 80.0% and 88.1%, respectively, indicating that acute infection may still be missed based on seronegative igm. the specificity negative predictive value and consistency rate of igg were 95.0%, 94.8% and 88.9%, respectively. that means patients can be diagnosed as covid-19 pneumonia base on seropositive igg. both seropositive antibodies demonstrate outstanding specificity and ppv, it suggest that seropositve igm and/or igg can help to establish the diagnosis of covid-19 pneumonia, especially in patients with a long course. to avoid misdiagnosis, patients with early seronegative antibodies should be retested after 10 days of onset. the data acquired from patients with suspected diagnosis demonstrate that the serological tests are reliable as they show high specifity, of which igm tests were up to 100% and igg tests were 95.0%. those testing results were in accordance with physician's judgments of the symptoms. there were 3 controls out of 60 were positive in igg tests. these 3 controls (one was weak positive) were healthcare providers, they didn't serve patients who diagnosed as confirmed or suspected covid-19 at that time. we think these should be false positive or community asymptomatic infection. in summary, detection of antibodies against covid-19 based elisa appears to be a valid approach to serodiagnosis of covid-19 pneumonia. the specific circulating antibody can be uniformly detected, therefore avoiding false-negative results due to sampling or potential absence of viruses in the respiratory system. as an emerging infection disease, the current population is generally susceptible to it and the background levels of serum specific antibodies are low. therefore, covid-19 pneumonia can be diagnosed base on seropositive of specific antibodies as an alterative to viral nucleic acid detection with clear advantages. the authors thank dr. xiang-ping yang at department of school of basic medicine, tongji medical college, huazhong university of science and technology for his review of our manuscript this work is funded by national natural science foundation of china (no. 81973990, 91643101), and science foundation of conflict of interest. the authors declare that there are no conflicts of interest coronavirus disease 2019 (covid-19) situation report-44. mar 04 2020. who real-time rt-pcr panel for detection 2019-novel coronavirus sensitivity of chest ct for covid-19: comparison to rt-pcr. radiology correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china sars and common viral infections detection of specific antibodies to severe acute respiratorysyndrome (sars) coronavirus nucleocapsid protein forserodiagnosis of sars coronavirus pneumonia a novel coronavirus associat ed with severe acut e respirat ory syndrome * specific igm and igg antibodies against sars-cov-2 from 216 serum samples of 85 confirmed covid-19 pneumonia cases were tested with elisa. serum samples were obtained at different time points after symptom onset key: cord-329290-vqvujry3 authors: kempker, russell r; kempker, jordan a; peters, marcia; rebolledo, paulina a; carroll, kelley; toomer, linda; wang, yun f (wayne); ray, susan m; hunter, mary title: loss of smell and taste among healthcare personnel screened for coronavirus 2019 date: 2020-06-28 journal: clin infect dis doi: 10.1093/cid/ciaa877 sha: doc_id: 329290 cord_uid: vqvujry3 among 283 symptomatic healthcare personnel (hcp) tested for sars-cov-2, 51 (18%) were positive. among those 51 hcp, self reported loss of smell and taste were present in 51% and 52.9%, respectively, with either present in 60.8%. these symptoms had high specificity (93% each, 96% for either) for a positive sars-cov-2 test. since first recognized in wuhan, china, in december 2020, coronavirus disease 2019 (covid-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has quickly spread throughout the world and was officially recognized as a pandemic on 11 march 2020 [1, 2] . the united states has since become the country with the highest number of reported cases and deaths. knowledge of covid-19 is rapidly increasing, including a better understanding of persons at high risk of acquiring infection and the most common presenting clinical manifestations. one particular high-risk group is healthcare personnel (hcp) given their frequent exposure to persons with covid-19. early reports have found high rates of hcp infection in china and italy, and a recent centers for disease control and prevention (cdc) report documented more than 9000 hcp with covid-19 in the united states [3] . given limited testing resources and the essential role of hcp in responding to covid-19, an early and efficient screening of this group is critical for maintaining the ongoing response to the pandemic. our aim in this study was to describe the most common and distinguishing clinical symptoms among hcp who underwent screening for covid-19, with the goal of enhancing future covid-19 symptom screening efforts. our health system's covid-19 screening program used redeployed hospital staff and was implemented through employee health services. the program was advertised through institutional emails to all hospital employees and medical staff and advised hcp with fever or symptoms (congestion, shortness of breath, cough) to use. two nurses operated a covid-19 advice line 24/7 and used a standardized symptom algorithm within the electronic health record (epic, verona, wi) to screen callers for consideration of further testing. hcp with symptoms consistent with a viral-like illness were triaged to the employee health services staff for a virtual clinical assessment and then scheduled for sars-cov-2 testing. testing was conducted monday through friday in an enclosed room near the emergency room and was retrofitted into a temporary medical unit. nasopharyngeal swabs were taken by a rotating team of grady health system-employed nurses and medical assistants specifically trained for the procedure and who were redeployed from other departments that had decreased clinical activities due to covid-19. each hcp filled out a paper or on-line screening data form (see supplementary materials) prior to testing. the symptom screen was based on cdc recommendations at the time with the major exception being additional questions on loss of smell and taste that were prompted by reports of these symptoms from our first few positive cases. sars-cov-2 polymerase chain reaction (pcr) testing was carried out using the abbott laboratories m2000 realtime (lake bluff, il) system in the grady memorial hospital microbiology laboratory. test results and back-to-work guidance were provided via a telephone call within 48-72 hours of testing by employee health services. analysis was completed with rstudio (version 3.6.3, r core team, 2020). r foundation for statistical computing (vienna, austria) was used to compare characteristics of those with and without a positive sars-cov-2 test result (see supplementary materials). study approval was obtained from the emory institutional review board and grady research oversight committee. during the first 4 weeks of covid-19 screening, 549 calls were received and 283 hcp were referred for and had testing performed; 51 (18%) had a positive sars-cov-2 test result. the majority of hcp tested were female (81%), worked in the hospital (83%), had patient contact (89%), and were under self-isolation (86%) at the time of testing. of note, 11 (21.6%) hcp with a positive test for sars-cov-2 were working at the time of testing. various types of hcp were tested, with nurses (33%) being the most common. slightly more than one-third (35%) reported contact with a patient with covid-19. the mean number of days with symptoms prior to testing was 5.7 and was similar throughout the study period (see supplementary materials). in comparing those with and without a positive sars-cov-2 test, there was no significant difference in demographic characteristics, including in patient contact (96% vs 88%, p = .14) and exposure to patients with covid-19 (33% vs 36%, p = .85). hcp with a positive sars-cov-2 test compared with those with a negative test had a higher mean number of symptoms and were more likely to have reported fever, chills, myalgia, and loss of smell or taste (table 1 ). in regard to the sensitivity of certain symptoms, the most sensitive symptoms for a positive sars-cov-2 test were fatigue (76.5%), cough (72.5%), chills (66.7%), and fever (62.7%). the sensitivity of self-reported loss of smell and taste was 51.0% and 52.9%, respectively, and the sensitivity of either loss of smell or taste was 60.7%. of note, slightly less than half (48.4%) of hcp with a positive sars-cov-2 test and either loss of smell or taste had nasal congestion. the most specific symptoms were loss of smell (93%) and taste (93%), and the presence of either symptom had a specificity of 96%. no other symptom had a specificity >70%. loss of smell and loss of taste were also the symptoms with the highest positive predictive values at 60% and 61%, respectively. the sensitivity, specificity, negative predictive value, and positive predictive value of each symptom are shown in table 1 . using a redeployed team of healthcare staff and an onsite testing facility, we were able to screen a large number of hcp and detect 51 cases of confirmed covid-19. importantly, we found the symptoms with the highest specificity and positive predictive value for a positive sars-cov-2 test to be loss of taste and/ or smell. we are the first to evaluate the sensitivity of loss of smell and taste in distinguishing symptomatic hcp with and without a positive sars-cov-2 test and support their recent inclusion to the list of symptoms associated with covid-19 provided by the cdc [4] . furthermore, our findings indicate loss of smell and/or taste may be useful to include in covid-19 screening algorithms. the cdc considers hcp to be a high-priority group for covid-19 screening given their high risk of exposure and their essential nature in combatting the pandemic. however, to date, there have been few reports of systematic covid-19 screening programs among hcp. our work provides an important example of the feasibility and yield of a hospital-wide screening program. the large number of cases detected highlights the value of screening hcp and, given that some were still working at the time of testing, the importance of detection to ensure individuals are provided proper care and counselled to self-isolate to limit potential workplace spread. our model of using redeployed staff allowed for the screening program to be rapidly implemented and to function efficiently. this method provided additional work opportunities for underused outpatient clinical staff, minimized the amount of training and onboarding needed, and engaged a team that was committed to the well-being of their colleagues. the high specificity and positive predictive value of loss of smell and/or taste for a positive sars-cov-2 test in our cohort highlights the utility of including these symptoms in covid-19 screening algorithms. in settings with limited testing capacity, the presence of these symptoms may help guide decisions on who to test. the association of loss of smell and/or taste with covid-19 was first reported in the media and more recently in research reports. cdc surveillance revealed a 16% prevalence of loss of smell or taste among more than 9000 hcp with covid-19. however, this is likely an underestimate given these symptoms were not part of the data collection form, but included as free text [3] . a cross-sectional study of patients already diagnosed with covid-19 found rates of loss of smell and taste of 68% and 71%, respectively, and a strong association with disease. however, limitations included a limited survey response, particularly among covid-19-negative patients, and potential recall bias [5] . two additional reports that used a specific smell questionnaire among patients with mild covid-19 found some level of olfactory and gustatory function loss in 86% and 88% of patients [6] , respectively, and any altered sense of smell or taste in 65% [7] . these results highlight the sensitivity of these symptoms, especially when obtained via a detailed inquiry. a major strength of our study was the inclusion of consecutive, symptomatic persons. this allowed us to determine the utility of loss of smell and taste in distinguishing covid-19 from other viral-like illnesses. the etiology of loss of smell and/ or taste is thought to be secondary to invasion of the olfactory epithelium and less likely due to nasal mucosal edema; notably, less than half of covid-19 patients with these symptoms had nasal congestion. in summary, our findings demonstrate the utility and need for hcp covid-19 screening and suggest that loss of smell and/or taste be added to symptom screening algorithms. loss of smell and/or taste are easy to obtain via self-report and, as we demonstrated, are very specific symptoms for covid-19. additional approaches to optimal hcp screening should be evaluated, including the implementation of serology testing. available data indicate that pcr testing is most accurate early in the course of the illness and decreases substantially after the first week, which may lead to missed cases [8, 9] . serology testing among our cohort may have revealed additional cases of covid-19 including among those with a negative sars-cov-2 test and loss of smell and/or taste. given that some hcp were working at the time of testing and the number of days from symptom onset to testing was high (median, 5.7 days) and did not change over time, initiatives to trigger earlier testing in hcp with symptoms are also needed. optimizing hcp screening for covid-19 will help protect essential front-line workers and sustain our healthcare systems during this pandemic. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china world health organization. who director-general's opening remarks at the media briefing on covid-19-11 characteristics of health care personnel with covid-19-united states covid-19): symptoms of coronavirus association of chemosensory dysfunction and covid-19 in patients presenting with influenza-like symptoms olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection sars-cov-2 viral load in upper respiratory specimens of infected patients antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 key: cord-332469-zegawla5 authors: li, wei; zhang, bo; lu, jianhua; liu, shihua; chang, zhiqiang; cao, peng; liu, xinhua; zhang, peng; ling, yan; tao, kaixiong; chen, jianying title: the characteristics of household transmission of covid-19 date: 2020-04-17 journal: clin infect dis doi: 10.1093/cid/ciaa450 sha: doc_id: 332469 cord_uid: zegawla5 background: since december 2019, sars-cov-2 virus has extended to most parts of china with more than 80 thousand cases and to at least 100 countries with more than 60 thousand international cases by march 15, 2020. here we applied household cohort study to determine the features of household transmission of covid-19. methods: total 105 index patients and 392 household contacts were enrolled. both index patients and household members were inspected by sars-cov-2 rt-pcr. the information of all recruited people was extracted from medical records and confirmed or supplemented by telephone interviews. the baseline characteristics of index cases and contact patients were described. secondary attack rates of sars-cov-2 to the contact members were computed and the risk factors for transmission within household were estimated. results: secondary transmission of sars-cov-2 developed in 64 of 392 household contacts (16.3%). the secondary attack rate to children was 4% comparing with 17.1% to adults. the secondary attack rate to the contacts within the households with index patients quarantined by themselves since onset of symptoms was 0% comparing with 16.9% to the contacts without index patients quarantined. the secondary attack rate to contacts who were spouses of index cases was 27.8% comparing with 17.3% to other adult members in the households. conclusion: the secondary attack rate of sars-cov-2 in household is 16.3%. ages of household contacts and spouse relationship with index case are risk factors for transmission of sars-cov-2 within household. quarantine of index patients at home since onset of symptom is useful to prevent the transmission of sars-co-2 within household. a c c e p t e d m a n u s c r i p t in december 2019, an increasing cases of patients with pneumonia of unknown etiology emerged in wuhan, a large city of 11 million people in central china [1] [2] [3] . the pathogen was soon identified as a novel coronavirus which is similar to the coronavirus of severe acute respiratory syndrome (sars-cov) and shares more than 79% of its sequence [4] . consequently, the novel virus was named as sars-cov-2 and its infection disease was announced as coronavirus disease 2019 (covid-19) by world health organization (who) [5] . with its rapid spread, the virus has extended to most parts of china with more than 80 thousand cases and as well as to at least 100 countries with more than 60 thousand international cases by march 15, 2020 [6] . despite the increasing number of reports about covid-19, the epidemic investigation of this disease is limited and little is known about the transmission probability and infectivity of sars-cov-2 in household. here, the retrospective data on household transmission of covid-19 were systemically analyzed and demonstrated certain epidemiological characteristics of covid-19 between people. data were collected from two local hospitals, zaoyang first people's hospital, 250 kilometers far away from wuhan, and chibi people's hospital, 150 kilometers away, between january 1 and february 20, 2020. eligible households were defined as the family only had one member, known as index patient, who had clear history of exposure to wuhan (departed from or travelled to wuhan) or people from wuhan or high-risk sites such as hospitals, supermarkets or railway stations within 14 days before illness onset and other family members had clear non-history of exposure to these; all the family members resided in one house before the index cases were hospitalized. index patients and infected contacts were confirmed by positive of their nasopharyngeal swab samples on sars-cov-2 rt-pcr [7] and uninfected household contacts were those who had no any symptom and at least twice negative on sars-cov-2 rt-pcr. all the family contacts were quarantined immediately after the index cases were confirmed for 14 days in special places designated by the local governments and monitored everyday by the health service personnels. the nasopharyngeal swab samples were collected at the beginning and the middle time of quarantine duration. the quarantined contacts who had symptoms were inspected at a c c e p t e d m a n u s c r i p t least four times by sars-cov-2 rt-pcr until they were positive. the information of all recruited people was extracted from medical records and confirmed or supplemented by telephone interviews. the last enrolled index case in this study was hospitalized in february 13, 2020 and the final date of followed-up was at march 1, 2020. household members were excluded if they lived less than 24 hours in the residence of the index cases. total 105 households with 392 family contacts were enrolled. secondary attack rate is defined as the proportion of infected household contacts (or household members except index patient) in the total number of household contacts. for categorical variables, the percentages of patients in each category were calculated. logistic generalized estimating equation model was applied to identify risk factors associated with the secondary attack rate while accounting for correlation of members within household [8] . the study included 105 index patients. the median age of the index patients was 51 years (25th to 75th percentile, 39 to 60 years) and 60 (57.1%) were male. of the 105 patients, 65 (61.9%) patients had direct history of exposure to wuhan, 13 (12.4%) patients had close contact with peoples from wuhan and another 27 (25.7%) patients had visited to high-risk sites within 14 days before onset of illness. of the 105 patients, 56 (53.3%) patients had fever at onset of illness, 31 (29.5%) cough, 20 (19%) fatigue, 12 (11.4%) chill, 6 (5.7%) dyspnea or anorexia, 5 (4.8%) dizziness or myalgia, 3 (2.9%) vomit or nausea, 2 (1.9%) diarrhea or rhinobyon and one patient had headache or palpitate. the period between onset of symptoms and hospitalization was 0 to 11 days. of the 105 patients, 12 (11.4%) patients waited 0 to 1 day for hospitalization after onset of illness, 34 (32.4%) waited 2-5 days and 59 (56.2%) waited 7-11 days. there were 14 (13.3%) index patients quarantined by themselves at home that was with mask, dieting separately and residing alone immediately since onset of symptoms (table 1) . a total of 392 elegible household contacts from the 105 family clusters were enrolled in this investigation. the median of household size was 4 (25th to 75th percentile, 3 to 6). the median of adult age was 46 (25th to 75th percentile, 32 to 58) and that of child a c c e p t e d m a n u s c r i p t was 6.5 (25th to 75th percentile, 4 to 11). there were 64 contacts infected with sara-cov-2. the median age of the secondary cases was 45 years (25th to 75th percentile, 30 to 57 years) and the median child age . of the 64 contact patients, 33 (51.2%) were male; 36 (56.3%) contact cases had fever at onset of illness, 11 (17.1%) cough, 4 (6.3%) chill or fatigue, 3 (4.7%) diarrhea and one contact case had dizziness or myalgia or sneeze or anorexia. there were 9 (14.1%) asymptomatic patients in total sars-cov-2 positive contacts and 3 nucleic acid negative contacts with symptoms, 2 adult with cough, one adult fatigue and one child cough. of the 64 contact patients, 4 (6.3%) cases were aged below 18 years and all were male, 12 (18.8%) cases in 18 to 30 years, 14 (21.9%) cases in 31 to 40 years, 9 (14.1%) cases in 41 to 50 years, 18 (28.1%) cases in 51 to 60 years and 7 (10.9%) cases above 60 years (table 2. ). the median of interval time, from symptomatic onset of index cases to symptomatic onset of household contacts, was 6 days (25th to 75th percentile, 4 to 10). the secondary attack rates to household contacts were evaluated in different conditions. the secondary attack to total 392 household contacts was 16.3%. when the data were stratified for analysis by the ages of contacts, the secondary attack rate to children (aged below 18 years) was 4% comparing with 17.1% to adult members (or 0.18, 95% ci 0.06 to 0.54, p=0.002). the secondary attack rate to the contacts in the household with index patients quarantined at home immediately since onset of symptom was 0% compared to 16.9% to the contacts in the households without index patients quarantined during the period between initiation of symptom and hospitalization (or 0, 95% ci 0.00 to 0.00, p=0.000). spouse relationship was another risk factor for the infection of sars-cov-2 to household contacts and the secondary attack rate to individuals who were spouses of index cases was 27.8%, compared to 17.3% to other members in the households (or 2.21, 95% ci 1.18 to 4.12, p=0.013). the gender, symptoms and the time between onset of illness of index patients and hospitalization were not related to the secondary attack rates of sars-cov-2 to household contacts (table 3 a c c e p t e d m a n u s c r i p t household model has more clear and fixed exposure of infection sources which makes it feasible to estimate the transmissibility and infectivity of prevalent virus [7, 8] . here, we found the general secondary attack rate of sars-cov-2 to contact members in households was 16.3%, which is the highest when comparing with those of two other coronaviruses related respiratory diseases, severe acute respiratory syndromes (sars) and middle east respiratory syndrome (mers) [9, 10] and is also higher than that of pandemic influenza a in 2009 with 13% [8] . this might contribute to the dramatically higher number of infected cases than the two other coronaviruses related diseases. as the time and chance of infection exposure to susceptible people within household model were more than that in community and population-level, the transmission probability in household may be stronger than that in community and population-level in most case [8, 10, 11] . although the household was different from community and large population, our results may exhibit a hint of transmission probability of sars-cov-2 in one aspect. sars-cov-2 [11] and the population distribution of covid-19 in ages were described by several other groups [13, 12] , there was no denominator number of peoples exposed to clear source of infection and the relative susceptibilities to sars-cov-2 for children and adults remain to be studied. in this report, the results showed secondary attack rate of sars-cov-2 to adults was dramatically higher than that to children which might reflect the different susceptibility to sars-cov-2 between children and adults and indicate adults were more susceptible to sars-cov-2 than children who aged below 18 years when they exposed themselves to the same sources of infection. this is similar with sars virus that infected most peoples aged above 18 years. however, the real causes leading to the different secondary attack rates between adult and children needed to be more explored and many factors could influence the secondary transmission of epidemic diseases in household, such as behaviors of contacts and occupations of members [10] . asymptomatic carriers exist in many infectious diseases [13] . in our data, we also found 9 cases in contact cases (14.1%). this is more than 10 times as that reported by other group in a large population-level [6] . in the household model, all the negative contacts were inspected by sars-cov-2 rt-pcr at least twice. however, in that retrospective study, the whole close contact population could not be scanned by the a c c e p t e d m a n u s c r i p t laboratory test, which may lead to missing many symptom-free cases to be statistically counted and this may contribute to inconsistencies with ours. in this case, there might be more asymptomatic carriers than we expected based on previous report. we knew asymptomatic carrier of sars-cov-2 could transmit virus to other people [14] , so more strategies may be needed to find out more asymptomatic cases in population. the effect of self-quarantine at home was also assessed in household model. of 105 index patients, 14 cases were quarantined by themselves at home immediately at onset of symptoms, that was with mask, repasting separately and residing alone. the results showed no infected contacts in the households with index cases who implemented quarantine immediately after appearance of symptoms, and so the secondary attack rate was zero. this indicated that home quarantine by themselves since onset of symptoms might make certain sense to prevent the transmission of sars-cov-2 in household. however, the power to make the conclusion for these was low since the number of cases was limited and large-scale investigation remained to be made. interestingly, the analysis of data stratified by spouses who were the husbands or wives of index cases, demonstrate that the secondary attack rate of sars-cov-2 to the spouses was significantly higher than that to other family members. this might be because the longer time of exposure to infection sources, the higher chance of illness for contacts and spouses might spend more time for each other to stay together than any other members they did with within household. m a n u s c r i p t a novel coronavirus from patients with pneumonia in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study outbreak of a novel coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin naming the coronavirus disease (covid-19) and the virus that causes it characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia household transmission of 2009 pandemic influenza a (h1n1) virus in the united states defining the epidemiology of covid-19 -studies needed household transmission of sars middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission novel coronavirus infection in hospitalized infants under 1 year of age in china clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series implications of asymptomatic carriers for infectious disease transmission and control presumed asymptomatic carrier transmission of covid-19 the authors would like to thank the two local hospitals, zaoyang first people's hospital and chibi people's hospital for supporting the data and also thank enrolled covid-19 patients and their contact family members. competing interests: we declare that we have no conflicts of interest.a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-329311-p68kr4ga authors: prebensen, christian; hre, peder l my; jonassen, christine; rangberg, anbjørg; blomfeldt, anita; svensson, my; omland, torbjørn; berdal, jan-erik title: sars-cov-2 rna in plasma is associated with icu admission and mortality in patients hospitalized with covid-19 date: 2020-09-05 journal: clin infect dis doi: 10.1093/cid/ciaa1338 sha: doc_id: 329311 cord_uid: p68kr4ga the clinical significance of sars-cov-2 rna in the circulation is unknown. in this prospective cohort study, we detected viral rna in the plasma of 58/123 (47%) patients hospitalized with covid-19. rna was detected more frequently, and levels were higher, in patients who were admitted to the icu and/or died. disease 2019 and evaluate the association between rna loads, disease severity and the sars-cov-2-specific antibody response. the coronavirus disease mechanisms study (covid-mech, nct04314232) recruited consecutive adult patients admitted to akershus university hospital with covid-19 confirmed by rt-pcr between march 18 th and may 4 th 2020. written informed consent was granted by study participants, or by next-of-kin if the patient was unable to consent. the study was approved by local regulatory authorities. the predefined primary endpoint was a composite of intensive care unit (icu) admission >24 hours and in-hospital mortality. clinical data were extracted from electronic patient records. routine biochemistry was taken at admission and study-specific samples of edta plasma and serum were taken at three time points; baseline (enrollment), day 3 (1 day) and day 9 ( 2 days) in patients who were still hospitalized (details in supplementary figure 1) . a c c e p t e d m a n u s c r i p t 4 sars-cov-2 rna was detected in plasma samples and upper respiratory tract swabs by rt-pcr and rna levels were estimated using the cycle threshold (ct) value. in pcr-positive plasma samples, rna was quantified and expressed as log10 copies/ml (details in supplementary methods file 1). upper respiratory samples were combined oropharyngeal and nasopharyngeal swabs, taken in the emergency department unless the patient had a positive rt-pcr from another laboratory within the preceding week. all rt-pcrs were performed as described by corman et al., targeting the viral envelope (e)-gene [2] . total antibodies against sars-cov-2 nucleocapsid (np) were quantified using the elecsys anti-sars-cov-2 test on the cobas e801 module (roche, penzberg, germany). igg antibodies directed against subunits s1 and s2 of the sars-cov-2 spike protein were quantified using the liaison sars-cov-2 s1/s2 igg assay and the liaison xl chemiluminescence analyzer (diasorin, saluggia, italy). data are presented as n (%), mean  sd and median [q1, q3]. differences between groups were assessed by t-tests, mann-whitney u tests, chi-square tests and spearman tests, as appropriate. all statistical analyses were performed in stata 16 (statacorp, college station, tx, usa). p-values <0.05 were considered statistically significant. plasma and serum samples were available from 123/135 (91%) patients, of whom 35 (28%) patients reached the primary endpoint. 31 patients were admitted to the icu; 29 received mechanical ventilation and four died. another four patients with do-not-intubate orders died on regular wards. all icu admissions and deaths were attributable to covid-19. clinical, biochemical, virological and a c c e p t e d m a n u s c r i p t 5 serological data are presented in table 1 (data on 12 patients without biobank samples in supplementary table 1) . sars-cov-2 rnaemia was detected in at least one sample in 58/123 (47%) patients, and in a significantly higher proportion of patients who were admitted to the icu or died (80% vs. 34%, p<0.001). rnaemia was detected in 48/123 (39%) patients at baseline, a median 0 [-1, 3] days before icu admission. 24 (41%) of patients with detectable rnaemia had ct values >38, below the quantitation limit of 2.70 log 10 copies/ml, and ct values were therefore used in this analysis. of note, the lowest ct value of 25.9 (corresponding to an rna load of 6.3 log 10 copies/ml) was observed in a female icu patient with a prolonged course of disease. rnaemia was significantly more frequent at all time points in patients who reached the primary endpoint, whereas rna loads were significantly higher at baseline and day 3 ( table 1 , supplementary figure 2a ). in a logistic regression analysis where nine patients with baseline samples taken after icu admission were excluded, baseline rnaemia and rna load were both significantly associated with icu admission and/or death (supplementary table 2 ). this association persisted after adjusting for age, sex and race (model #1) and bmi, diabetes mellitus and symptom duration (model #2). there was no correlation between days from symptom onset and rnaemia frequency or rna load at baseline. figure 2c) . there was no difference in the titers of total ig or igg at any timepoint between patients who reached the primary endpoint or not. there was no correlation between baseline rnaemia, rna loads or upper respiratory ct values and subsequent antibody titers. while four patients had low or undetectable antibody titers out to 20+ days after symptom onset, there was no association between a lack of an antibody response and the primary endpoint, baseline rnaemia and rna loads, or admission upper respiratory ct values. in this prospective study of patients hospitalized with covid-19 we detected sars-cov-2 rnaemia in 47% of included patients, and a significantly higher frequency of rnaemia and higher rna loads in and similarly found that rnaemia was associated with icu admission and hospital mortality [3] . collectively, this supports the possible utility of sars-cov-2 rnaemia as a prognostic marker in covid-19. with potent antiviral agents in the pipeline, early markers of severe disease will facilitate the identification of patients who may benefit the most from these new therapeutics, particularly as early initiation tends to be a critical factor in antiviral drug efficacy [4] . severe disease and high mortality [5, 6] . on the other hand, negative upper respiratory pcrs are not uncommon in patients with severe covid-19 [7] , and high rna loads are frequently detected in individuals with few or no symptoms [8] . furthermore, differences in sampling techniques and specimen type, pcr targets (orf1ab vs. e gene), and variations in preanalytical conditions may contribute to discrepant findings. we found no evidence that viral rna in plasma or the upper respiratory tract influenced development of sars-cov-2-specific antibodies during acute infection. several retrospective studies have found higher levels of igg in patients with more severe covid-19 [9, 10], but other investigators, like us, have detected no such association [11] . larger prospective studies with longterm follow-up data will be required to determine the significance of sars-cov-2-specific antibodies in acute infection and subsequent re-challenge. this was a single center study, which may limit the generalizability of our findings. while patients were recruited consecutively, we were unable to recruit and sample all patients admitted with covid-19 in the study period and cannot exclude selection bias. the time between baseline sampling and icu admission was frequently short, largely due to severe clinical disease at presentation. we expect this to be common, which may limit the useful prognostic time window of a marker such as rnaemia. no correction for multiple comparisons was made, potentially increasing the risk of type i statistical errors, and our findings should be replicated in other cohorts. in conclusion, we found a high proportion of sars-cov-2 rna in the plasma of patients hospitalized with covid-19, and a significantly higher frequency and level of plasma rna in patients who were admitted to the icu or died. sars-cov-2 rnaemia may be a useful prognostic marker in covid-19. the pathophysiological significance of circulating viral rna must be ascertained by future studies. m a n u s c r i p t a c c e p t e d m a n u s c r i p t multiorgan and renal tropism of sars-cov-2 detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr high frequency of sars-cov-2 rnaemia and association with severe disease impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza a(h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients impact of sars-cov-2 viral load on risk of intubation and mortality among hospitalized patients with coronavirus disease viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases virological assessment of hospitalized patients with covid-2019 a c c e p t e d m a n u s c r i p t key: cord-348036-yub2cqz6 authors: lighter, jennifer; phillips, michael; hochman, sarah; sterling, stephanie; johnson, diane; francois, fritz; stachel, anna title: obesity in patients younger than 60 years is a risk factor for covid-19 hospital admission date: 2020-04-09 journal: clin infect dis doi: 10.1093/cid/ciaa415 sha: doc_id: 348036 cord_uid: yub2cqz6 nan a c c e p t e d m a n u s c r i p t dear editor, risk factors for infectious disease severity are determined by the pathogen, host and environment [1] . covid-19 disease, caused by sars-cov-2 infection includes a spectrum of illness; from asymptomatic infection [2] to severe pneumonia characterized by acute respiratory injury in about 20% of patients presenting to medical care [3] . the risk factors associated with disease severity, included increased age, diabetes, immune suppression and organ failure [3] . recognition of risk factors for morbidity and mortality is important to determine prevention strategies as well as to target high-risk populations for potential therapeutics. we performed a retrospective analysis of bmi stratified by age in covid-19-positive symptomatic patients who presented to a large academic hospital system in new york city. patients presented to the ed with signs of respiratory distress were admitted to the hospital. critical care was defined based on intensive care accommodation status or invasive ventilator documentation in our electronic health record. patients who were pcr-positive for covid-19 during march 4, 2020-april 4, 2020 were extracted from our electronic health record system and analyzed with a chisquare wald test using sas v9.4 (sas institute, care nc). of the 3,615 individuals who tested positive for covid-19, 775 (21%) had a body mass index (bmi) 30-34, and 595 (16% of the total cohort) had a bmi >35. there were 1,853 (51%) patients discharged from the ed, 1,331 (37%) were admitted to the hospital in acute care and 431 (12%) were either directly admitted or transferred to the icu during admission. during analysis we found significant difference in admission and icu care only in patients <60 years of age with varying bmis (table 1) patients aged <60 years with a bmi between 30-34 were 2.0 (95% 1.6-2.6, p<0.0001) and 1.8 (95% ci 1.2-2.7, p=0.006) times more likely to be admitted to acute and critical care, respectively, compared to individuals with a bmi <30 (table 1) a c c e p t e d m a n u s c r i p t years were 2.2 (95% ci 1.7-2.9, p<.0001) and 3.6 (95% ci 2.5-5.3, p=<.0001) times more likely to be admitted to acute and critical care compared to patients in the same age category who had bmi <30. though patients aged <60 years are generally considered a lower risk group of covid-19 disease severity, based on data from our institution, obesity appears to be a previously unrecognized risk factor for hospital admission and need for critical care. this has important and practical implications, where nearly 40% of adults in the us are obese with a bmi >30 [4] . the bmi range of individuals in this study appears representative of the nation, as 36% of the patients have a bmi >30. there is geographic variation in reported mortality, as south korea, china and italy report case fatality rates of 0.8, 2.3 and 7.2, respectively [5] and regional risk factors such as prevalence of smoking, pollution or aging population has been cited. unfortunately, obesity in people <60 years is a newly identified epidemiologic risk factor which may contribute to increased morbidity rates experienced in the us. there are no financial disclosures to report on any of the authors m a n u s c r i p t ecological theory to enhance infectious disease control and public health policy estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention prevalence of obesity among adults, by household income and education -united states case-fatality rate and characteristics of patients dying in relation to covid-19 in italy a c c e p t e d m a n u s c r i p t key: cord-330951-k54e3lbu authors: pollett, s; rivers, c title: social media and the new world of scientific communication during the covid19 pandemic date: 2020-05-12 journal: clin infect dis doi: 10.1093/cid/ciaa553 sha: doc_id: 330951 cord_uid: k54e3lbu the human and social toll of the covid19 pandemic has already spurred several major public health ‘lessons learned’, and the theme of effective and responsible scientific communication is among them. we propose that twitter has played a fundamental – but often precarious role in permitting real-time global communication between scientists during the covid19 epidemic, on a scale not seen before. here, we discuss three key facets to twitter-enabled scientific exchange during public health emergencies, including some major draw-backs. this discussion also serves as a succinct primer on some of the pivotal epidemiological analyses (and their communication) during the early phases of the covid19 outbreak, as seen through the lens of a twitterfeed. and the theme of effective and responsible scientific communication is among them. the expansion of the outbreak has demanded a rapid response from public health authorities; fundamental epidemiological and scientific evidence has been acquired at break-neck speed to support those decisions. the demanding pace and large volume of covid19 science generated in the last three months, however, has made timely scientific communication through the conventional route of published biomedical journals at best challenging, and at worst obsolete. twitter has an estimated global user network of 330 million monthly users, including an extensive network of scientists and epidemiologists who frequently use this media for scientific exchange [4, 5] . we propose that twitter has played a fundamental -but often precarious -role in permitting real-time global communication between scientists during the covid19 epidemic, on a scale not seen before. here, we discuss three key facets to twitter-enabled scientific exchange during public health emergencies, including some major draw-backs. this discussion also serves as a succinct primer on some of the pivotal epidemiological analyses (and their communication) during the early phases of the covid19 outbreak, as seen through the lens of a twitter-feed. we do not cover the other major roles of twitter and other social media during this epidemic, including transmission of rapid situational awareness reports, advisories, and public education from formal public health agencies and normative bodies [6, 7] . similarly, concerns of malignant misinformation about covid19 deliberately spread through this medium are beyond the scope of this commentary [8] . m a n u s c r i p t twitter accelerated the rapid, global dissemination of the first whole genome sequence of sars-cov-2 from a consortium led by fudan university, shanghai, to the global science community approximately 10 days after the first alerts of the sars-cov-2 outbreak [9]. this sequence data permitted development of a pcr diagnostic assay, the protocol of which was disseminated mere days later through twitter [10]. between jan 11-18, the first genomic analyses of viral genomes sequenced from chinese cases, and then initial thailand cases, were posted in real-time to twitter as with other outbreaks, early estimation of epidemic parameters during the first month of the covid19 epidemic has been critical to predict the epidemic trajectory and inform decision-making. a c c e p t e d m a n u s c r i p t as highlighted in an early 2020 nature microbiology editorial, global scientists openly reprimanded a group who published a genomic sars-cov-2 analysis through twitter but failed to properly acknowledge the source of this molecular data [37] . such open critique through this medium helps enable codes of conduct around epidemic sequence data sharing [38] . real-time rebuttal, coupled with supporting pre-print analyses, led to fast rejection of an invalid scientific conclusion that snakes were a probable animal reservoir for sars-cov-2, a claim that had led to widespread misinformation [39, 40] . similarly, prompt corrections over journalist misinterpretations of supposed pangolin origins to the sars-cov2 outbreak has been valuable. in this way, twitter has facilitated vital counter-narratives from the scientific community during these and other instances of controversial scientific communication, be they claims of the zoonotic origins of sars-cov2, alarmist interpretation of upper-end 0 estimates, or confusion on whether particular public health policies were grounded on goals of 'herd immunity'. twitter has and continues to serve as a valuable medium to discuss the caveats and future directions in applying infectious disease models in covid19 decision-making [41] [42] [43] [44] . still, twitter remains the doubleedged sword of rapid scientific communication during the ongoing covid19 pandemic. as advocated on twitter itself, scientists will need to exercise great care in their communication using this and other social media to share their research as this outbreak unfolds throughout 2020 [45] . how to fight an infodemic a c c e p t e d m a n u s c r i p t key: cord-340579-cvze15cj authors: dudley, joseph p; lee, nam taek title: disparities in age-specific morbidity and mortality from sars-cov-2 in china and the republic of korea date: 2020-03-31 journal: clin infect dis doi: 10.1093/cid/ciaa354 sha: doc_id: 340579 cord_uid: cvze15cj we analyzed ageand sex-specific morbidity and mortality data from sars-cov-2 pandemic in china and republic of korea (rok). data from china exhibit a gaussian distribution with peak morbidity in the 50-59 years cohort, while the rok data have a bimodal distribution with highest morbidity in the 20-29 years cohort. there are many uncertainties regarding the clinical epidemiology of the sars-cov-2 virus that is currently spreading worldwide in a global pandemic whose ultimate impacts are still uncertain, but which appears based on observed impacts to have the potential to overwhelm the medical surveillance and medical treatment infrastructure of even the world's most affluent countries. there is a need to gain greater understanding of the highest risk populations for infection and serious disease from the sars-cov-2 virus to support the development and implementation of effective public health surveillance and mitigation efforts, and minimize the adverse effects of the current covid-19 pandemic in countries worldwide [1] . control & prevention (kcdc) [2] , and the china centers for disease control [3] to compare the agespecific rates of morbidity and mortality from recent epidemics in china and the republic of korea, and gain insights into the potential for variation in public health impacts of the covid-19 pandemic in different countries. we identified major disparities in the age-specific and sex-specific rates of mortality from sars-cov-2 in china and the rok. data from china (n = 44,672 as of 11 february 2020) exhibit a symmetrical gaussian configuration, with peak morbidity among individuals in the 50-59 year age cohort ( figure 1 ). the sex-specific morbidity ratio among confirmed cases was near parity, with a slight male bias (male 51%; female 49%). there was a high degree of difference in sex-specific case fatality rates, with fatality rates much higher among males (2.8% cfr) than females (1.7% cfr). data from rok (n = 7,755 as of 11 march 2020) exhibited a skewed bimodal distribution with the highest rate of confirmed sars-cov-2 infections among individuals in the 20-29 year age cohort (30%), and a second peak in the 50-59 year age cohort (19%) (figure 1 ). there is a high degree of difference in the sex-specific rates of infection from sars-cov-2 in the rok, with female cases (62%) outnumbering males by a factor of nearly 2:1 ratio (females: 62%; males: 38%). the data on fatalities from confirmed sars-cov-2 infections in the rok exhibit a reversed bias ratio to that among cases, with the case fatality rate among males more than twice as high as that among females (cfr: males 1.19%; females 0.52%). investigations of age-specific and sex-related differences in morbidity and mortality from emerging diseases can provide important tools for identifying populations at highest risk for surveillance, monitoring and intensive medical interventions, and provide insights into geographic, genetic or cultural factors that may influence the spread of pandemic disease viruses such sars-cov-2, within and among different countries and continents [4] [5] . the available data indicate that the epidemiological characteristics of sars-cov-2 infections appear to distinctly different from those of typical human coronaviruses and the recently-emerged zoonotic coronaviruses (sars-cov, mers-cov), zoonotic influenza viruses (e.g., h5n1, h7n9, h792), seasonal influenza viruses, and recent pandemic influenza viruses [7] . while many patients developed influenzalike illness that progresses to pneumonia or acute respiratory distress syndrome (ards) resulting in hospitalization and/or death, a large spectrum of clinical presentations have been documented including a growing number of reported cases with mild symptoms and asymptomatic infections [8] . asymptomatic infection has been reported from adults and children, but the proportion of truly asymptomatic infections remains uncertain [9] [10] . a recent report documented rt-pcr detection of sars-cov-2 in fecal samples from an asymptomatic child, which persisted for 26 days following the estimated date of exposure [11] . the most commonly reported symptoms in order of relative frequency include fever and dry cough (>70%) and fatigue (>35%), dyspnea (<20%), sore throat (<15%), headache (<15%), myalgia or arthralgia (<15%), chills (<15%), nausea or vomiting (5%), nasal congestion (<5%), diarrhea (<4%), and conjunctivitis (<1%). people with symptomatic covid-19 infections usually become symptomatic within 1-2 weeks following infection (mean incubation period 5-6 days, range 1-14 days). the majority of people infected with covid-19 virus have mild disease and recover. approximately 15% of laboratory confirmed patients develop severe disease with pneumonia and/or dyspnea, of whom about 7% progress to respiratory failure, septic shock, or multiple organ failure). individuals at highest risk for severe disease and death include people aged over 60 years and individuals with chronic disease. symptomatic disease appears to be relatively rare and typically mild in children and adolescents [7] . we hypothesize that differences in public health intervention practices and age-related sociocultural factors may be significant factors mediating the observed marked disparities in the age-specific and sexspecific rates of infection from confirmed cases in china and korea. the most striking anomaly in the rok data appears to be the relatively high proportion of cases among the 20-29 year age group (29% of all cases), which may be attributable in part to lower rates of compliance among individuals in this age group with social distancing and self-quarantine recommendations issued by korean health authorities. religious affiliations and practices have been an important factor for the sars-cov-2 epidemic in the rok, because approximately 61% of all confirmed cases as of 11 mar 2020 have epidemiological links to the shincheonji religious community, a korean-based sect with satellite churches in china and at least 24 other countries, with some 200,000 members in rok (~0.3% of the rok population of 51.5 million persons) [12] . lack of adherence to social-distancing and self-quarantine recommendations appears to be a key factor in the high ratio of confirmed sars-cov-2 infected individuals associated with the shincheonji religious community also. observational data collected throughout the course of the epidemic in the rok indicates much lower rates of compliance with rok government social distancing recommendations by individuals in the young adults and teenagers in the 15-29 year age cohorts, which could help explain the relatively higher rates of infection among the 10-19 and 20-29 year age cohorts in the rok data. the reported data on confirmed cases and fatalities from the sars-cov-2 indicate highly significant the available epidemiological and observational data from the rok suggests that reduced rates of compliance with social distancing and self-quarantine recommendations among different sectors of the population -especially the younger adult and juvenile age cohorts --may have a significant impact on the age-specific rates of morbidity and mortality within the population as a whole. duty to plan: health care, crisis standards of care, and novel coronavirus sars-cov-2. nam perspectives discussion paper the updates on covid-19 in republic of korea as of 11 march novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china age-specific and sex-specific morbidity and mortality from avian influenza a(h7n9) age-specific infection and death rates for human a(h5n1) avian influenza in egypt middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications clinical characteristics of coronavirus disease 2019 in china epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-ncov) outbreak presumed asymptomatic carrier transmission of covid-19 transmission of 2019-ncov infection from an asymptomatic contact in germany detection of novel coronavirus by rt-pcr in stool specimen from asymptomatic child, china. emerg infect dis figure 1. comparison of age-specific morbidity and mortality rates among reported confirmed cases from china and republic of korea figure 1 the authors declare no conflicts of interest acknowledgments ian m. mackay provided review and comments on a preliminary draft, source reference citation and tabular summary data on reported cases from china. donald j. kosiak provided review and comments on draft manuscript. the authors commend and thank the korea cdc for their tremendous efforts in tracking the course of the covid-19 pandemic, and for making detailed epidemiological data on cases confirmations publicly available. this is a model that other countries should follow. no corporate or institutional funding support was received for this work. key: cord-339004-49dkucxd authors: yu, ignatius tak-sun; qiu, hong; tse, lap ah; wong, tze wai title: severe acute respiratory syndrome beyond amoy gardens: completing the incomplete legacy date: 2014-03-01 journal: clin infect dis doi: 10.1093/cid/cit797 sha: doc_id: 339004 cord_uid: 49dkucxd the temporal and spatial distributions of the 2003 severe acute respiratory syndrome (sars) outbreak in amoy gardens of hong kong was reexamined using all confirmed cases. the outbreak actually extended to nearby residential complexes. airborne spread was the most likely explanation, and the sars coronavirus could have spread over a distance of 200 m. the temporal and spatial distributions of the 2003 severe acute respiratorysyndrome (sars) outbreak in amoy gardens of hong kong was reexamined using all confirmed cases. the outbreak actually extended to nearby residential complexes. airborne spread was the most likely explanation, and the sars coronavirus could have spread over a distance of 200 m. keywords. sars; outbreak; airborne. this year (2013) is the 10th anniversary of the pandemic of severe acute respiratory syndrome (sars) [1] , and we take this opportunity to update the medical community on what actually happened in the largest community outbreak in the amoy gardens in 2003. in 2004, we analyzed the temporal and spatial distributions of the initial 187 cases in 7 blocks (a-g) of amoy gardens, a residential building complex with 19 buildings, where the largest community outbreak of sars took place. coupled with computational fluid dynamics modeling on the 3-dimensional spread of a hypothetical plume of virus-laden aerosols, our analyses supported the probability of an airborne spread of the sars virus in that outbreak [2] . a year later, we reported the temporal-spatial distribution of sars among inpatients in a major nosocomial outbreak and concluded that the spread of sars in the ward was consistent with airborne transmission [3] . at the same time, a canadian group of researchers reported the detection of sars coronavirus in 2 air samples obtained from a room occupied by a patient with sars, which provided the first experimental confirmation of viral aerosol generation by a patient with sars, supporting the possibility of airborne transmission [4] . an earlier report on transmission of sars on aircraft also supported airborne spread [5] . it is now widely accepted that the sars coronavirus could be transmitted through aerosols under certain circumstances in medical facilities [6] , but fear of panic and political blame led to the reluctance of various health authorities in admitting airborne spread of sars in the community. there was also a query if sars spread beyond the amoy gardens through the air in 2003 [7] . in general, respiratory droplet spread can occur only through direct contact or at close distance (approximately 1 m), is less efficient, and has a lag time between generations of cases that is influenced by the incubation period and the infectious period, whereas in airborne spread, many cases can be affected within a short period of time by a common source and at distances much greater than 2 m. information on all confirmed cases of sars in hong kong was obtained from the centre for health protection, department of health, in hong kong, including date of symptom onset, occupation, and residential address. subjects were divided into 3 groups according to their proximity to the common source of sars coronavirus present in the amoy gardens: (1) amoy gardens residents, (2) where the amoy gardens was located. epidemic curves were produced for each group and compared. the spatial distribution of all cases in amoy gardens and surrounding residential buildings was located on a map, focusing on cases with symptom onset during the period 24-29 march 2003, that is, 5-10 days after the index patient visited a unit in block e of amoy gardens on march 19 and used the toilet there [2] . the index patient was receiving renal dialysis in the prince of wales hospital during the largest nosocomial outbreak [3] , had diarrhea, and was subsequently found to have very high viral load. the temporal distributions of dates of symptom onset for cases in the 3 groups during the period 20 march to 10 april 2003 are depicted in figure 1 . the peak of the epidemic curve for amoy gardens residents occurred on 24-25 march 2003. the numbers of cases in the surrounding buildings were much smaller, but a small peak could be seen on 25 march 2003. in contrast, no peaks of the epidemic curve among residents elsewhere in kt district were noted, and the number of cases was small. the proportion of cases with symptom onset during the period 24-29 march was 76.4% (253/331), 55.3% (57/103), and 16.2% (16/99), respectively, for amoy gardens residents, residents in the surrounding residential buildings, and residents elsewhere in kt district. the spatial distributions of all cases in amoy gardens and surrounding residential buildings are shown in figure 1 . amoy gardens had the largest number of cases (n = 331); only 12 of 331 cases in the amoy gardens occurred in the other 12 blocks (blocks h-s) outside the ring consisting of blocks a-g reported by us in 2004 [2] . a substantial number of cases also occurred in residential buildings near the amoy gardens (n = 103). in the lower ngau tau kok estate (lntke), a public housing estate with 14 large apartment buildings, 48 cases were reported. all but 1 case concentrated in the 7 buildings (blocks 8-14) situated directly downwind (southwest) from blocks a-g of amoy gardens. to the west of the lntke, across a major highway and railroad, is a huge residential complex (telford gardens) comprising 41 buildings where 6 cases were reported, 5 of which were from buildings directly facing lntke. furthermore, 14 cases were reported in another private housing estate (lee kee building) with 4 buildings about 30 m northwest of buildings c and d of amoy gardens. the next housing estate (wang kwong building) in the northwest direction with 3 buildings reported 8 cases. farther northwest, 3 cases were reported from the next housing estate of 3 buildings (jade field gardens) and 5 cases from the last housing estate with 8 buildings (tak bo gardens) in that direction (about 200 m from block e of amoy gardens). if only cases with symptom onset during 24-29 march were included, the directional localization of infected subjects became clearer, with an apparent gradient to the north/northwest (figure 2 ). no cases occurred in choi ha estate to the northeast of amoy gardens, and only 1 case occurred in upper ngau tau kok estate and 2 cases in lok wah estate to the south and southeast. the temporal similarity of the epidemic curves between surrounding buildings and amoy gardens suggested that the majority of cases in these 2 groups arose from the same common-source outbreak, and could not be adequately explained by person-to-person spread alone. it was impossible for the index patient in the amoy gardens outbreak to come into close contact with so many secondary cases within a short time, and a lag time of several days would have been expected before secondary sars patients in amoy gardens developed symptoms and then became infective [8] . common sources of contaminated food or water were not reported, and the only mode of spread that could adequately explain this commonsource outbreak was airborne transmission. analysis on the spatial distribution of cases showed strong directional preference, with residential buildings located to the north, west, and southwest of amoy gardens having many more cases than those to the east, southeast, and south, especially during the period 24-29 march. the prevailing northeasterly wind at the time could explain the spread to lntke and then telford gardens on the west/southwest of blocks a-g of amoy gardens. the spread to the residential buildings located to the north/northwest was in line with our earlier computational fluid dynamics modeling that showed a portion of the hypothetical virus-laden plume passing through the space between blocks c and d and spreading toward the northwest [2] . no cases occurred during the period 24-29 march in the 2 housing estates (kai tai court and kai yip estate) about 300 m northwest of telford gardens, which suggested that the spread through the wind did not extend that far, possibly due to too much dilution over the distance. considering that cases in telford gardens and tak bo garden with symptom onset during 24-29 march were most likely resulting from the common source originating in block e of amoy gardens, the distance of airborne transmission in that outbreak could be >200 m. the probable long-range airborne transmission of sars in the community should sound an alarm to health authorities of the world, and now is the right time for all to rethink the strategy for managing future emerging infectious diseases. infectious disease scientists should keep an open mind to all possible routes of transmission by objectively analyzing the available data, instead of emphasizing too much their historical understanding of models for person-to-person transmission. our results should be valid, as our dataset included all reported sars cases that were subsequently confirmed. although a few affected persons might not have stayed in the reported residential address, this should not cause serious misclassification. in conclusion, the airborne spread in the largest community outbreak of sars in 2003 actually extended beyond the amoy gardens and affected residential buildings >200 m from the source. admitting such long-distance transmissions through the air and wind would enable governments, public health authorities, and communities to be more adequately prepared for the next possible outbreak from any airborne virus. summary table of sars cases by country evidence of airborne transmission of the severe acute respiratory syndrome virus temporal-spatial analysis of severe acute respiratory syndrome among hospital inpatients detection of airborne severe acute respiratory system (sars) coronavirus and environmental contamination in sars outbreak units transmission of the severe acute respiratory syndrome on aircraft supplement c: preparedness and response in healthcare facilities, public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) version 2/3 evidence of airborne transmission of sars clinical progress and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study acknowledgments. the authors thank the centre for health protection of the department of health in hong kong for providing the data for this study.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-338899-qt17jhg0 authors: lakshmi, vemu; neeraja, mamidi; subbalaxmi, m. v. s.; parida, m. m.; dash, p. k.; santhosh, s. r.; rao, p. v. l. title: clinical features and molecular diagnosis of chikungunya fever from south india date: 2008-05-01 journal: clin infect dis doi: 10.1086/529444 sha: doc_id: 338899 cord_uid: qt17jhg0 an epidemic of chikungunya fever of unprecedented magnitude occurred in many parts of india in early 2006 after an interval of 33 years, and there has been a resurgence in some parts of south india since june 2007. the article highlights clinical manifestations of infection and various molecular tests that were used for diagnoses of chikungunya virus infection. of particular interest is the real-time loop-mediated isothermal amplification (rt lamp) assay, which is rapid and cost-effective and can be adopted at ill-equipped laboratories. clinical symptoms were characterized by a triad of fever, rash, and severe rheumatic manifestations. rt lamp identified 20 additional chikungunya virus—positive cases, compared with reverse-transcriptase polymerase chain reaction. chikungunya virus was isolated from 20 randomly selected samples. genotyping of the virus isolates revealed that the east central south african genotype of chikungunya virus was the etiologic agent of this epidemic. molecular diagnosis is an important tool to identify such new vectorborne viral illnesses. emerging viral infections have become a serious problem in recent years. emergence or reemergence of severe arboviral hemorrhagic fevers caused by mosquitoborne viruses, such as dengue virus and chikungunya (chik) virus, have been frequently reported in the indian subcontinent in the past few years. from the clinical perspective, these infections have similar clinical manifestations and are difficul to distinguish from one another. because the outcomes of these infections vary on the basis of the infecting agent (dengue has a high mortality rate), they pose a diagnostic dilemma for the clinician. therefore, there is a need for a means of definitiv diagnosis and identificatio of the viral agent to prognosticate the outcome. the causative agent chik virus, a single-stranded, positive sense rna, enveloped virus, is a member of the genus alphavirus of the togaviridae family. it is generally transmitted from primates to humans via aedes aegypti and aedes albopictus mosquitoes [1, 2] . chik infection produces a self-limiting illness in humans that is often characterized by sudden onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia, and severe and very painful polyarthralgia, which lasts for 1-10 days. however, arthralgia may persist for months to years [1, 3] . as is the case for most alphaviruses, detection of chik virus depends on isolation of the virus in blood specimens obtained from viremic patients or in infected tissue specimens obtained from blood-feeding arthropods, which are time-consuming. molecular diagnostic tools, such as the conventional rt-pcr, are available for the study of chik virus replication in virus culture supernatants or clinical samples [4, 5] . we report clinical observations and laboratory investigations involving virus isolation methods and molecular assays performed for 296 clinically suspected cases of chik fever. of particular interest was the applicability of a novel method of gene amplificatio called real-time loop-mediated isothermal amplifica tion (rt-lamp) as a rapid, sensitive, and specifi real-time method to detect and quantify chik virus in the acute phase of the infection. the study included 296 patients with a history of sudden onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia, and severe and very painful polyarthralgia suggestive of chik infection. patients either reported directly or were referred to nizam's institute of medical sciences (hyderabad, india) for treatment from regions in and around hyderabad, andhra pradesh, south india, during the period march-december 2006. there was no sampling bias or any attempt to specially recruit patients. the study was approved by the institutional ethics committee of nizam's institute of medical sciences (ec/ nims/675/2006). written informed consent was obtained from each patient. acute-phase serum samples were obtained during days 1-7 after the onset of symptoms. two sets of whole-blood specimens were collected from all 296 patients. one set was used for virus isolation, which was conducted using molecular assays with vacutainer edta tubes (bd biosciences); the other set underwent elisa with sst vacutainer tubes (bd biosciences). plasma and serum specimens were aliquoted in sterile vials and stored at ϫ80њc at the department of microbiology department at nizam's institute of medical sciences until testing. the samples were transported under cold chain to the virology laboratory at the defense and research establishment (gwalior, india), where all the assays and virus isolations were performed. serologic testing. sixty-fiv of 296 patients reported for follow-up. serum specimens obtained from these 65 patients were tested for the presence of chik virus-specifi igm and igg antibodies using an in-house dipstick elisa kit [6] . because symptoms of chik fever mimic those of dengue fever, a panel of 107 of 296 serum samples obtained from patients with clinical features similar to those of chik or dengue fever was included in the study. in addition, a panel of 20 serum samples obtained from healthy individuals without any signs and symptoms of chik or dengue fever was included as a negative control. virus isolation. virus isolation was attempted in c 6/36 cell lines from 32 rt-pcr-positive plasma samples that were randomly selected during the outbreak. virus isolation was performed using the virus adsorption technique [7] . in brief, a confluen monolayer of cells grown in a 25-cm 2 culture flas was adsorbed with 0.5 ml of inoculum at 37њc for 2 h. after adsorption, the inoculum was replenished with 8 ml of maintenance medium supplemented with 2% fetal bovine serum. suitable mock-infected cell controls were also incubated for comparison of cytopathic events. cells were incubated at 37њc and were observed daily for cytopathic effects. after observation of 80%-100% cytopathic effects, the infected culture supernatant was clarifie by light centrifugation at 2000 rpm for 10 min, which was further purifie by sucrose gradient ultracentrifugation. the isolated virus was confi med to be chik virus by rt-pcr. molecular assays. all 296 plasma samples were tested for the presence of chik virus-specifi rna by rt-pcr and rt-lamp. positive and negative controls were included in each run of the assays, and all precautions to prevent cross-contamination were observed. for rt-pcr, rna was extracted using the qiaamp viral rna mini kit (qiagen). one-step rt-pcr was performed using the access quick rt-pcr kit (promega), in accordance with the manufacturer's protocol, employing primer pairs targeting the e1 gene designed from the nucleotide sequence of the reference s27 strain (genbank accession number af490259; ck 13, tta cat cac gtg cga ta c; ck-14, ctt tc tct cag gg tgc gac ttt). the amplificatio was performed in a 50-ml total reaction volume with the promega access quick one-step rt-pcr kit, with 50 pmol of each forward and reverse primer and 2 ml of extracted viral rna, in accordance with the manufacturer's instructions. the thermal profil of rt-pcr was 48њc for 45 min and 94њc for 2 min, followed by 35 cycles of 94њc for 30 s, 54њc for 30 s, and 72њc for 30 s and a fina extension at 72њc for 10 min. rt-lamp was performed at a total 25-ml reaction volume using the loopamp rna amplificatio kit (eiken chemical). the real-time monitoring was accomplished by incubating at 63њc for 60 min in a loopamp real-time turbidimeter (la-200; teramecs). real-time monitoring of the rt-lamp amplificatio of chik virus template was observed through spectrophotometric analysis by recording the optical density at 400 nm every 6 s, with the aid of the loopamp real-time turbidimeter (la-200; teramecs). the cutoff value for positivity for the real-time rt-lamp assay was determined by taking into account the time of positivity (in min), at which point the turbidity increases to more than the threshold value (which was fixe at 0.1, which is 2 times more than the mean turbidity value for the negative controls of several replicates). after incubation at 63њc for 60 min, 10-ml aliquots of rt-lamp products were examined by electrophoresis on 3% nusieve 3:1 agarose gel (bma) in tris-borate buffer, followed by staining with ethidium bromide and visualization on a uv transilluminator at 302 nm. to facilitate the fiel application of the rt-lamp assay, the monitoring of rt-lamp amplificatio was also performed with naked-eye inspection. after amplification tubes were inspected for white turbidity by the naked eye after a pulse spin to deposit the precipitate in the bottom of the tube. the inspection for amplificatio was also performed by observing a color change after the addition of 1 ml of sybr green i dye to the tube. positive amplificatio is indicated by green fluo escence, which is permanent and which can be stored for recording purposes [7] . the rt-pcr-positive amplicons were subjected to doublestranded sequencing with big dye terminator cycle sequencing ready reaction kit on an abi 310 sequencer (applied biosystems). the genotype of the chik virus, based on the partial e1 gene sequence, was determined by nucleotide sequencing and compared with 30 other globally diverse chik isolates. a dendrogram was constructed by pair-wise comparison of 340 nucleotide sequences of partial e1 gene (positions 10254-10503, with respect to the s27 genome), which classifie all isolates into 3 different genotypes. the phylogenetic tree was constructed with the neighbor-joining method, with a bootstrap analysis of 1000 replicates, using mega software, version 2.1 [8] . the chik fever epidemic affected male and female patients at a ratio of 1:1.6. the most affected age group was persons aged 31-40 years (figu e 1). during the acute phase of infection, which lasted for 7-10 days, the most common symptoms among 296 patients were fever (temperature, 38.5њc-40њc) and severe arthralgia and arthritis, which affected the fingers wrists, toes, ankles, and knee joints (table 1). the chronic phase of infection was characterized by severe joint pain, which severely limited the patients' ability to walk and perform everyday tasks. almost 10% of cases reported experiencing prolonged arthralgia (duration, 13 weeks). chik infection has an important economic impact in many tropical countries, and because of the lack of specifi symptoms, the infection cannot be differentiated from dengue or yellow fever [1, 9] . a chik fever epidemic is characterized by its sudden disappearance for a considerably long period from a particular geographic area before its resurgence. this has been well documented in the republic of the congo and indonesia. however, in early 2005, a major epidemic of chik fever started in many indian ocean island nations, and in late 2005, it started spreading to several parts of india, after a hiatus of epidemic activity of nearly 32 years. this recent outbreak of chik infection in many parts of southern india is a point of major concern. this was the largest and most severe epidemic, affecting 11,000,000 persons in andhra pradesh, maharastra, and karnataka states of southern india and spreading to several new areas, with huge public health and administrative concerns to control the epidemic [10] . although the resurgence of chik fever was anticipated, this epidemic is considered to be unprecedented, owing to the magnitude of morbidity and geographical distribution. in several villages, 190% of inhabitants were found to be affected [7] , with an estimated figu e of 1.3 million cases to date [11] . in 2006, outbreaks of chik fever were reported from 194 districts in 12 states across india. the clinical manifestations of cases in the current outbreak match the known description of the disease. analysis of the 2006 outbreak suggested that the increased severity of disease may have been associated with a change in the genetic sequence, altering the virus coat protein, which potentially allowed the virus to multiply more easily in mosquito cells [12] . laboratory diagnosis is critical to establish the diagnosis and to initiate a specifi public health response. the alphavirus species can be characterized by hemagglutination inhibition, elisa, complement fixation and neutralization of viral infectivity using reference serum samples [1, 9] . serodiagnosis rests on demonstrating a 4-fold increase in chik virus igg antibody titer between the acute-and convalescentphase serum samples. because obtainment of paired serum samples is usually not practical, the demonstration of igm antibodies specifi for chik virus in acute-phase serum specimens is done. a chik-positive viral culture, coupled with neutralization by reference serum, is taken to be definitiv proof of the presence of chik virus. pcr results for e1 and c genome either singly or together constitute a positive result for chik virus [13] . in the present study, the detection of chik virus rna in 48.6% of samples by rt-pcr and in 55.4% of samples by rt-lamp, as well as the detection of igm antibodies in 21.5% of samples, confi med that the causative agent of this epidemic was chik virus. all 132 patients who had clinically suspected chik virus but whose rt-pcr and rt-lamp results were negative presented 17 days after the onset of fever; this may be the reason for the negative test results. the peak number of positive pcr results occurred on day 2 of illness (figu e 3). the rt lamp assay is a novel nucleic acid amplificatio method developed by eiken chemical that has the potential to replace pcr because of its simplicity, rapidity, specificit , and cost-effectiveness [14] [15] [16] . the rt-lamp assay has emerged as a powerful gene amplificatio tool for rapid identificatio of microbial infections and is being increasingly used by various investigators for rapid detection and typing of emerging viruses, such as the west nile, severe acute respiratory syndrome, dengue, and japanese encephalitis viruses [17] [18] [19] . the lamp method is cost-effective, because it requires only 1 type of dna polymerase with strand displacement activity. in the present study, the 1-step, single-tube, real-time accelerated rt-lamp assay was standardized by targeting the immunodominant e1 gene for rapid and real-time detection of chik virus. in our study, the rt-lamp assay uncovered 20 additional cases of infection, which were detected by naked eye or with a uv lamp. these samples had negative rt-pcr results, thereby proving the high sensitivity of rt-lamp. there were a few mismatches in the rt-pcr primers (i.e., 1 base in the forward primer and 2 bases in the reverse primer), but they did not affect the test's sensitivity [20] . all of the rt-pcr-positive samples also yielded positive rt-lamp results. the rt-lamp allows rapid, realtime detection of chik virus in acute-phase serum samples, without requiring sophisticated equipment, and has potential usefulness for clinical diagnosis and surveillance of chik virus in developing countries. real-time quantificatio of the chik virus level can also be performed by rt-lamp with use of a loop turbidimeter. isolation of chik virus from 20 of 32 rt-pcr-positive clinical samples further confi med that the infection was due to chik virus. previous phylogenetic studies showed that strains of chik virus were clustered into 3 distinct groups on the basis of origin: west africa, central/south africa, and asia [21] . the sequence of chik virus was directly determined from clinical samples without risk of altering the genome by in vitro passaging. mo-lecular phylogenetic analysis revealed that all of these new indian chik virus strains were very closely related to analogous strains from indian ocean island nations and that they formed a distinct clade in ecs african genotype. these new strains are reported to harbor many unique molecular features, making them more virulent and evolutionarily more competent [22] . the clustering of older indian isolates (including the last outbreak isolates) into the asian genotype indicates that the cause of this outbreak was the sudden introduction of the ecs genotype into the telangana region of andhra pradesh in southern india, rather than in situ evolution of existing strains. because the period of our study was march-december 2006, which is later than the period studied by prasanna et al. [23] and abu barkar et al. [24] , to the best of our knowledge (after an extensive search of the pubmed database), we submit that this is the firs report of ecs african genotype of chik virus as the causative agent of an unprecedented epidemic. a clustering of cases among members of the same family (40%) was observed in our study, which is in concordance with the findin that direct human-to-human transmission can occur as a consequence of high viral loads in patients, as was demonstrated in southern france [21] . although the viremia is transient, the concentration of virus is sufficien to infect feeding vector mosquitoes [25] . that the rt-pcr and rt-lamp results were positive during the period of peak viral load (i.e., days 1 and 2 of fever) also confi ms this fact. chik infection is a self-limited illness, with joint symptoms and signs usually lasting for months and occasionally for у1 year [25] . however, deaths due to chik infection are rare [25] . indiscriminate use of antibiotics and nonsteroidal anti-inflam matory drugs (especially aspirin) can contribute to thrombocytopenia, gastrointestinal bleeding, and vomiting. this may lead to prerenal acute renal failure and dehydration. these can indirectly contribute to mortality due to chik fever [26] . a high morbidity rate with no mortality was noted in our study. this is in contrast to the finding from kerala, southern india, where the cause of death for 3 patients with chik fever was probably an underlying illness and was not related to chik infection [27] . young adults (age, 21-40 years) were the most affected persons, and there was a preponderance of female patients in our study ( , by fisher's exact test); these finding are in p ! .001 concordance with the finding of the study from reunion island [28] . some patients (4.7%) were so disabled that they required hospitalization, but the number of such cases was negligible, and such cases mostly involved elderly patients. the chronic phase of disease resolved over a few months, extending up to 6 months. some patients improved after receiving short-term steroid treatment. because no specifi antiviral therapy exists for chik infection, treatment consists of supportive care, including administration of analgesics and anti-inflammato y medication for joint symptoms. persons with febrile illness that is suspected to be due to chik virus should avoid mosquito exposure for at least 7 days after the onset of illness, to reduce the likelihood of transmitting chik virus to local mosquitoes, which might then transmit the virus to other humans [24] . although the number of cases being reported has decreased, this epidemic may still be continuing and spreading. therefore, continuous surveillance is warranted to monitor the spread of infection and to track the possible evolution of the virus during the epidemic. the natural history of chik fever is not fully understood. although mortality is rare or infrequent, early diagnosis and vector control will play an important role in preventing the outbreak of epidemics in the future. intraoutbreak studies point toward recent changes in the viral genome that have facilitated the rapid spread and enhanced pathogenecity of infection [29] . the lack of herd immunity, as evidenced from available studies [30, 31] , appears to be the simplest attributable factor. also, the reasons for the current outbreak and the causes behind reemergence of the virus in india have to be further assessed. molecular diagnosis is an important tool to identify new vectorborne viral illnesses, such as chik fever, at an early stage. public health measures need to be improved to prevent such epidemics in the future. there is also an immediate need for an effective vaccine for chik infection. the arboviruses: epidemiology and ecology complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site the alpha viruses combined detection and genotyping of chikungunya virus by specifi reverse transcriptionpolymerase chain reaction detection of west nile and japanese encephalitis viral genome sequences in cerebrospinal flui from acute encephalitis cases in karachi genome microevolution of chikungunya viruses causing the indian ocean outbreak rapid and real-time detection of chikungunya virus by reverse transcription loop-mediated isothermal amplificatio assay molecular evolutionary genetics (mega) software development of monoclonal antibody based antigen capture elisa to detect chikungunya virus antigen in mosquitoes re-emergence of chikungunya virus in india failure to control mosquitoes has led to two fever epidemics in india chikungunya doctor ndtv health information on chikungunya communicable diseases branch. chikungunya fever, laboratory diagnosis of chikungunya fevers. geneva: world health organization detection of loop mediated isothermal amplificatio reaction by turbidity derived from magnesium pyrophosphate formation accelerated reaction by loop mediated isothermal amplificatio using loop primers loop-mediated isothermal amplificatio of dna development and evaluation of a novel loop mediated isothermal amplificatio method for rapid detection of severe acute respiratory syndrome corona virus real-time reverse transcription loop mediated isothermal amplificatio for rapid detection of west nile virus evaluation of a dipstick elisa and a rapid immunochromatographic test for diagnosis of dengue virus infection east central south african genotype as the causative agent in reemergence of chikungunya outbreak in india novel chikungunya virus variant in travelers returning from indian ocean islands genome microevolution of chikungunya viruses causing the indian ocean outbreak chikungunya outbreaks caused by african genotype reemergence of endemic chikungunya update: chikungunya fever diagnosed among international travelers-united states chikungunya fever: clinical manifestation and management infectious diseases surveillance update chikungunya outbreak in reunion: epidemiology and surveillance emergence of chikungunya virus in indian subcontinent after 32 years: a review serological survey in madras city with special reference to chikungunya serosurvey of chikungunya antibody in calcutta metropolis potential conflict of interest. all authors: no conflicts key: cord-317092-5qba9jiq authors: singh, tulika; heston, sarah m; langel, stephanie n; blasi, maria; hurst, jillian h; fouda, genevieve g; kelly, matthew s; permar, sallie r title: lessons from covid-19 in children: key hypotheses to guide preventative and therapeutic strategies date: 2020-05-08 journal: clin infect dis doi: 10.1093/cid/ciaa547 sha: doc_id: 317092 cord_uid: 5qba9jiq the current pandemic of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of coronavirus disease 2019 (covid-19), reveals a peculiar trend of milder disease and lower case fatality in children compared to adults. consistent epidemiologic evidence of reduced severity of infection in children across different populations and countries suggests there are underlying biologic differences between children and adults that mediate differential disease pathogenesis. this presents a unique opportunity to learn about disease modifying host factors from pediatric populations. our review summarizes the current knowledge of pediatric clinical disease, role in transmission, risks for severe disease, protective immunity, as well as novel therapies and vaccine trials for children. we then define key hypotheses and areas for future research that can use the pediatric model of disease, transmission, and immunity to develop preventive and therapeutic strategies for people of all age groups. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) originated in wuhan, china in december 2019, and was deemed a public health emergency of international concern by the world health organization. 1 sars-cov-2 is the etiological agent of the disease known as coronavirus disease 2019 (covid19) , which is characterized by fever, cough, dyspnea, and progression to acute respiratory distress syndrome (ards). in the four months since its identification, sars-cov-2 has led to more than 3 million cases and 228,000 deaths globally. 2 sustained community-based spread is constraining healthcare resources, shutting down economies, and leading to unprecedented governmental recommendations for quarantining and social distancing to limit transmission. while these measures are necessary to slow the rate of new infections, they have been highly disruptive to society and other preventative and therapeutic approaches are urgently needed. surprisingly, epidemiological evidence across countries consistently reveals that children experience less severe disease and lower case fatality from covid-19 than adults. 1, 3, 4 this trend suggests that there are underlying biological differences between children and adults that could inform the development of therapeutics, and preventative measures. recent cohort studies indicate that only up to 6% of infected children experience severe disease, whereas up to 26% of adult cases progress to severe illness requiring icu admission. 5, 6 notably, a similar trend of mild disease and low mortality rate in children was observed during the severe acute respiratory syndrome (sars-cov-1) outbreak in 2003 and middle east respiratory syndrome (mers)-cov outbreak in 2012, indicating that this pattern is driven by common virologic features across coronaviruses (cov). 7, 8 also, varicella disease is similarly known to be milder in young children compared to infants and adults. 9 in contrast, most other respiratory viruses, such as influenza and respiratory syncytial virus, cause more severe disease in young children compared to middle-aged adults 10 . this presents a unique opportunity to learn about disease-a c c e p t e d m a n u s c r i p t 4 modifying host factors to inform our understanding of cov pathogenesis across age groups. understanding differences in children's immunity, host cellular factors required for virus replication, and physiology can provide insights into the correlates of protection from sars-cov-2 and other covs. in this review, we summarize current pediatric-specific knowledge on clinical disease, transmission, risks for severe disease, protective immunity, and novel therapies and vaccines in trial. importantly, we identify key unanswered questions in translating this evidence towards the development of preventive and therapeutic interventions for all ages (table 1) . currently available clinical descriptions of covid-19 consistently describe milder symptoms in children than that of adults. while children constitute 22% of the united states population, they only represent 1.7% of sars-cov-2 infections identified to date, consistent with estimates from china. 11 yet, as more pediatric studies have become available, it is clear that children from birth to 18 years can be infected with sars-cov-2. 6, 12 infected children appear to be less symptomatic, and thus less likely to be tested for the virus in the setting of limited diagnostic capacity. while definitions of clinical severity vary among studies, there are consistently fewer severe or critical cases among children than adults. in a retrospective review of over 2,000 pediatric cases in china, only 6% of cases were severe (112 cases) or critical (13 cases). 6 moreover, in a case series of 36 children in china from whom sars-cov-2 was detected, 28% were asymptomatic. 13 in contrast, severe and critical cases represent up to 19% and 26% of adult cases reported in china and italy, respectively, and occur mostly in people >60 years of age. 1, 4 a c c e p t e d m a n u s c r i p t 5 the most common symptoms of covid-19 include fever and cough, with fewer patients experiencing shortness of breath, upper respiratory symptoms, vomiting, diarrhea, myalgias, and fatigue. 11, 12 interestingly, only 56% of symptomatic children had fever and 54% had cough, while fever and cough were identified in 71% and 80% of adults, respectively. 11 laboratory and radiographic abnormalities are also less common in children. while lymphopenia, elevated creactive protein (crp), and abnormal coagulation tests are common in adults and correlate with disease severity, there are no consistent laboratory abnormalities across pediatric studies. 14 however, laboratory abnormalities that more closely reflect those of adults have been reported in children >5 years of age and adolescents. 13, 15 in both adults and children, ground glass opacities and "patchy shadows" were the most common abnormalities on chest computed tomography. 16 altogether, the differences in symptoms and disease severity between children and adults with covid-19 imply that there are potential immunological or host factors that modulate disease in children. children less than 15 years of age are primarily exposed to sars-cov-2 through close contact with a sick family or household member, although exposure may also occur with travel to an endemic area or contact with other infected individuals. 11 while transmission primarily occurs through aerosolized droplets and fomite contact, there is concern that fecal-oral transmission may also occur, particularly in children. in epidemiological investigations, viral rna was detected in the stool of 8 of 10 children who tested positive for the virus via nasopharyngeal swab. 17 moreover, virus was detected in stool up to 27 days after admission, compared with up to 15 days via nasopharyngeal swab and at higher magnitude of viral rna detected in stool as compared to nasopharyngeal samples, however more studies are needed to determine if detection of viral rna correlates to infectious virus in stool. 17 notably, recent reports identified viable virus in fecal samples from adult patients. 18 given the large proportions of asymptomatic a c c e p t e d m a n u s c r i p t 6 pediatric infections, lower severity of disease, and potential risk of fecal-oral transmission, it is highly likely that children have a distinct role in population transmission. development of reliable and specific serological tests for sars-cov-2, such as those based on binding of serum antibodies to the viral spike protein, are important for accurate detection of rates of infection in children 19 . the possibility of vertical transmission remains of concern for maternal and neonatal health. in a case series of 33 neonates born to mothers with covid-19 pneumonia, 3 presented with early onset of neonatal infection identified by detection of the virus by pcr in nasopharyngeal samples, and are suspected cases of perinatal transmission. 20 also, amongst other cohorts, 17 infants born to sars-cov-2 positive mothers did not demonstrate evidence for vertical transmission 21, 22 . however, elevated sars-cov-2 igm antibodies detected in serum taken within two hours of birth from three newborns, despite negative testing of nasopharyngeal samples by pcr 21,23,24 is also suggestive of in utero sars-cov-2 exposure. 21, 23, 24 nevertheless, these cases could represent false-positive igm testing, as has been reported frequently with serological testing for other viruses. 25 thus far, there is no report of detection of sars-cov-2 in amniotic fluid or breast milk, and it is unclear if vertical transmission occurs when pregnant women become infected during the first or second trimester of gestation. 21 maternal infection can also lead to severe symptoms in the mother, which can result in birth asphyxia or premature birth. 21 in sars-cov-1, there was a higher case fatality among pregnant women and reported cases of miscarriage, spontaneous abortion, preterm birth and intrauterine growth restriction. 26 further research is needed to understand the impact of sars-cov-2 infection on maternal and fetal health. while children represent a minority of severe covid-19 cases, a third of the reported severe cases and more than half of the critical cases were among children less than one year of a c c e p t e d m a n u s c r i p t 7 age. 6 children less than 1 year old also had the lowest percentage of asymptomatic cases as compared to older children. 6 an interesting observation in adults is that slightly higher rates of severe disease have been reported in men than women. 27 similarly, of the >4000 pediatric cases reported in the us and china, 57% were male; however, there are currently no reports of sex differences related to disease severity in children 6, 11 further analysis is required to determine whether a sex bias exists in severe pediatric sars-cov-2 infections. future studies will need to continue examining sex and age-related differences in covid-19 severity as this might provide insights into host factors that mitigate severe disease outcomes. moreover, studies should consider whether physiologic changes during puberty underlie age-dependent disease modifying factors in children 28 . the presence of medical comorbidities, such as hypertension, diabetes, chronic pulmonary disease, and cardiovascular disease is another risk factor for severe disease in adults 29 , and the relative lack of comorbidities in children may contribute to the disparate covid-19 severity between the age groups. of the few reports of severe covid-19 disease in children, all three critical cases had a significant underlying or concurrent medical condition, including acute lymphoblastic leukemia (all), hydronephrosis, and intussusception. 30 however, it should be noted that none of the five severe cases had significant comorbidities. 30 given the low prevalence of severe and critical disease in children, it is difficult to determine the contribution of pre-existing comorbidities to covid-19 severity. specifically, underlying medical issues such as prematurity, chronic lung disease, congenital heart disease, asthma, and even lung injury from vaping and smoking, may result in an increase in the risk for severe covid-19 disease. intriguingly, there are few reports of severe disease in immunocompromised patients with covid-19 despite receipt of immunosuppressive agents and chemotherapies. while data are limited to small cohort studies, adult renal transplant recipients tended to have a typical a c c e p t e d m a n u s c r i p t 8 covid-19 course, while adults with malignancy had more severe disease if they had recently received chemotherapy or underwent surgery. 31, 32 of immunocompromised sars-cov-2 infected children, the aforementioned child with all developed critical disease, but only mild to moderate disease has been observed in pediatric liver transplant recipients. 30, 33 the surprisingly mild course of covid-19 in immunocompromised patients could allude to the substantial role that the host immune system plays in the development of severe disease. protection from severe disease in children may be related to lower expression of host factors required for viral replication, and to differences in the magnitude and timing of innate or adaptive immune responses. host factors: sars-cov-2 uses the angiotensin-converting enzyme 2 (ace2) as a cell entry receptor and the cellular transmembrane protease serine 2 (tmprss2) to activate the spike (s) viral protein for membrane fusion. 34 ace2 modulates vasoconstriction to maintain homeostasis and is expressed in the oral mucosa, respiratory tract, and intestine. 35,36 lower ace2 expression in the lungs of children as compared to adults could contribute to the observed differences in disease pathogenesis across these groups. 37 however, given the large variability in human ace2 expression profiles, further studies are required to confirm differences across age groups. 37 there are also age-dependent differences as lungs develop throughout childhood 38 . in particular, processes that impact the course of lung pathology and respiratory distress such as inflammation, apoptotic activation, surfactant secretion, alveolar fluid clearance, and tissue repair mechanisms differ in children compared to adults. 38 for example, a regulator of lung morphogenesis that is lower in childhood, nuclear factor kappa-light-chainenhancer of activated b cells (nf-b), plays a pathologic role in inflammatory diseases and should be evaluated as a protective host factor in pediatric versus adult sars-cov-2 infections. 38, 39 indeed, even outside the context of sars-cov-2 infections, rates of ards are a c c e p t e d m a n u s c r i p t 9 lowest in children and increase with age, suggesting a role for protective host factors in the lungs of children. 40, 41 innate and adaptive immunity: th1 responses are thought to be important for immune protection against sars-cov-1 since increased th2 cytokines were identified in patients with fatal disease. 42 however, excess th1 pro-inflammatory cytokine responses and circulating neutrophil levels are also associated with increased disease severity and delays in regulatory and repair responses. 43, 44 in fact, over expression of serum il-6 is associated with severe disease and mortality due to sars-cov-2 infection suggesting that aging-related inflammation may contribute to disease severity in elderly. 45 whereas, children who recovered from sars-cov-1 infection demonstrated elevated plasma il-1β but not tnf-α or il-6 early in infection, suggesting a less destructive disease pathology. 46 descriptions of lung pathology from sars-cov-1 and sars-cov-2 fatalities reveal that macrophages are the predominant leukocyte infiltrate in the alveoli 43, 47 . higher prevalence of macrophages in the alveoli may be due to prolonged il-6 inflammation, in combination with monocyte chemoattractant protein-1 (mcp-1) expression, which induces a transition from neutrophil activation in early inflammation to monocyte accumulation in late inflammation 48 . interestingly, lower levels of il-6 and mcp-1 are observed in the lungs of children who survive ards compared to adults. 37 although neutrophils are associated with lung pathology during ards, the role for lung neutrophils in covid-19 severity remains unclear. 49 neutrophil depletion in rodent models of respiratory viral infections such as sars-cov-1, influenza, and respiratory syncytial virus leads to worse clinical outcomes and higher levels of viral replication, suggesting that neutrophils may serve a protective function during these infections 38, [50] [51] [52] [53] . thus, the role for neutrophils and macrophages in sars-cov-2 infections needs to be evaluated further and compared between children and adults. effective immune responses to cov require regulated th1 immunity for viral control and infected cell killing, followed by regulatory signaling that mediate tissue repair 54 . intriguingly, children a c c e p t e d m a n u s c r i p t 10 experience less leukopenia during sars-cov-2 infection than adults and have a relatively higher level of circulating lymphocytes compared to neutrophils, which may contribute to better viral control during acute infection. 5, 12, 55 thus, milder sars-cov-2 infection in children may be driven by intrinsically lower levels of inflammation, higher lymphocyte to neutrophil ratio in blood, and less predominantly monocytic infiltration than adults. recent reports have demonstrated that neonates less than 1 year of age (<1 year) are more susceptible to severe covid-19 disease compared to older children (1-18 years). 6 dynamics of t cell-mediated immunity may contribute to the increased covid-19 severity in adults and neonates (<1 year) compared to the milder disease observed in children (<18 years). for example, virus-specific cd8+ t cells play an important role in viral clearance by directly killing infected cells, but excess cytolytic activity can also mediate lung pathology 56 . the observed increase in lung pathology in both infants <1 year and older adults may be due to inappropriate levels of t cell activity. indeed, in infants <1 year t cell activation is decreased and effector responses are characterized by th2 cytokine secretion as infants transition from tolerogenic fetal immunity 57 . in contrast, higher inflammation associated with aging can lead to t cell exhaustion, which is linked with severe covid-19 disease. 58 in comparison, children between 1-18 years may experience an intermediate level of t cell activation, leading to milder sars-cov-2 disease. 59 also, an age-dependent increase in lung prostaglandin production may play a role in sars-cov-2 pathogenesis. for example, in mice lung prostaglandin concentrations correlated with decreased dendritic cell migration and t cell responses and greater sars-cov-1 induced lung pathology with age. 60 further examination of children t cell immunity during sars-cov-2 infection compared to adult responses is required. our understanding of protective humoral responses to cov infections comes from prior studies of sars-cov-1, and ongoing studies on the current sars-cov-2 pandemic. typically, neutralizing antibody responses against the immunodominant s viral protein are elicited after a c c e p t e d m a n u s c r i p t 11 two weeks of infection and can protect from challenge in animal models. 61 yet, high magnitude and early (< 2 week) peak neutralizing antibody responses were associated with more severe disease in sars-cov-1 infection, indicating antibody responses may also be related to disease pathology. 62 moreover, the sars-cov-2 s protein contains neutralizing and non-neutralizing epitopes and is 76% identical to sars-cov-1 s at the amino acid level. 63 heterologous strains, has led to eosinophilic lung immunopathology. 76, 77 intriguingly, young mice showed less immunopathology as compared to aged mice, indicating that age of vaccination may impact safety profile. though it is unclear whether lung immunopathology reflects enhanced disease in humans, whole virus vaccine platforms must be carefully evaluated for safety. lack of vaccine candidates with a proven safety and/or immunogenicity profile represents a gap in translating these technologies to pediatric populations during a pandemic. while rapidly testing candidates, it will be crucial to consider the earliest possible stage for inclusion of children in vaccine trials. a key question for vaccine development in the current pandemic is the possibility of reinfection with sars-cov-2. prior studies indicate that reinfection may be possible after several years, since sars-cov-1 neutralizing antibody titers reduced substantially 3 years after exposure and virus-specific memory b cells were undetectable 6 years after infection. 78, 79 further, virus-specific memory t cells were undetectable by 6 years post infection in 40% patients who recovered from sars-cov-1 infection. 79 therefore, it will be important to assess if a c c e p t e d m a n u s c r i p t 13 sars-cov-2 immunity in children lasts longer than that of adults, which would indicate that childhood represents an opportune period for vaccination to elicit life-long protection. also, differential waning of vaccine immunity in adults and children should be evaluated to optimize age of vaccination and develop boosting strategies to provide long term protective immune responses. two leading antivirals are currently being tested in patients with covid19. remdesivir is an intravenously delivered investigational antiviral that that is being tested in several randomized controlled clinical trials globally, largely in adults with moderate or severe covid-19. 80 remdesivir is a nucleoside analog that inhibits cov replication by terminating the rna genome transcription. 81 assessments in children are underway to determine optimal pediatric dosing. another option being tested is hydroxychloroquine, an approved oral antimalarial drug that is also used for rheumatoid arthritis and systemic lupus. while hydroxychloroquine demonstrates high antiviral activity in-vitro, underpowered clinical trials have indicated virologic control but no significant effect on clinical outcomes in patients with severe disease. 82 in addition to antivirals, passive immunization strategies using convalescent plasma and purified immunoglobulins to limit virus replication and abrogate disease progression are under investigation. reports of successfully treating critically ill covid-19 patients with convalescent plasma from recovered individuals has enabled approval for emergency use in the us for cases of serious and life threatening covid-19. 83 meta-analysis of this therapy for sars-cov-1 suggests that this intervention appears safe and reduces mortality. 84 however, since antiviral potency of plasma may vary by donor, it is important to determine the characteristics of plasma that support efficacy and optimal prognosis. for example, poor treatment outcomes for sars-cov-1 patients were observed when convalescent plasma intervention was administered during pcr-positivity and before the 14 th day of illness. 85 these observations allude to the relationship a c c e p t e d m a n u s c r i p t 14 between viral dynamics and igg-mediated pathology that may differ between adults and children. the current covid-19 pandemic has resulted in more than 3 million cases worldwide, and the lack of protective vaccines and specific antiviral therapies to prevent severe disease has resulted in more than 228,000 deaths 2 . a pattern of milder covid-19 in children in compared to adults offers a unique opportunity to identify protective host and immunologic factors within pediatric populations and apply findings to the design of interventions for all ages. in this review, we evaluated recent reports on the pathology and immunity to sars-cov-2 infection and offered several hypotheses for how these features may differ in children versus adults, and how they may differentially modulate disease in these populations. further understanding of the pathogenesis of sars-cov-2 infection in children may provide important insights and guide development of therapeutic strategies and vaccines as we collectively strive to generate approaches to reduce the public health burden of sars-cov-2 pandemic. a c c e p t e d m a n u s c r i p t 15 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention an interactive web-based dashboard to track covid-19 in real time severe outcomes among patients with coronavirus disease 2019 (covid-19) -united states sorveglianza integrata covid-19 in italia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china. pediatrics severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines middle east respiratory syndrome coronavirus disease in children chapter 22: varicella, epidemiology and prevention of vaccine-preventable diseases mortality associated with influenza and respiratory syncytial virus in the united states coronavirus disease 2019 in children united states sars-cov-2 infection in children clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study clinical characteristics of coronavirus disease 2019 in china laboratory abnormalities in children with novel coronavirus disease 2019 clinical and ct features in pediatric patients with covid-19 infection: different points from adults characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding covid-19: gastrointestinal manifestations and potential fecal-oral transmission a serological assay to detect sars-cov-2 seroconversion in humans. medrxiv neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china. jama pediatr clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia antibodies in infants born to mothers with covid-19 pneumonia possible vertical transmission of sars-cov-2 from an infected mother to her newborn immunoglobulin m for acute infection: true or false? pregnancy and perinatal outcomes of women with severe acute respiratory syndrome clinical progression of patients with covid-19 in shanghai sex differences in pediatric infectious diseases prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis clinical features of severe pediatric patients with coronavirus disease 2019 in wuhan: a single center's observational study cancer patients in sars-cov-2 infection: a nationwide analysis in china covid-19 in kidney transplant recipients the facts during the third epidemic -d'antiga --liver transplantation -wiley online library sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis age-dependent differences in pulmonary host responses in ards: a prospective observational cohort study mechanisms of acute respiratory distress syndrome in children and adults age-associated changes in basal nf-κb function in human cd4+ t lymphocytes via dysregulation of pi3 kinase incidence and outcomes of acute lung injury incidence and outcomes of pediatric acute lung injury cell responses to whole sars coronavirus in humans lung pathology of fatal severe acute respiratory syndrome neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019 novel coronavirus in the early stage clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intensive care med inflammatory cytokine profile in children with severe acute respiratory syndrome pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer interleukin-6 and chronic inflammation contribution of neutrophils to acute lung injury neutrophils do not impact viral load or the peak of disease severity during rsv infection neutrophils ameliorate lung injury and the development of severe disease during influenza infection neutrophils promote mononuclear cell infiltration during viral-induced encephalitis neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice immune system development during early childhood in tropical latin america: evidence for the age-dependent down regulation of the innate immune response virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection evolution of the immune system in humans from infancy to old age evaluation of nucleocapsid and spike protein-based elisas for detecting antibodies against sars-cov-2. medrxiv differences between pediatric and adult t cell responses to in vitro staphylococcal enterotoxin b stimulation age-related increases in pgd 2 expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice serological responses in patients with severe acute respiratory syndrome coronavirus infection and cross-reactivity with human coronaviruses 229e, oc43, and nl63 neutralizing antibody response and sars severity immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov hepatitis b immunisation induces higher antibody and memory th2 responses in new-borns than in adults impact of baseline covariates on the immunogenicity of a quadrivalent (types 6, 11, 16, and 18) human papillomavirus virus-like-particle vaccine early development of broadly neutralizing antibodies in hiv-1-infected infants infant hiv type 1 gp120 vaccination elicits robust and durable anti-v1v2 immunoglobulin g responses and only rare envelope-specific immunoglobulin a responses hiv exposed infants vaccinated with a mf59/rgp120 vaccine have higher magnitude anti-v1v2 igg responses than adults immunized with the same vaccine with record-setting speed, vaccinemakers take their first shots at the new coronavirus. science (80-) rapid development of an inactivated vaccine for sars-cov-2 rapid development of a synthetic dna vaccine for covid-19 under rev the covid-19 vaccine development landscape sars-cov-2 vaccines: status report a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus. poehlmann s duration of antibody responses after severe acute respiratory syndrome lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial treatment of 5 critically ill patients with covid-19 with convalescent plasma the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis use of convalescent plasma therapy in sars patients in hong kong none. a c c e p t e d m a n u s c r i p t 21 m a n u s c r i p t 24  will antivirals suppress viral load in vivo as well as lower clinical pathology?  in which target population will the vaccine be most effective and durable?  what will be the effect of pre-existing immunity and maternal antibody on the vaccine?  can children respond most effectively to this vaccine, and will infancy be the optimal timing to achieve lifelong protection?  how soon can we include vulnerable populations including pregnant women, neonates, and children in the vaccine development process to optimally tailor vaccine design to these populations?  can we leverage understanding of protective pediatric immunity and pathophysiology to guide design of therapeutic targets and vaccines? key: cord-344038-20n74z3o authors: han, mi seon; seong, moon-woo; heo, eun young; park, ji hong; kim, namhee; shin, sue; cho, sung im; park, sung sup; choi, eun hwa title: sequential analysis of viral load in a neonate and her mother infected with sars-cov-2 date: 2020-04-16 journal: clin infect dis doi: 10.1093/cid/ciaa447 sha: doc_id: 344038 cord_uid: 20n74z3o we report changes in viral load over time in a 27-day old neonate with covid-19 who presented with fever, cough, and vomiting. sars-cov-2 rna was detected in the nasopharynx, oropharynx, stool, saliva, plasma, and urine. the highest viral rna copies in nasopharynx decreased over time while viral load in stool remained high. the coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) started in china and rapidly spread worldwide, resulting in a pandemic [1] . as covid-19 cases surge, the number of children with covid-19 is also on the increase. since the report of the first pediatric case in korea, 619 cases aged <20 years (6.46% of the total cases) with covid-19 have been reported, as of march 29, 2020 [2, 3] . limited reports from china described the clinical manifestation of children with to be mild, and asymptomatic infections are not uncommon [4, 5] . however, it is difficult to infer whether neonates under 28 days of age with covid-19 follow a similar clinical course because the immune system in early life is unique. currently, only three descriptive studies on neonates with covid-19 have been reported, and to our knowledge, none have investigated viral dynamics in infected neonates [6] [7] [8] . in this study, we described the clinical manifestation of covid-19 in a neonate and her mother, and further analyzed the viral load kinetics of sars-cov-2 in clinical specimens from different sources. 4 a 27-day old neonate and her mother were diagnosed with covid-19 and hospitalized at seoul metropolitan government-seoul national university (smg-snu) boramae medical center on march 8, 2020. their medical records including symptoms and signs, laboratory examination, results of sars-cov-2 tests, radiologic findings, and management were reviewed. the exposure route to sars-cov-2 was described based on the report by the local government and the history taken from the mother. rna of the clinical specimens was extracted by using the magna pure 96 dna and viral the 27-day old baby girl was born by vaginal delivery on february 11, 2020 at 38 weeks and 6 days' gestation with a birth weight of 3.73 kg. the neonate lived at her grandparents' house with her parents and two older siblings. she was directly breastfed from birth. on march 2, 2020, both of her grandparents started to cough and noticed sputum. on march 4, 2020, her mother reported sputum production and a sore throat, followed by chills and myalgia on the next day when the neonate developed nasal stuffiness. the baby's father reported chills and a sore throat on the same day and was confirmed with covid-19 on march 7, 2020. accordingly, the remaining family members were all tested for covid-19, and the neonate and her mother along with her grandparents were confirmed with the diagnosis. the two older siblings tested negative. as the neonate and her mother had not left home since her birth, sars-cov-2 seemed to be transmitted from one of the family members, the source of whose infection remains unknown. on march 8, 2020, the neonate was hospitalized as she was too young and her mother was admitted in the same isolation room to take care of her. on admission, the neonate had mild fever of 37.6°c and nasal stuffiness. blood pressure was 82/53 mmhg, heart rate 145/min, respiratory rate 62/min, and spo 2 95%. whole body jaundice was observed on physical examination. lung sounds were clear on auscultation and the abdomen was soft with normoactive bowel sound. her jaundice was presumed to be breast milk jaundice, which spontaneously resolved within 2-3 days. the neonate developed a fever up to 38.4°c along with tachycardia from the 2 nd hospital day and fever lasted for two days. she also had increased frequency of vomiting. from the 3 rd hospital day, she started to have a mild cough 6 yet did not show any signs of respiratory difficulty and was stable without requiring oxygen. no lung lesions were observable on her chest radiographs serially taken on the 1 st , 3 rd , and 5 th hospital days. the neonate's laboratory examination was unremarkable (see supplementary table) . no organisms grew on blood culture and urinalysis was normal. as the neonate remained well, no antiviral or antibacterial agents were administered. she fed well and continuously gained weight. the neonate's mother remained afebrile and the only complaint she had was a sore throat with mild sputum production. laboratory findings were normal and serial chest radiographs were unremarkable. as both the neonate and her mother's viral test results were negative from two consecutive nasopharyngeal swab specimens collected ≥24 hours apart, they were discharged home on march 26, 2020. sars-cov-2 rna was detected in the neonate's clinical specimens from several sources including the nasopharynx, oropharynx, plasma, urine, stool, and saliva specimens ( figure 1a ). at the early stage of the infection, the viral load was highest in the nasopharynx (1.2 x10 10 copies/milliliter) followed by oropharyngeal swab (1.3 x10 8 copies/milliliter). the viral load in the respiratory specimens gradually decreased with time and was undetectable after 17 days from the onset of symptoms. notably, the sars-cov-2 rna in the stool sample remained high (range, 1.7 x10 6 -4.1 x10 7 copies/milliliter) until the 18 th day since the onset even though the neonate's gastrointestinal symptoms improved. the neonate also excreted the virus in urine at relatively low rna copy numbers for more than 10 days. the viral load of the mother's respiratory and stool specimens was approximately 100-fold lower than that of the neonate's on the 10 th day from symptom onset ( figure 1b) . the mother's plasma and urine specimens were tested negative for sars-cov-2. the virus was also not detected in her breast milk. 7 this study described the viral load kinetics of a neonate, the youngest covid-19 patient in korea as of march 29, 2020, and her mother. the neonate was febrile and sars-cov-2 rna was detected in all of her clinical specimens, with high viral loads in the respiratory and stool samples. her mother had mild symptoms with sars-cov-2 rna detected in the respiratory and stool specimens at low titers. fortunately, the neonate as well as her mother recovered well without antiviral therapies. limited reports on neonates with covid-19 have been published, all from wuhan, china [6] [7] [8] . a 17-day-old neonate had mild fever, sneezing, intermittent vomiting, and diarrhea [6] . the four other neonates were diagnosed with covid-19 shortly after the birth from mothers confirmed with covid-19, and it remains unclear whether the cases were from intrauterine transmission or not [7, 8] . the neonates had mild symptoms and their clinical outcomes were favorable. an interesting finding in this study is that sars-cov-2 rna was detected in all of the neonate's clinical specimens, including blood, urine, stool, and saliva along with the upper respiratory tract specimens. in comparison, although exposed to the same infection source, only the mother's respiratory and stool specimens were positive for sars-cov-2 and at a much lower viral load. these findings suggest that covid-19 could be systemic in neonates, affecting multiple organs, including the kidney and the gastrointestinal tract. only approximately 1-15% of the adult patients with covid-19 had rnaemia, and no child with rnaemia has been reported so far [5, 9, 10] . to fight off virus infections in the absence of maternally transmitted igg antibody, neonates must rely exclusively on their immature innate immune system and their own, also immature, t cells [11] . this makes them vulnerable to viral infections, including sars-cov-2. although previously reported neonates with 8 covid-19 went through favorable clinical courses, careful monitoring on this specific population at high risk is still needed until more data are available. recent studies have reported that sars-cov-2 rna could be detected in different types of clinical specimens other than respiratory tract samples [9] . especially, stool samples could be positive for sars-cov-2, irrespective of the presence of gastrointestinal symptoms, and remain positive even for one month [12] . the viral load in this neonate's stool specimen world health organization. coronavirus disease (covid-19) situation report-51 first pediatric case of coronavirus disease 2019 in korea korea centers for disease control & prevention. the updates of covid-19 in republic of korea sars-cov-2 infection in children a case series of children with 2019 novel coronavirus infection: clinical and epidemiological features first case of neonate infected with novel coronavirus pneumonia in china a case report of neonatal covid-19 infection in china neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china detection of sars-cov-2 in different types of clinical specimens clinical features of patients infected with 2019 novel coronavirus in wuhan, china development of immunity in early life prolonged presence of sars-cov-2 viral rna in faecal samples key: cord-350686-q2bu7o4i authors: bilder, christopher r; iwen, peter c; abdalhamid, baha title: pool size selection when testing for sars-cov-2 date: 2020-06-16 journal: clin infect dis doi: 10.1093/cid/ciaa774 sha: doc_id: 350686 cord_uid: q2bu7o4i nan m a n u s c r i p t dear editor-pooling samples has been proposed in multiple articles as an efficient way to test for sars-cov-2 [1] [2] [3] [4] . in particular, yelin et al [1] showed that sars-cov-2 can be detected in pools with up to 32 samples and potentially in pools of 64 samples. they concluded that "this pooling method can be applied immediately in current clinical testing laboratories." however, this research [1] and similar research of others [2] [3] missed answering a very important question: how does one choose the most efficient pool size relative to sars-cov-2 prevalence in samples? without answering this question, laboratories cannot fully benefit from pooling. our correspondence provides the answer so that laboratories can increase their testing capacity to its fullest potential. the efficiencies from pooling samples occur when pools test negative. in general, the probability of a negative pool ( ) is given by = (1 -) for a prevalence ( ) and pool size ( ) [5] . for example, the most efficient pool size is four samples when prevalence is 10% (calculation to be discussed shortly). this will lead to 66% of the pools testing negative on average, resulting in three tests saved for each negative pool. on the other hand, choosing a pool size too large can be very inefficient. by changing the size to 32 samples in our example, only 3% of the pools will test negative. we subsequently show that there are no benefits from using this pool size with this prevalence. similar inefficiencies occur as well when selecting pool sizes that are too small. yelin et al [1] identified a range of pool sizes that appear to not compromise testing sensitivity. from this range, one needs to determine the optimal pool size to perform testing most efficiently. statistical research has shown in general that this is the pool size that minimizes the average number of tests on a per capita basis ( ) when testing a continuous series of samples, where is a c c e p t e d m a n u s c r i p t a mathematical function of prevalence [5] [6] [7] . separate testing of each sample corresponds to = 1, and pooling is more efficient when < 1. expressions for are available [5] [6] [7] , and the optimal pool size can be approximated by the next integer larger 1/√ [8] or found exactly [9] [10] . the authors report no conflicts of interest. m a n u s c r i p t evaluation of covid-19 rt-qpcr test in multisample pools pooling of samples for testing for sars-cov-2 in asymptomatic people sample pooling as a strategy to detect community transmission of sars-cov-2 assessment of specimen pooling to conserve sars cov-2 testing resources comparison of group testing algorithms for case identification in the presence of test error the objective function controversy for group testing: much ado about nothing group testing for identification the blood testing problem a shiny app for pooled testing key: cord-322082-80ym2rsq authors: monto, arnold s; fukuda, keiji title: lessons from influenza pandemics of the last 100 years date: 2020-03-01 journal: clin infect dis doi: 10.1093/cid/ciz803 sha: doc_id: 322082 cord_uid: 80ym2rsq seasonal influenza is an annual occurrence, but it is the threat of pandemics that produces universal concern. recurring reports of avian influenza viruses severely affecting humans have served as constant reminders of the potential for another pandemic. review of features of the 1918 influenza pandemic and subsequent ones helps in identifying areas where attention in planning is critical. key among such issues are likely risk groups and which interventions to employ. past pandemics have repeatedly underscored, for example, the vulnerability of groups such as pregnant women and taught other lessons valuable for future preparedness. while a fundamental difficulty in planning for the next pandemic remains their unpredictability and infrequency, this uncertainty can be mitigated, in part, by optimizing the handling of the much more predictable occurrence of seasonal influenza. improvements in antivirals and novel vaccine formulations are critical in lessening the impact of both pandemic and seasonal influenza. outbreaks of seasonal influenza are perennial occurrences in the temperate zones. their impact on morbidity and mortality is highly variable but in some years can occur at levels that nearly disrupt the functioning of healthcare systems [1, 2] . during such seasonal outbreaks, questions usually center around the severity and how well the vaccine is protecting. but, regardless of disruptions, their impact is quickly forgotten. by contrast, pandemics of influenza occur much less often but are viewed as more threatening because of their relative unfamiliarity and potential for catastrophic impact. even a century later, much of the concern stems from recognition of the sheer number of deaths attributable to the 1918 influenza pandemic. while estimates of death have varied greatly, recent scholarship, largely based of previously omitted data from lower-income countries, such as india, has revised global estimates upwards [3] now, the estimate of 50 million deaths is generally used as an overall global estimate, constituting nearly 3% of the world's population at the time [4] . pandemics are caused only by type a viruses. the current classification of a subtypes was developed in 1980 based on molecular evidence indicating that the previous nomenclature needed revision with, in addition, the inclusion of neuraminidase (na) [5] . table 1 shows the terminology used pre-1980 and the current terminology. years listed are either the start of virologically confirmed pandemics or consensus dates reflecting when it was thought that a new subtype had emerged based on serology [6] [7] [8] [9] . influenza viruses were first isolated in the 1930s, and the etiology and timing of previous activity were based on testing of sera from individuals who had lived through the period in question. this approach, termed "seroarcheology, " resulted in occasional controversy. most identified the 1889 influenza as caused by a2 viruses and postulated that a3 viruses had started to circulate in 1902, with no recognized pandemic occurrence. persons who lived through the 1918 pandemic were found to have antibodies against "swine" influenza viruses, now designated as a(h1n1). the more recent reconstruction of that virus confirms the overall validity of the seroarcheologic technique [10, 11] . the most remarkable epidemiological feature of the 1918 pandemic was the unexpectedly high mortality among those aged 20-39 years [3] . theories to explain this pattern abound but most involve an aberrant immune response [12] . one recent hypothesis postulates that prior infection of children in the 1889 pandemic rendered them particularly susceptible by immunologic imprinting to reinfection in 1918 when they were in their late 20s [13] . current evidence suggests that older individuals may have actually been protected in 1918. this is in contrast to the traditional belief in the w-shaped epidemic curve, in which the high mortality in the elderly was a result of the erroneous inclusion of seasonal disease from the early months of 1918 [14] . figure 1 shows the age-specific mortality in philadelphia where the pandemic shut down the city and peaked at a weekly annualized rate of 140 deaths per 1000 population [3, 15, 16] . another often overlooked but constant feature of all pandemics is the high mortality in the very young experiencing their first influenza infection [14] . these observations indicate that understanding the positive and negative effect of prior influenza exposure is critical [17] . other observations of relevance to planning efforts are indications of the usefulness of nonpharmaceutical interventions in mitigating community impact [18] . the susceptibility of pregnant women was well documented; it should not have been such a surprise during the 2009 influenza pandemic when it was rediscovered [3] . sudden death has often been emphasized as a feature of 1918, but it took, on average, 9 or more days for death to occur [19] (figure 2 ). this stresses the need for health systems to have the surge capacity necessary to handle patients with the more typical prolonged illness regardless of the severity of a pandemic. a proportion of the deaths were associated with bacterial complications. the global increase in antibioticresistant organisms is another major vulnerability [20] . most pre-1980 lists of influenza pandemics included one in 1947 despite lack of documentation of global outbreaks ( table 1 ). in that year, seasonal vaccine became ineffective and it was thought that a new subtype named "a prime" had emerged [21, 22] . this understanding was important in developing the doctrine of original antigenic sin. it is now understood that an intrasubtypic reassortment occurred in 1947, which resulted in a major antigenic change of the a(h1n1) viruses [23] . a new subtype, a(h2n2), actually emerged in 1957 when 3 gene segments coding for b these strains were identified by serology, but the specific identification is in dispute. some have the 1889 virus as h3n8 but without a different subtype identified starting in 1902, a year when there was not a clear pandemic [6] [7] [8] [9] . hemagglutinin (ha), na, and an internal component moved from an avian virus into the circulating a(h1n1) virus through genetic reassortment [24, 25] . the resulting a(h2n2) virus, which was called "asian influenza" since it emerged from china, totally replaced the a(h1n1) viruses. since this was the first true pandemic since 1918, there was immediate concern about its potential impact and great relief when it was found to resemble seasonal influenza with morbidity highest in children and mortality at the extremes of age [26, 27] (figure 3 ). in the united states, the virus emerged in the spring of 1957, but outbreaks intensified only after schools in the southern united states opened in august, underscoring the importance of children in dissemination [28] . although vaccine was available in the united states late in the first wave, it had to be reformulated because of subpotency and standardization issues, concerns still being addressed [29] ( figure 4 ). with little vaccine available, attention was paid to other ways to reduce transmission. a controlled experiment conducted at the veterans administration hospital in livermore, california, demonstrated reduced transmission from the use of ultraviolet lights [30] . that tantalizing observation has been used recently to strengthen the suggestion that small-particle aerosol transmission of influenza viruses is of importance. the a(h2n2) period lasted only 11 years until mid-1968. in july of that year, a major outbreak in hong kong signaled that another reassortment event had occurred [31] . avian influenza genes, one coding for ha and the other an internal component, replaced the existing counterparts in the circulating a(h2n2) virus; the na gene was not replaced [24, 25] . emergence of a(h2n2) and a(h3n2) viruses and later events led to the concept that "novel" influenza viruses are most likely to come from east asia. at the time, it was conjectured that reassortment (or "shift") of avian and human influenza viruses occurred in a nonhuman "mixing vessel" because humans were believed not to have the right cellular entry receptors for avian influenza viruses. pigs have receptors for both human and avian influenza viruses, and since influenza viruses replicate in these animals they were considered to be the mixing vessel [32] . this was further supported by the observation that humans, poultry, pigs, and wild birds live in close proximity in east asia, providing ample opportunity for reassortment to occur there. the a(h3n2) pandemic exhibited the same patterns of morbidity and mortality as the earlier a(h2n2) pandemic. in terms of reasons for emergence of a pandemic variant after only 11 years, it is of interest that the last outbreak of a(h2n2) in 1967-1968 was extensive, as measured by pneumonia and influenza (p and i) mortality. this indicates that a considerable percentage of the population still remained susceptible to a(h2n2) [33, 34] . however, the new a(h3n2) virus completely replaced the previous subtype, and its variants, more than 50 years later, have been responsible for the greatest proportion of mortality from influenza viruses. the first a(h3n2) pandemic wave occurred in the united states in midwinter 1968-1969 at a time typical of seasonal influenza but which in some parts of the world was delayed. there has been speculation that the delay was a result of protection from the unchanged na. even in the united states, contemporaneous studies showed reduction in infection in those with higher anti-na titers, indicating an independent protective effect beyond anti-ha [35] . the role of anti-na remains an issue in present-day efforts to improve vaccine [36] . in january 1976, an outbreak of severe influenza occurred at the us military's fort dix, new jersey. the causative virus was surprisingly found to be a variant of swine influenza, now recognized to be an a(h1n1) virus [37] . since previous serologic studies had shown that the 1918 pandemic was probably caused by swine influenza (table 1) , there was strong concern that the fort dix outbreak could herald another severe pandemic [38] . in the united states, vaccine production was begun after liability concerns of the manufacturers had been addressed. even though no further human outbreaks were detected, mass vaccinations were begun and stopped only when a relationship between the vaccine and guillain-barré was identified. this "affair" has been studied extensively in terms of potential pitfalls in pandemic response and decision making [39] . in the following year, a different a(h1n1) virus, one that had been circulating before 1957, was identified [40] . transmission of this virus, termed "russian influenza" since the reports first came from the far east of the soviet union, was unexpected because the virus had not been detected for 20 years. infections were widespread, generally mild, and limited to younger individuals; residual protection was nearly complete in older individuals [41] . this event has never been considered a true pandemic because so much of the world's population was not susceptible and because of the uncertain origin of the virus. the re-emerged a(h1n1) virus remained in persistent circulation worldwide along with a(h3n2) viruses and continued to evolve until it disappeared in 2009. before this, when a new a subtype began circulating, it completely replaced the previous one. the concept that avian influenza viruses could not directly infect humans ended in 1997 when avian a(h5n1) viruses spread directly from poultry to humans, causing a small but highly important outbreak in hong kong. this event, which raised global concern, resulted in the deaths of 6 of 18 patients with documented infection [42, 43] . once control measures, especially culling of poultry, were put into place, new cases abruptly stopped. no further human cases were detected until, in 2003, when, in conjunction with die-offs of poultry, the spread of a(h5n1) to humans occurred, mainly in southeast asia [44] [45] [46] . most human infections were the result of contact with poultry, but examples of limited human-to-human transmission were documented [47] . because human cases were often severe and resulted in respiratory failure and death, there was high global concern that a pandemic of this virus would be severe if sustained human-to-human transmission occurred [48] . the nature of the threat, arising in animals but directly of concern to humans, highlighted the generally poor coordination and often rigid separation between animal and human health authorities at national and international levels, as well as a general lack of national planning. the adoption of new international health regulations in 2005, which was strongly influenced by the emergence of sars (severe acute respiratory syndrome) and the re-emergence of a(h5n1) in 2003, constituted a major step forward [49] . in the united states, there was particular attention directed to nonpharmaceutical interventions, a result of the recognition that pandemic-specific vaccines would be available relatively late and that influenza-specific antiviral drugs, while important, would be limited in quantity. there were discussions as to whether the use of antivirals might be able to contain human transmission of an emerging virus at the source; these plans were mainly predicated on the emergence occurring in asia [50, 51] . continuing concern about outbreaks of avian influenza was interrupted when the first pandemic of the 21st century unexpectedly started in mexico in 2009 [52] . as a result of intrasubtypic reassortment, the a(h1n1) variant involved was antigenically highly distinct from previously circulating influenza a(h1n1) viruses [53] . the previous prevailing dogma was that pandemic influenza was the result of the emergence of a new virus subtype. however, the subsequent global spread indicates that a pandemic is better defined by the global population's immunological susceptibility and antigenic distance of the new virus from other influenza viruses, rather than rigid applications of virologic rules involving antigenic shift [54] . the 2009 a(h1n1) virus was associated with lower attack rates in older individuals, presumably because of prior exposure to older a(h1n1) viruses. its spread in north america in the spring extended quickly to other parts of the world, highlighting the importance of air travel in accelerating dissemination. in the united states, the spring wave slowed with the beginning of school summer vacations only to pick up again as schools opened in the autumn, reconfirming the importance of children in transmission. this most recent pandemic has been extensively documented. severe disease developed in a small proportion of healthy adults, many of whom had no underlying conditions, which was reminiscent of 1918 but at a much smaller scale [55] . particularly vulnerable groups included indigenous populations, well-documented in canada in the first spring wave [56] . this was not observed in the second wave, most likely related to modifications in response, including careful employment of antivirals. the association of severity with pregnancy was another clear reminder of the 1918 pandemic. a newly observed risk was morbid obesity [57] . the new pandemic virus completely replaced the prior circulating seasonal a(h1n1) but cocirculation of the influenza a(h3n2) virus continued. a societal issue of considerable importance, which is essential to address for future pandemics as well as for seasonal influenza planning efforts, was the perception promoted by some that the 2009 pandemic was a "fake" pandemic [58, 59] . the claim, amplified by social media, was that the public health response was a conspiracy by governments and the world health organization (who) to benefit the sale of influenza vaccines. the overall pattern of mortality, which was less extensive than in 20th-century pandemics, was an important component [55] . but perhaps the more fundamental observation is that the accusations were consistent with a broader erosion of trust within society. the need to focus on communications and trust building in all phases of a pandemic is an essential lesson for improving planning for pandemics and responding to seasonal influenza. during the past 100 years since 1918, each of the 4 influenza pandemics has presented both common and unique challenges. none has been predictable in terms of timing, location of onset ,or the causative influenza virus. those that started in 1957 and 1968 had the most similar morbidity and mortality patterns, with severe complications and deaths that were highest at the extremes of age. the practical consequences for planning are the need to direct interventions to cover such groups while recognizing that other groups may also be at a higher than usual risk [60] . the age groups most at risk may be the same as in seasonal influenza but may not. questions about "severity" are to be expected early, but determining such levels is particularly challenging. the impact on morbidity and mortality may differ, and perception of severity may also differ widely depending on place and time. during the start of events, the information available to health authorities is often limited and highly uncertain. nonetheless, severity assessments are likely to be important for justifying the use of nonpharmaceutical interventions, such as closing schools and restricting population movement. such actions, which apparently had an effect in 1918, are socially disruptive and likely to be divisive. reducing impact may benefit from using more resources early while communicating the uncertainties involved and the consequences of inaction. since 1957, vaccine has always been available late, often after the first wave. in 2009, the current system of virus sharing through frameworks already established at who worked well but vaccine was still not available widely nor equitably. new technologies, such as a universal vaccine, may eventually change this situation but not in the near term. the prepandemic use of vaccines containing known potential pandemic viruses, often with adjuvants, has been proposed, but there are significant uncertainties in choosing what viruses might go into such a vaccine or for taking the inherent risks [61, 62] . preparing for, and responding to, a pandemic is a complex phenomenon, combining science, societal beliefs, practical operational considerations, and political will. some countries and regions have continued to update plans, but others have not. this is a reflection, in part, of uncertainties following the 2009 pandemic but also what has been termed "pandemic fatigue. " the latter issue has been made worse by the repeated recognition of the pandemic potential of different avian influenza virus variants that have infected humans [63] [64] [65] . given this context, it is important to recognize that seasonal influenza occurs every year and many of the essential control measures for pandemics are based on those used for seasonal influenza. it is critical to avoid viewing pandemic and seasonal influenza as unrelated. seasonal influenza is a cause of significant morbidity and mortality, and the vaccine supply used for seasonal influenza sets, in a real-world sense, the production capacity for a pandemic. some countries that will want access to pandemic vaccine do not consider seasonal influenza as a priority. this will limit their capacities to vaccinate their most vulnerable subpopulations in a pandemic, even if vaccine is available. this situation is especially true of lower-resource countries, and continued efforts to document the impact of seasonal influenza and, concomitantly, to develop the health system capabilities needed to support a pandemic response remain high priorities. determining the possible reduction in seasonal severe disease from the use of vaccine can be evaluated in a vaccine probe study in which the vaccine is given under controlled conditions to young children in underresourced areas, similar to studies that documented the need for pneumococcal vaccine [66] . the need for all countries to have and use vaccine in a pandemic is an issue of the equitable distribution of resources on both a national and global scale. scientific advances have positioned the world to respond better to both seasonal and pandemic threats of influenza. however, to make the most of such advances before the next pandemic will still require consistent attention and both scientific and political leadership. potential conflicts of interest. a. s. m. reports consulting fees from sanofi, seqirus, and roche, outside the submitted work. k. f. 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an avian influenza pandemic world health organization. international health regulations strategies for containing an emerging influenza pandemic in southeast asia containing pandemic influenza at the source critically ill patients with 2009 influenza a(h1n1) in mexico antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans what is a pandemic? preliminary estimates of mortality and years of life lost associated with the 2009 a/h1n1 pandemic in the us and comparison with past influenza seasons canadian critical care trials group h1n1 collaborative. critically ill patients with 2009 influenza a(h1n1) infection in canada who working group for risk factors for severe h1n1pdm infection. risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis report of the review committee on the functioning of the international health regulations (2005) in relation to pandemic (h1n1) 2009. 2011 (fineberg report). available at response to the 2009 pandemic: effect on influenza control in wealthy and poor countries novel framework for assessing epidemiologic effects of influenza epidemics and pandemics investing in immunity: prepandemic immunization to combat future influenza pandemics a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases human infection with influenza h9n2 human infection with a novel avian-origin influenza a (h7n9) virus h6 influenza viruses pose a potential threat to human health vaccines as a tool to estimate the burden of severe influenza in children of low-resourced areas key: cord-331719-uijwq8gx authors: russek-cohen, estelle; feldblyum, tamara; whitaker, kathleen b.; hojvat, sally title: fda perspectives on diagnostic device clinical studies for respiratory infections date: 2011-05-01 journal: clin infect dis doi: 10.1093/cid/cir056 sha: doc_id: 331719 cord_uid: uijwq8gx two pathways are described for submission to fda for clearance of a diagnostic device: a premarket application (pma), which can lead to approval of a diagnostic device, and a premarket notification, which can lead to clearance. the latter is often called a 510(k), named for the statute providing for this path. recent fda clearance of molecular-based multiplex panels represents the beginning of a new era for the diagnosis of respiratory infections. the ability to test for multiple pathogens simultaneously, accompanied by the increasing availability of molecular-based assays for newly recognized respiratory pathogens will likely have a major impact on patient care, drug development, and public health epidemiology. we provide a general overview of how fda evaluates new diagnostics for respiratory tract infections and the agency’s expectations for sponsors developing new tests in this area. two pathways are described for submission to fda for clearance of a diagnostic device: a premarket application (pma), which can lead to approval of a diagnostic device, and a premarket notification, which can lead to clearance. the latter is often called a 510(k), named for the statute providing for this path. recent fda clearance of molecular-based multiplex panels represents the beginning of a new era for the diagnosis of respiratory infections. the ability to test for multiple pathogens simultaneously, accompanied by the increasing availability of molecular-based assays for newly recognized respiratory pathogens will likely have a major impact on patient care, drug development, and public health epidemiology. we provide a general overview of how fda evaluates new diagnostics for respiratory tract infections and the agency's expectations for sponsors developing new tests in this area. the food and drug administration (fda) has regulatory responsibility in the united states for review and oversight of the products that form the backbone of modern medical practice. fda premarket and postmarket activities are administratively organized around centers, with the center for drug evaluation and research (cder), center for biologics, evaluation, and research (cber), and center for devices and radiological health (cdrh) bearing most of the responsibility for patient diagnosis and treatment. cdrh, through the division of microbiology devices, in the office of in vitro diagnostic device evaluation and safety has regulatory responsibility for in vitro diagnostic devices (ivds) intended for use in the diagnosis of almost all infectious diseases, including respiratory tract infections. the specific regulations that guide fda are defined under title 21 of the code of federal regulations (cfr); in this overview, we highlight both the elements of the cfr relevant to in vitro microbiology diagnostic devices as well as how fda approaches the review of microbiology device applications for respiratory infections. (specific regulations cited are included in brackets when referenced.) ivds are defined as ''reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae.for use in the collection, preparation, and examination of specimens from the human body'' [21 cfr 809.3] . similar to drug and biological products, new in vitro devices must undergo fda review prior to marketing. for a new ivd, both ''safety'' and ''effectiveness'' of a device must be demonstrated. the broad criteria for evaluating the safety and effectiveness of new ivd are also defined by regulation: requirements for marketing also include the need for labeling that includes, among other requirements, product performance characteristics ''as appropriate . . . describing such things as accuracy, precision, specificity, and sensitivity'' [21 cfr 809.10]. the following discussion provides an overview of how fda approaches these requirements during the development and review of a new microbiology diagnostic device. fda regularly issues guidances in an effort to make the fda submission and review process more consistent. guidances are not rules but provide fda's current thinking on a topic. guidances may be applicable to all of fda (eg, see [1] ), to a center within fda, to a class of products, or tied to a novel product (often called special controls guidance; eg, see [2, 3] ). some are initially issued as draft guidances where the public has an opportunity to comment for a prespecified period. in this article, we provide references to several fda guidances. the fda drug approval process is more familiar to physiciansadequate and well-controlled trials to establish safety and efficacy [21 cfr 314.50] are required as the standard for regulatory approval, usually conducted as randomized, comparative trials. in contrast, study designs used to support device applications are typically not randomized, controlled trials; most often, clinical performance is documented by a single prospective multicenter study in the intended use population, supported by extensive nonclinical analytical studies. for diseases of low prevalence, prospective studies may not be feasible. one major difference between drugs and devices is the existence of two distinct pathways for the marketing of devices: devices may be ''cleared'' through the 510(k) process when a new device is determined to be substantially equivalent to a previously marketed product. devices for entirely new indications or higher-risk intended use are ''approved'' by the premarket application (pma) process. it is important that clinical and analytical requirements for submissions are more aligned with the technology and the clinical settings for use of the device rather than the specific pathway used to go to market. some devices are novel, but may be considered to be moderate risk in the context of how they are used. these devices may still may be cleared via the 510(k) process using what is called a de novo 510(k). however, the potential for a de novo pathway should be discussed with fda well in advance of a formal submission. essential elements considered in the review of a new in vitro diagnostic device for a respiratory pathogen are outlined in table 1 and discussed in more detail below. how diagnostic devices are evaluated is strongly influenced by the intended use (iu) and the risks associated with that use. the proposed iu for a device is an explicit statement of the analyte that the device is measuring or detecting (eg, a specific organism or a biomarker for that organism, and the clinical disease resulting from infection). the iu also describes how the results are reported: qualitative assays most often report whether a specific pathogen is detected or not (eg, culture or direct antigen tests); less commonly (for respiratory pathogens), the results may be quantitative (eg, viral load assays or antibody titers). the specimen type (also referred to as the sample matrix) that can be tested is also an important component of the iu of the device. typical specimens for an upper respiratory tract infection diagnostic device are nasal swabs, nasopharyngeal swabs, nasal washes; or for lower respiratory tract specimens, bronchoalveolar lavage or sputum. less obvious matrices for respiratory pathogens include urine or gastric lavage specimens. evaluation of an analyte in a specific matrix may be critical for a proposed iu as test performance may significantly vary across different matrices. the setting (and/or timing) where test specimens should be collected may also be part of the iu; for example, some devices (including several rapid influenza tests) are cleared for point-ofcare settings (ie, used near the patients while under the auspices of a laboratory with professional laboratorians). timing may be equally critical to test performance; for example, sensitivity of certain rapid antigen tests may drop dramatically as an illness evolves. new devices must be compared with a benchmark for establishing whether or not a specific target condition is present. fda recognizes two major situations for assessing the diagnostic performance of new qualitative diagnostic tests: when a clinical reference method is available or when a comparator other than a reference method is used. in the former circumstance, a clinical reference method is considered to be the best available method for establishing the presence or absence of the target organism [1] but the reference method cannot incorporate results reported by the new device; for example, a new enzymelinked immunosorbent assay (elisa) could not be used as one of the diagnostic criteria for the reference method in a study used to evaluate the performance of the assay. confusion may sometimes arise when distinguishing between analytical and clinical reference method since these may be identical or separate in different contexts; for example, the performance characteristics for a new assay for group a streptococcus against an analytical reference method such as culture on appropriate media may only be valid in the clinical setting of pharyngitis, since group a streptococcus may be a normal colonizer and performance may be different in the setting of colonization versus infection. in contrast, the analytical and clinical reference methods for influenza a are identical since the presence of influenza a is always considered abnormal. determining the appropriate clinical reference methods may be particularly complex in the setting of multiplex assays for diseases with multiple etiologies such as community-acquired pneumonia, and where two or more possible pathogens may be isolated. newly identified organisms introduce different challenges in selecting the appropriate reference method; for example, more recently identified pathogens such as metapneumovirus may be difficult to isolate or confirm by traditional methods such as culture. because a clinical reference method cannot include the new device in its ascertainment, methods such as polymerase chain reaction (pcr) followed by bidirectional dna sequencing may be necessary to confirm test performance. similar concerns exist for assays that differentiate subtypes of novel influenza, a disease where diagnostics are essential but where relatively few clinical specimens are available for confirming performance. there are recent fda guidances that are useful in aiding companies in developing valid comparators. one is a draft guidance on establishing the performance of an influenza ivd [4] , whereas the other two guidances are special controls guidances: one for multiplex devices for respiratory viral pathogens [2] and the other for multiplex devices for influenza a subtyping [3] . another common issue that arises in clinical studies for new devices is result discordance; that is, when discrepancies occur between the result with the new device and the reference method. the objective of a pivotal study is to ascertain performance of the new device. retesting discrepant samples tends to result in an overly optimistic view of performance of the new device. if the new device is truly better than the current clinical reference method, companies should consider contacting fda to discuss an alternative comparator prior to beginning their pivotal trial. clinical studies for evaluating new devices should support the intended use of the device and be conducted in a manner that will yield results consistent with how the device would be integrated into us clinical practice. new devices under study may include foreign sites depending on the specific analyte and the specific intended use for the device; however, clinical studies with foreign sites must include a justification for why similar performance is expected in the united states. study protocols should include clear inclusion and exclusion criteria, case report forms, and testing procedures. sample sizes should be sufficient to ensure that the clinical performance is acceptable in its iu setting. see section on assay performance characteristics: statistics 101 for diagnostic devices for further discussion. the interpretation of results from a new investigational device should be determined prior to conducting 'pivotal' clinical studies. for a qualitative assay, this includes the criteria for scoring device results (eg, as either analyte detected, analyte not detected, equivocal, or invalid). invalid results may occur when one or more test parameters fail to meet the expected criteria. for equivocal results, rules for retesting specimens should be specified. assay performance characteristics: statistics 101 for diagnostic devices sensitivity/specificity. the simplest approach to describing diagnostic performance characteristics is when every test result can be described qualitatively (eg, analyte detected or not detected), and where a reference method exists. for a prospectively enrolled study where patients meet the inclusion/exclusion criteria for the iu of the device, agreement of the test results with the reference method establishes sensitivity and specificity of the test device. sensitivity is the probability that the new device will have a positive test result given that the clinical reference method is positive; it is estimated in a clinical study as the fraction of patients that are positive by the new test among those that are positive by the clinical reference method. for example, in a study of novel h1n1 influenza, it is the percent of patients that both test positive by the new device and are positive via the reference assay divided by the number of patients positive by the reference assay. low test sensitivity reflects less security with the results of a negative test; that is, patients with the disease may be falsely labeled as negative by the new assay. (this fundamental consideration, i.e., -equating ''analyte not detected'' with the absence of disease [or the analyte]-is a common misinterpretation that bedevils the clinical use of devices with imperfect sensitivity.). specificity is the converse; that is, the probability that the new device will have a negative test result given that the clinical reference method is negative. specificity is estimated in the pivotal study by the fraction of subjects that are negative by the new test among those that are negative by the clinical reference method. higher specificity yields greater confidence in a positive result. it is important to recognize that although patients included in specificity calculations do not have the disease of interest, in most cases they are still symptomatic (depending on the specific iu for the test); asymptomatic subjects would not be considered part of the iu population for such a device and therefore would not be appropriate for use in a specificity calculation. there are examples where a new device is not compared with a reference but to a previously cleared/approved device. in this setting, sensitivity and specificity cannot be estimated due to the lack of known infected status. (for example, a benchmark reference standard for aspergillosis infection may include galactomannan as part of the definition of aspergillosis infection in certain populations, even in the absence of invasive procedures to document infection.). identical statistical calculations can be used in this setting but are interpreted as being positive and negative percent agreement rather than sensitivity and specificity; these considerations are also described in fda guidance [1] . percent agreement estimates may be misleading as similar devices can both be in error; for example, 100% agreement between two similar devices would be unlikely to indicate the new device has perfect clinical sensitivity/specificity were the clinical truth available. there may be significant challenges with estimating the sensitivity of a new assay if the prevalence of the disease being tested for is low. as table 2 illustrates, estimates of sensitivity are dependent both on the true underlying sensitivity and sample size. in a clinical study with only 5 specimens positive for a specific pathogen (via the reference method) and an observed sensitivity of 5/5 (100%) results in a lower confidence bound of only 55.6%, meaning diagnostic performance is very uncertain. even for a test with perfect sensitivity, at least 35 patients with the condition of interest would be needed to yield a 95% lower bound greater than 90%. in a pivotal study for devices to detect more common conditions, it is not uncommon to see a sample size of 50 or more patients that are positive via the reference method. fda considers the observed sensitivity and specificity and their respective 95% lower confidence bounds as part of device performance. when sensitivity, specificity, and prevalence are known, one can calculate positive and negative predictive values (ppv and npv, respectively). ppv and npv ask an alternative question: given a positive or negative test result, what is the probability that the patient has (or does not have) the condition being tested for? in a prospective study, this would be estimated as the percentage of patients with the condition of interest from all patients that test positive; similarly, the negative predictive value is estimated as the percentage of patients without the condition of interest among those that test negative. the relationship between sensitivity, specificity, prevalence, ppv, and npv is illustrated in table 3 . as can be seen, ppv and npv are expressed as percentages; ideally, both will be close to 100%. the importance of test prevalence is illustrated in table 4 where, despite high sensitivity and specificity, test utility is extremely limited in the context of ruling in a diagnosis as the ppv of the test result is still only 8%, underscoring the value of tests as an adjunct to diagnosis rather than the sole means for diagnosis. however, tests may still have substantial clinical value even when only the npv is very high. a high npv indicates that if the patient tests negative, it is very unlikely that the disease is present and the physician may want to pursue further work up to obtain a diagnosis. however, the formulas for npv and ppv in table 3 are inappropriate when prevalence is unknown (e.g., when representative cases and controls are selected for study). instead, a more complex set of formulas found in pepe [5] provides a means of estimating npv and ppv under various scenarios for prevalence. assuming one believes reasonable estimates of sensitivity and specificity are available, these equations can be used to illustrate what impact prevalence has on ppv and npv: a lower limit of a two-sided 95% score confidence interval (see [1] ). the lower confidence bound is influenced by the number of specimens with pathogen of interest and how good the device is. fn 1 tn 1 fp) 5 100% x (n 1 /n) , positive predictive value (ppv) is 100% x tp/(tp 1 fp), negative predictive value (npv) is 100% x tn/(tn 1 fn) . in these equations, prevalence is assumed known (ie, measured without error). so if we had a device with a sensitivity of 90% and a specificity of 90% as in table 4 , but the prevalence of the target condition of interest was now 10% rather than the 1% in table 4 , the ppv would be 0.50 or 50% rather than 8.33% as in table 3 , and npv would be 0.99 or 99%. npv is negligibly lower than in table 3 . when a clinical study only assesses agreement to another previously cleared device rather than to a reference method, reporting npv and ppv would be inappropriate. prospective studies in the iu population provide the best estimate of real-world performance for a diagnostic device. however, clinical performance of devices for detecting rare conditions, such as potential bioterrorism agents (eg, anthrax) may be impossible to ascertain. low prevalence often mandates the use of retrospective or banked specimens as clinical specimens for new devices. with banked specimens and/or enriched studies, prevalence usually cannot be estimated. (retrospective samples that were prospectively archived [e.g., all patients meeting a prespecified inclusion/exclusion criteria are included] can still be representative provided storage conditions do not impact the assay results. however, banked [repository] specimens may not be representative and should be considered separately.). as an alternative, the ppv and npv can be calculated for a set of plausible prevalence estimates so that the clinical impact of a different disease prevalence can be understood. it is also important to appreciate that including a nontrivial proportion of banked specimens or substantial enrichment of specimens can change the disease spectrum in the study, which in turn changes the estimates of sensitivity and specificity and not just prevalence; in this situation, sensitivity, specificity, ppv, and npv could all be biased and misleading. given the critical role of study design in determining performance characteristics for a new test, companies are strongly encouraged to discuss their study proposals with fda prior to initiation of studies essential for device clearance or approval. to evaluate the performance of the assay in multiple settings, precision studies must be conducted to confirm the reproducibility of assay results. these studies usually include multiple days or multiple runs across clinical sites. studies should be designed to capture all major sources of variability, including site-to-site variation, the effect of different operators, and instrument-specific variability. detailed guidance regarding precision studies for in vitro diagnostic devices is available in the clinical and laboratory standards institute (clsi) guideline ep5-2a [6] . for qualitative assays, test panels used for precision/reproducibility studies should not only include both positive and negative samples, but particularly samples close to assay cutoffs; that is, thresholds for distinguishing positive from negative or equivocal. for quantitative assays, panels should include samples that are at the extremes of the analytical measurement range as well as values near key clinical decision points. analytical sensitivity (limit of detection) for each detected pathogen (analyte) and each specimen type (ie, matrix) need to be assessed [2-4, 7, 8] . analytical specificity, including crossreactivity with other possible respiratory pathogens, should be assessed, as well as interference by common substances such as blood, medications, and so forth. for some quantitative assays, it may be necessary to rule out a possible hook effect with high positive samples. for studies utilizing banked specimens to establish clinical or analytical performance, it would be necessary to provide evidence that storage conditions do not impact assay results. other analytical studies may be requested depending on the type of technology used. a recent major advance for respiratory in vitro diagnostic testing has been fda clearance of multiplex devices that can test for multiple pathogens simultaneously. the technical definition of a multiplex assay is ''two or more targets simultaneously detected via a common process of sample preparation, target or signal amplification, allele discrimination, and collective interpretation'' [7] . essentially, a multiplex assay takes a single sample and then provides more than one output (eg, adenovirus present or absent and influenza a present or absent). there are several known hurdles to developing a multiplex assay with many pathogens, as illustrated by the multiplex respiratory viral infection panel example [2] . adequate performance must be demonstrated for each viral pathogen included adding a high-risk pathogen such as severe acute respiratory syndrome (sars) coronavirus to a multiplex assay would mean submitting a pma for approval. thus, the regulatory path may be a consideration when designing a multiplex device. an fda special controls guidance for respiratory viral panel multiplex devices has been recently published [2] and addresses some of these issues. fda also publishes special control guidance on devices detecting new pathogens, which can be either standalone tests or part of multiplex assays. the clinical laboratory improvement amendments of 1988 (clia) defines categorization for a subset of devices sufficiently simple to use in a clia-waived settings, thus in essence permitting the use of these diagnostic tests outside of a clinical laboratory [9] . (a clia-waived test is different from ''home use devices'' [eg, pregnancy tests] which can be used with no medical oversight, or ''point-of-care'' tests [eg, tests performed in an emergency room, which remains under the auspices of the hospital laboratory]. ''point-of-care'' tests may or may not be clia-waived depending on the specific device.) upon application by a sponsor, fda has the authority to designate a specific in vitro diagnostic test as a waived test. waived tests such as urine dipsticks or group a streptococcal throat swab tests have widespread clinical use due to their availability at sites such as physician offices or commercial pharmacies. devices being considered for a clia waiver are first subject to the same regulatory process as other diagnostic devices; that is, the device must demonstrate it has adequate performance characteristics in the hands of a trained laboratorian. however, beyond the demonstration that the device has adequate performance characteristics, the device must perform well in a clia-waived setting. this can be done either after a device is cleared or approved, or the initial study of the device can be designed to support a clia waiver application, which can be submitted after clearance/approval is granted. as part of an application for a clia waiver, sponsors are required to include studies to show the outcomes of not following the ''instructions for use'' provided with the device (e.g., prolonging the incubation of reagents, adding reagents in the wrong order, etc). these are studies designed to ensure that the device is robust and simple enough to operate without a trained laboratorian. by this definition, a sample that required centrifugation for processing, for example, would not be eligible for a waiver. the study participants in a clia-waiver study represent end users typical of those who would perform the test in a waived setting (eg, nurses or aides in a doctor's office or nursing home). three or more sites should be included in the study, with 1-3 users per site for a minimum of 9 intended users. testing must also include specimens with results close to the test cutoff values (ie, specimens that may be the most challenging to nonlaboratorians). detailed information regarding the design of studies for a clia waiver application, including the minimal performance criteria acceptable for a clia-waived device, is described in ''fda guidance for industry and fda staff: recommendations for clinical laboratory improvement amendments of 1988 (clia) waiver applications for manufacturers of in vitro diagnostic devices'' [9] . it is highly recommended that sponsors seeking to market a device for use in a waived setting consult with fda early in the regulatory process, especially for new products under development. it is important to note that clia-waiver requirements are very stringent; sponsors should consider the value of device use in clia-waived settings before pursuing the waiver process. most pivotal studies for ivds do not involve managing the patient based on the result of the investigational device; thus, ivd companies are rarely required to have an investigational device exemption (ide) to start their pivotal study. however, fda center for devices and radiological health, including the division of microbiology devices, provides advice to sponsors on whether an ide is required and other issues regarding the pre-ide submission consultative process. given the extensive number of diagnostic indications, the evolving technologies, and potential regulatory pathways, fda strongly recommends that companies consider participating in the pre-ide submission process early in the device development cycle and especially prior to conducting pivotal studies. the pre-ide process allows fda to evaluate if the proposed study designs are appropriate to support the iu of a device. for new instrumentation that have not been reviewed by the fda, documentation of hardware, software, and manufacturing processes will need to be assessed, and could be topics for discussion in a pre-ide submission [10, 11] . as an aid to sponsors developing new diagnostic devices, fda posts summaries of the review decisions for cleared devices on the fda web site (http://www.accessdata.fda.gov/scripts/cdrh/ cfdocs/cfpmn/pmn.cfm). companies may want to examine these summaries for recently cleared devices that are similar to the ones they are proposing to submit. in addition, substantial advice concerning the device development process is available through published fda guidance [12] . the previous discussion has only briefly reviewed some of the concerns manufacturers of new in vitro diagnostic tests should consider during their development. the extensive number of diagnostic indications, evolving technologies, increasing use of multiplex instruments in microbiology, and potential regulatory pathways mandate careful planning by sponsors when developing new diagnostic assays. it is essential that sponsors plan for clinical and analytical studies that consider the device performance needed to support the proposed intended use. the use of appropriate statistical methods in both planning and evaluating these studies is also highly recommended. interactive dialogue with fda during the device development process is strongly encouraged. fda guidance for industry and fda staff: class ii special controls guidance documents: respiratory virus panel multiplex nucleic acid assay fda guidance for industry and fda staff: class ii special controls guidance document: testing for detection and differentiation of influenza a virus subtypes using multiplex nucleic acid tests deviceregulationandguidance/guidancedocuments/ucm180310 fda draft guidance: establishing performance characteristics of in vitro diagnostic devices for the detection or detection and differentiation of influenza viruses the statistical evaluation of medical tests for classification and prediction 2a: establishment of precision of quantitative measurement procedures a: verification and validation of multiplex nucleic acid assays a: protocols for determination of limits of detection and limits of quantitation fda guidance for industry and fda staff: recommendations for clinical laboratory improvement amendments of 1988 (clia) waiver applications for manufacturers of in vitro diagnostic devices fda guidance for industry and fda staff: guidance for the content of premarket submissions for software contained in medical devices fda guidance for industry, fda reviewers and compliance on off-the-shelf software use in medical devices fda draft guidance for industry and fda staff: in vitro diagnostic (ivd) device studies-frequently asked questions diagnostic device clinical studies d cid potential conflicts of interest. all authors: no conflicts. key: cord-348392-e35cd9sg authors: moraleda, cinta; serna-pascual, miquel; soriano-arandes, antoni; simó, silvia; epalza, cristina; santos, mar; grasa, carlos; rodríguez, maria; soto, beatriz; gallego, nerea; ruiz, yolanda; urretavizcaya-martínez, maría; pareja, marta; sanz-santaeufemia, francisco josé; fumadó, victoria; lanaspa, miguel; jordan, iolanda; prieto, luis; belda, sylvia; toral-vázquez, belén; rincón, elena; gil-villanueva, nuria; méndez-echevarría, ana; castillo-serrano, ana; rivière, jacques g; soler-palacín, pere; rojo, pablo; tagarro, alfredo title: multi-inflammatory syndrome in children related to sars-cov-2 in spain date: 2020-07-25 journal: clin infect dis doi: 10.1093/cid/ciaa1042 sha: doc_id: 348392 cord_uid: e35cd9sg some clusters of children with a multisystem inflammatory syndrome associated with sars-cov-2 infection (mis-c) have been reported. we describe the epidemiological and clinical features of children with mis-c in spain. mis-c is a potentially severe condition that presents in children with recent sars-cov-2 infection. in the last weeks, some clusters of children with a multisystem inflammatory syndrome (mis-c) linked to sars-cov-2 infection have been described in the united kingdom, france, italy and usa, among other countries. 1,2 this syndrome shares features of kawasaki disease, toxic shock syndrome and macrophage activation syndrome. 3 some of these children tested positive for sars-cov-2 real-time reverse-transcriptase polymerase chain reaction (rt-pcr) and/or had a positive serological response for this infection. the specific link with sars-cov-2 remains unclear. in spain, this phenomenon has also been observed. in this case series, we intended to describe the epidemiological and clinical features of children with mis-c in spain. this is a case series of children with mis-c associated with sars-cov-2 enrolled in the epidemiological study of covid-19 in children of the spanish society of pediatrics (epico-aep), from march 1 st to june 1 st, 2020. epico-aep is a multicenter national study aiming to describe the covid-19 in spanish children. children younger than 18 years with infection due to sars-cov-2 and attended at 49 hospitals were included in this registry. inclusion criteria included positivity in real-time polymerase chain reaction (rt-pcr) positive, igm or igg in lateral-flow rapid test, elisa or immuno chemiluminescence serology (see table 1 ), or severe disease suggestive of mis-c and recent household contact with a confirmed patient with covid-19. m a n u s c r i p t 6 by june 1st, 312 patients had been attended in the 49 hospitals, and 252 participants were hospitalized. of them, 181 (72%) were admitted due to causes directly or likely related to sars-cov-2. the remaining 71 (28%) were admitted due to causes not related with sars-cov-2, but were screened and found to be infected with sars-cov-2. a total of 31/252 (12%) children were diagnosed as mis-c and/or kawasaki disease by their physicians. weekly admissions of children with mis-c and children with other clinical presentations associated with covid-19 were recorded ( figure 1 ). the peak of mis-c cases was one month after the peak of admissions for other covid-19 related reasons and decreased afterward. median age and interquartile range were 7. 6 [4.5;11.5] a c c e p t e d m a n u s c r i p t 7 rash or bilateral non-purulent conjunctivitis, or muco-cutaneous inflammation signs were found in 21/31 (67%) patients; hypotension or shock in 15/31 (48%), features of myocardial dysfunction 25/31 (80%) consisting of pericarditis, valvulitis, arrhythmias or coronary abnormalities in 19/31 (61%); 6 (19%) additional children had only an elevation of a biochemical marker of heart dysfunction (nt-probnp); evidence of coagulopathy (specifically, elevated d-dimers) was found in 29/30 (97%), and acute gastrointestinal problems (diarrhea, vomiting, or abdominal pain), in 27/31 (87%). no other apparent microbial cause of inflammation as sepsis or staphylococcal or streptococcal shock syndrome was found. the patient who does not include who criteria was a 12-month old girl. the criterion she did not meet was the elevation of inflammatory markers. she was cohousing with a covid-19 patient. she was on chronic oral treatment with steroids due to a chronic idiopathic interstitial lung disease. she presented with 6 days of fever, shortness of breath and cardiogenic shock (ph=7.2). she had lymphopenia (1,100 cells/mm 3 ). she was diagnosed with cardiogenic shock. echocardiography showed left ventricle dilatation above +2.6 z-score for age and sex, and ejection fraction of 55%. enterovirus infection was ruled out with pcr in nasopharyngeal aspirate. she received 10 days of remdesivir. although she had low inflammatory markers, this fact was attributed to the long-term immunosuppressive therapy with steroids. she had a coinfection with human metapneumovirus (hmpv). she was treated for misc with intravenous immunoglobulin (ivig) and steroids. she was on long-term oral steroids due to pulmonary interstitial disease, which may avoid the rising of acute-phase reactants. a c c e p t e d m a n u s c r i p t 8 twenty (65%) patients needed admission to the pediatric intensive care unit, and 6/31 (19%) invasive mechanical ventilation. cardiac complications consisted of myocardial dysfunction (15/31; 48%), pericardial effusion (6/31; 19%) ; valvular dysfunction (9/31; 29%), arrhythmias (7/31; 23%) and coronary abnormalities (3/31; 10%, among them 1 aneurysm). four patients (13%) had renal failure. two (6%) patients received remdesivir and 7/31 (23%) lopinavir/ritonavir. a total of 21/31 (68%) children received corticosteroids: 19 of these received methylprednisolone (13 patients received doses of 1 to 2.5 mg/kg/day; 2 patients boluses of 8 and 30 mg/kg/day for 3 days; 4 had dosing not available), 20/31 (65%) patients received 2 gr/kg of intravenous immunoglobulin (ivig) and 13/31 (42%) patients received both ivig and corticosteroids. all but three patients received broadspectrum antibiotics. one patient with acute leukemia and bone marrow transplant died, and one 6-month-old patient developed anterior-descendant coronary aneurysm (z-score +9). this patient was an infant with down syndrome, who presented with 5 days of fever, shortness of breath and shock due to myocardial dysfunction. he had a a positive rt-pcr for sars-cov-2 at diagnosis and coinfection with hmpv, probnp=9,968 pg/ml and troponin i=34.1 ngr/ml. he developed valve insufficiency, renal failure, coronary aneurysm, and eventually had 50 days of fever despite treatment for infection (antiviral treatment with 2 days with lopinavir/ritonavir, hydroxychoroquine, cefotaxime, vancomycin and meropenem, micafungine) and for kawasaki disease (igiv and steroids). the rest of the patients recovered without sequels. a c c e p t e d m a n u s c r i p t 9 in this registry, entry criteria was covid-19 disease, differently from the previous reports that include patient without sars-cov-2 1,3 . previous reports raised discussion as some children with mis-c or kawasaki disease lacked evidence of infection with sars-cov-2. disease triggered by other causes may have been included within those reports. our data strongly support the idea that not only there is a temporal association with sars-cov-2, but also a microbiological association. in this report, only 1 patient without microbiological or serological evidence of sars-cov-2 was included, but he had a strong epidemiological link. there is a possibility that not all mis-c cases are microbiologically related to sars-cov-2, because rt-pcr and serology do not have 100% sensitivity and specificity. that is why we have included a patient with negative tests and with recent contact with a patient with covid-19, according to who definition of mis-c. limitations of this study include that some cases without microbiological, serological or epidemiological link may not have been included in this registry. a c c e p t e d m a n u s c r i p t 10 sars-cov-2 could be a relevant trigger for a delayed cytokine storm and an inflammatory condition, with potentially severe consequences. 6 coinfections as hmpv may be present and might play a role in triggering the immune response. it is possible that some particular patients with special features -as chronic immunosuppressive treatment influencing inflammatory markersmay have misc but not fulfill all who criteria. mis-c is a potentially severe condition that presents in some children after sars-cov-2 infection. until herd immunity or a vaccine are available, physicians should be aware of this severe condition in children during covid-19 epidemics. more studies are necessary to clarify the physiopathology of this syndrome and its adequate treatment. [sp]=100%), n=4; immunochemoluminescence diasorin tm sars-cov-2 s1/s2 igg, s=97%, e=98%, n=1; elisa in-house total antibody test, included within solidarity ii trial, ongoing and results pending, n=6; rapid test biozek tm , igm (s=85%, sp=96%), igg (s=99.9%, sp=88%), n=3; immunoassays elecsys sars-cov-2 cobas tm , total antibodies, s=84%, sp=100%, n=2. demographic male 18/31 (58%) a c c e p t e d m a n u s c r i p t m a n u s c r i p t a c c e p t e d m a n u s c r i p t 20 figure 1 enrique otheo (hospital ramón y cajal) sara guillén (hospital de getafe) julia jensen (hospital infanta cristina), paula vidal (hospital infanta elena) hyperinflammatory shock in children during covid-19 pandemic. the lancet european centre for disease prevention and control. paediatric inflammatory multisystem syndrome and sars-cov-2 infection in children system syndrom ecdc: stockholm an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study. the lancet primera ronda estudio nacional de seroprevalencia covid-19 an insertion unique to sars-cov-2 exhibits superantigenic character strengthened by recent mutations. biorxiv hemoglobin (g/dl), worst value 31/31 (100%) we thank all the patients and families for their participation in this cohort, the staff members who cared for them at their personal risk in this time of epidemic and gathered data from them in the pediatrics wards, pediatric intensive care units, cardiology units, and all the involved units. we acknowledge the rest of the epico team: rut del valle, ana barrios (hospital infanta sofia), sara a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-348478-ho89o8mj authors: pawlotsky, jean-michel title: sars-cov-2 pandemic : time to revive the cyclophilin inhibitor alisporivir date: 2020-05-15 journal: clin infect dis doi: 10.1093/cid/ciaa587 sha: doc_id: 348478 cord_uid: ho89o8mj december 2019 saw the emergence of a new epidemic of pneumonia of varying severity, called covid-19, caused by a newly identified coronavirus, sars-cov-2. no therapeutic option is available to treat this infection that has already killed more than 235,000 people worldwide. this viewpoint summarizes the strong scientific arguments supporting the use of alisporivir, a non-immunosuppressive analogue of cyclosporine a with potent cyclophilin inhibition properties that has reached phase 3 clinical development, for the treatment of covid-19. they include the strong cyclophilin dependency of the lifecycle of many coronaviruses, including sars-cov and mers-cov, and preclinical data showing strong antiviral and cytoprotective properties of alisporivir in various models of coronavirus infection, including sars-cov-2. alisporivir should be tested without delay on both virological and clinical endpoints in patients with or at-risk of severe forms of sars-cov-2 infection. a c c e p t e d m a n u s c r i p t 3 december 2019 saw the emergence of a new epidemic of pneumonia of varying severity that started in the chinese city of wuhan [1] . the agent responsible for the epidemic was very rapidly identified [2] . it is a coronavirus, hitherto unknown member of the orthocoronavirinae subfamily, genus beta-coronavirus, forming a separate clade within the sarbecovirus subgenus (lineage b) to which also belongs the virus responsible for the despite initial measures aimed at preventing its spread, the disease is now pandemic, with active hotspots of infection in western europe and north america. the typical clinical presentation of covid-19 is an acute respiratory illness with fever and respiratory symptoms, including cough and shortness of breath. the disease may aggravate and necessitate oxygen therapy and, especially in patients with risk factors (such as overweight, hypertension, diabetes or cardiac disease), transfer to an intensive care unit and mechanical ventilation. milder forms may be limited to runny nose or sore throat, while an unknown proportion of patients remains asymptomatic. non-respiratory, sometimes severe manifestations have also been described, including taste and/or olfactory disorders, as well as gastrointestinal, neurological, cardiovascular and/or ocular symptoms [1, 3] . at the time of writing this viewpoint, more than 3. several mechanisms have been identified as potential targets for direct-acting antivirals and host-targeted agents against sars and the middle-east respiratory syndrome coronavirus (mers-cov) [5] . however, these hypotheses could not be clinically evaluated, because these two epidemics spontaneously stopped their progression. empirical attempts have been made in the context of the covid-19 epidemic to repurpose drugs that are approved or have reached late clinical developmental stages in other indications. the combination of the antiretroviral drug lopinavir with ritonavir failed to demonstrate antiviral efficacy in patients with severe covid-19 disease [6] . chloroquine, an anti-malarial drug, bears significant antiviral properties against sars-cov-2 in vitro [7] . chloroquine has been tested in patients with covid-19, alone or in combination with the anti-bacterial drug azithromycin, with thus far contradictory results, probably due to the heterogeneity of the populations studied (mild to severe, early to late disease) and methodological flaws [8] . camostat mesylate was also recently suggested to act as a sars-cov, mers-cov and sars-cov-2 entry inhibitor in vitro [9] , but no clinical data are available. the nucleotide analogue remdesivir showed antiviral efficacy against sars-cov and mers-cov viruses in both in vitro and animal models [10] [11] [12] [13] . remdesivir was recently reported to inhibit the sars-cov-2 lifecycle in vitro [7] . modest but significant results from a randomized clinical trial were recently released: the administration of remdesivir was associated with a significantly shortened median time to recovery (11 vs 15 days) and a trend towards less frequent death (8.0% vs 11.6%, p=0.059) [14] . however, a yet unpublished chinese study reported negative results with this compound. overall, the antiviral treatment of covid-19 remains to be found. a c c e p t e d m a n u s c r i p t 5 a very credible therapeutic option for sars-cov-2 is the use of cyclophilin inhibitors. cyclophilins are host peptidyl-prolyl cis-trans isomerases. they catalyze the interconversion of the two energetically preferred conformers (cis and trans) of the planar peptide bond preceding an internal proline residue [15, 16] [17] . cyclophilins also play a very important role in the lifecycle of many coronaviruses. it has indeed been shown that the lifecycles of human coronaviruses 229e (hcov-229e) and nl-63 (hcov-nl63), responsible for mild respiratory infections in humans, of feline infectious peritonitis coronavirus (fpiv), responsible for a fatal disease in cats, and of sars-cov were highly dependent on cyclophilin a (and possibly also cyclophilin b for fpiv) [18] [19] [20] [21] [22] . the n protein of sars-cov binds strongly to cyclophilin a and this binding could facilitate cell invasion [19, 23] . interestingly, the sars-cov and mers-cov virions bring with them quantities of cyclophilin a sufficient for the achievement of their lifecycle, allowing them to compensate for a defect in cell production in their target cells [24] . in this context, it is not because of its strong immunosuppressive properties, csa cannot be considered as a therapeutic option for sars-cov-2 infections. there are, however, alternative molecules capable of strongly inhibiting the functional activity of cyclophilins without exerting any immunosuppressive effects. this is the case for the non-immunosuppressive analogue of csa alisporivir (debio 025). alisporivir was developed by the swiss company debiopharm, initially for the treatment of chronic hcv infection, as the hcv lifecycle is strongly dependent on cyclophilin a. alisporivir induced a decrease of hcv replication of the order of 3 to 4 logs in patients infected with all hcv genotypes, with a very high barrier to resistance since the drug target is a host protein [27, 28] . administered for 14 days to 24 weeks as a monotherapy, alisporivir was well tolerated. headache, nausea, fatigue and a few cases of reversible hyperbilirubinemia were the most common adverse events in patients treated with the highest dose of the drug [28] [29] [30] [31] . hyperbilirubinemia was not observed in previous studies with alisporivir at oral dosages up to 1,200 mg/day for 10 days [28] . we have shown that the combination of alisporivir and ribavirin is also very well tolerated for several weeks in hcv-infected patients [31] . alisporivir has entered phase 3 clinical evaluation for the treatment of chronic hepatitis c, in combination with pegylated interferon alpha and ribavirin, under license with novartis. however, in april 2012, this phase 3 program was interrupted due to the occurrence of 3 cases of acute pancreatitis, including one fatal case, occurring several weeks after the onset of treatment in a trial. overall, there were 7 cases of acute pancreatitis among over 2,000 patients included in the alisporivir clinical development program, with equal frequency in patients receiving pegylated interferon alpha with or without alisporivir (0.35% vs 0.41%, respectively) [32] . pancreatitis is a classic complication of interferon alpha and the direct responsibility of alisporivir in the reported cases has not been formally a c c e p t e d m a n u s c r i p t 7 established, especially since no case has occurred with alisporivir alone or with alisporivir and ribavirin in other studies [28, 31] . the current situation is that a non-immunosuppressive csa analogue endowed with powerful cyclophilin inhibitory properties and having reached phase 3 clinical development, which is well tolerated for several weeks of administration alone or in combination with ribavirin, exists. strong arguments suggest that alisporivir will have antiviral efficacy against sars-cov-2 infections. first, the lifecycles of many coronaviruses, including sars-cov, are strongly dependent on cyclophilins. secondly, experimental results, including findings from our laboratory, indicate that alisporivir inhibits the replication of hcov-229e, hcov-nl63 and mhv at low micromolar concentrations and without cytotoxic effect in vitro [18, 33, 34] . thirdly, we observed that alisporivir fully abolishes the cytopathic effect of hcov-229e in cell culture (unpublished data). a recent study showed that alisporivir blocks the lifecycle of sars-cov and mers-cov in different cellular models of infection [34] . however, treatment of a mouse model of sars-cov infection with alisporivir and ribavirin did not lead to a reduction in infection-related mortality or weight loss compared to untreated mice [34] , emphasizing the importance of a careful design of clinical trials to maximize the clinical effect of the antiviral treatment. alisporivir inhibits all cyclophilins, including cyclophilin d, an essential component of the mitochondrial permeability transition pore (mptp). cyclophilin d inhibition has been shown to be beneficial in heart, kidney and hepatic disorders in which abnormal mptp opening plays a major pathogenic role [36, 37] . whether similar protection of lung tissues will be achieved by alisporivir in order to improve the clinical course of covid-19, in the presence or in the absence of a significant antiviral effect, must be assessed. therefore over 235,000 patients already died from sars-cov-2 infection on the day of writing this viewpoint. if alisporivir proves to have an antiviral and/or clinical effect, we will, finally, have a first-line therapeutic solution to the current pandemic, as well as, possibly, a weapon against the future emergence of other coronaviruses. in the absence of an effect, we will have advanced scientific knowledge and, most importantly, we will have no regrets. clinical features of patients infected with 2019 novel coronavirus in wuhan, china a novel coronavirus from patients with pneumonia in china clinical features of covid-19 coronaviruses: drug discovery and therapeutic options a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro rethinking the role of hydroxychloroquine in the treatment of covid-19 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases the cyclophilins the role of immunophilins in viral infection human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine aderivatives including alisporivir nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a genetic deficiency and polymorphisms of cyclophilin a reveal its essential role for human coronavirus 229e replication feline coronavirus replication is affected by both cyclophilin a and cyclophilin b the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus cyclophilins and cyclophilin inhibitors in nidovirus replication suppression of feline coronavirus replication in vitro by cyclosporin a cyclosporin a inhibits the replication of diverse coronaviruses the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro the cyclophilin inhibitor debio-025 shows potent anti-hepatitis c effect in patients coinfected with hepatitis c and human immunodeficiency virus the cyclophilin inhibitor debio 025 combined with peg ifn alpha-2a significantly reduces viral load in treatment-naive hepatitis c patients alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis c virus genotype 2 or 3 infection randomised clinical trial: alisporivir combined with peginterferon and ribavirin in treatment-naive patients with chronic hcv genotype 1 infection (essential ii) influences of cyclosporin a and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229e replication alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model inhibition of sars-cov-2 infection by the cyclophilin inhibitor alisporivir (debio 025) mitochondrial permeability transition: a molecular lesion with multiple drug targets the author is grateful to drs abdelhakim ahmed-belkacem and slim fourati for helpful comments.conflict of interest disclosure: the author has served as an advisor for abbvie, gilead, glaxosmithkline, merck, regulus and siemens healthcare. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-326297-0r9pex1o authors: hartmann, stacy; rubin, zachary; sato, heidi; oyong, kelsey; terashita, dawn; balter, sharon title: coronavirus 2019 (covid-19) infections among healthcare workers, los angeles county, february may 2020 date: 2020-08-17 journal: clin infect dis doi: 10.1093/cid/ciaa1200 sha: doc_id: 326297 cord_uid: 0r9pex1o across the world, healthcare workers (hcw) are at a greater risk of infection by the novel coronavirus 2019 (covid-19) due to the nature of their work. the los angeles county department of public health (lac dph) set out to understand the impact of covid-19 on healthcare facilities and hcws by tracking and analyzing data from case-patient interviews of hcws. as of may 31st, over three months into the pandemic, nearly 5,500 positive hcws were reported to lac dph, representing 9.6% of all cases. cases reported working in 27 different setting types, including outpatient medical offices, correctional facilities, emergency medical services, etc., with the highest proportion from long-term care facilities (46.6%) and hospitals (27.7%). case-patients included both clinical and non-clinical roles, with nearly half (49.4%) of positive hcws being nurses. over two-thirds of hcws (68.6%) worked at some point during their infectious period and nearly half (47.9%) reported a known exposure to a positive patient and/or co-worker within their facility. overall, compared to all lac cases, hcws reported lower rates of hospitalization (5.3% vs. 12.2%) and death (0.7% vs. 4.3%) from covid-19. there are many factors that increase hcws risk of infection, including high risk work environment, limited supply of personal protective equipment, and even pressure to help and work during a pandemic. in response to these data, lac dph created resources and provided guidance for healthcare facilities to best protect their patients and staff during the covid-19 pandemic. a c c e p t e d m a n u s c r i p t 3 los angeles county (lac) is a jurisdiction of over 10 million residents, served by 4,228 licensed healthcare facilities and thousands more non-licensed healthcare settings (1) . multiple reports (2, 3, 4) have demonstrated that healthcare workers (hcw) in lac, both medical providers and laboratories are mandated to report all covid-19 positive cases to lac dph. all lac residents who test positive for covid-19 are interviewed by lac dph using a standardized form that identifies if the case-patient worked in a high-risk environment, such as a healthcare setting. each case-patient was contacted three times to interview. in addition, outside jurisdictions email lac dph when they identify a case-patient working in a lac high-risk environment. occasionally, additional hcw cases were also identified during the course of covid-19 outbreak investigations and direct communication from healthcare facilities. hcws were defined as any person working or volunteering in a licensed or non-licensed healthcare settings, including hospitals and skilled nursing facilities, as well as outpatient practices, mental health facilities, emergency medical services, etc. hcws included both clinical staff that interacted directly with patients and non-clinical staff that worked in the healthcare industry but did not provide direct clinical care to patients. in addition, hcws providing care in a non-healthcare settings, a c c e p t e d m a n u s c r i p t 4 such as school or correctional facility nurses, or caregivers in senior living facilities, were included. all hcws, including staff, contractors, licensed independent practitioners, and volunteers, were included in analysis. from case-patient interviews and/or emailed reports, lac dph recorded occupational setting, occupational role, date of symptom onset, date last worked, known exposure, and if hospitalized for each hcw. a case-patient was determined to have worked during their infectious period if the date last worked was after, the same, or within 48 hours prior to the date of symptom onset. exposure was split into two categories: healthcare and non-healthcare exposure. healthcare exposure was defined as contact with a confirmed case while working in a healthcare setting. due to limitations of the interview formatting, exact exposure was not always stated and a "not specified" option was added for each category. if the case-patient did not work more than three weeks prior to symptom onset date, they were considered to have a non-healthcare exposure. the extended time period, three weeks instead of two, was used to ensure the case-patient really had no healthcare exposure before even mild symptom onset. in addition, if covid-19 death report forms identified an hcw, their information was tracked, along with co-morbidities. hcws that were not interviewed or where minimum information, occupational setting and role, was not provided by the reporting jurisdiction or facility were removed from analysis. through may 31 st , 2020, 57,118 confirmed covid-19 cases in lac were reported, of which interviews were conducted for approximately 60%. in this time, 5,458 confirmed hcws were reported to lac dph, representing 9.6% of all lac cases. after removing hcws with incomplete information, 5,118 hcws were included for analysis. these hcws were reported from 27 different healthcare setting types (table 1) . nearly half of all confirmed cases (46.6%) worked in a long-term a c c e p t e d m a n u s c r i p t 5 care setting, including skilled nursing facilities (snfs), assisted living, and other senior residential communities. over one-fourth of case-patients worked in a hospital (27.7%), including general and long-term acute care hospitals. hcws from medical offices comprised 6.9% of the case-patients. all other settings (ex. home health, correctional facilities, emergency medical services, etc.) accounted for less than 4% each of the total hcw cases. case-patients were identified among a range of occupational roles (table 2 ), but nurses (including registered nurses, licensed vocational nurses, and certified nursing assistants) accounted for nearly half of all cases (49.4%). caregivers in the home or within long-term care facilities were the second most common role (5.8%). case-patients included both clinical hcws, such as medical assistants (3.6%) and physicians (2.6%), and non-clinical roles, such as administrators (4.3%), environmental services (3.2%), and food services (2.9%). hcws reported symptom onset dates between february 13 th and may 31 st (figure1), with two peaks on april 6 th (2.2%) and april 20 th (2.0%). although may onset cases are likely still to be added from those interviewed in june, lac is seeing a decline in hcw cases. at the end of april, hcws represented 12.8% of all lac cases, whereas only 9.6% at the end of may. when asked if they had a known exposure to covid-19, (table 3) , healthcare exposures within their facility accounted for nearly 44%, including contact with either a positive patient, co-worker, or both. non-healthcare exposures, including infected family members or friends, or travel within 14 days of illness, was reported by 11.3% of cases. the remaining 45.1% were unknown exposures. using their reported date last worked, table 4 provides information on when hcws stopped working relative to their symptom onset. nearly two-thirds worked during their infectious period, either on the day of symptom onset (24.2%), after symptom onset (22.4%), or within 48 hours prior to m a n u s c r i p t 6 symptom onset (17.6%). over 12% reported not working more than 48 hours before symptom onset. the last day worked was unknown for 17.3% of hcw cases because one or more dates were missing. more than 6% of hcws reported being completely asymptomatic. at the time of interview, 5.3% of hcws reported requiring hospitalization due to covid-19. as of may 31 st , there were 40 (0.7%) deaths among hcws with confirmed covid. compared to the overall median age of reported hcws, 42 years old (range 17 to 85), the median age of hcws who died was higher at 60 years old (range 32 to 75). twenty percent of those who died were older than 65 and 86.6% had a known co-morbidity. the covid-19 pandemic has placed immense pressure on healthcare infrastructure and hcws. it is likely that hcws in lac, and across the world, have been disproportionately infected with covid-19 compared to the general public due to high rates of exposure in healthcare facilities, limited availability of personal protective equipment (ppe) nationwide, and delayed understanding of the risk of asymptomatic transmission of covid-19. pressure to work during the pandemic, lack of paid sick leave, and staffing shortages may have led many hcws to work while symptomatic. though our data are unable to capture the nature of the precise exposures of hcws, nearly half knew of an exposure to patients and/or co-workers within their facility. this is similar to reports from china and italy which found one patient could be the source of infection for 10 hcws (5) . although lac numbers are still higher than reports from china, which reported only 4% of cases were hcws (6), lac is seeing a decline in hcw cases. this is likely due to improved availability of ppe, better institutional infection control practices, and adoption of universal source control, all helping to additionally, widespread testing is a major factor in understanding and controlling the spread of covid-19. testing of hcws has been a priority from the outset of the pandemic in the setting of limited access to testing, but as testing capacity expanded, free city and county testing sites opened to those with symptoms at the end of march, prioritizing high risk individuals. in mid-april, all hcws could get tested and by the beginning of may, any lac resident could get tested, regardless of symptoms (9). expansion of testing could account for the decreased proportion of hcw casepatients. it is also likely to increase the number of asymptomatic case-patients, leading to lac dph needing to better understand the risks of asymptomatic transmission. compared to the general lac case-patientss (10), hcw case-fatality rates and hospitalization rates are much lower; 0.7% and 5.3% for hcws compared to 4.3% and 12.2%, respectively, for lac overall. the significant difference in severity of covid-19 presentation and mortality seen in hcws compared to the general population case-patients is likely an artifact of the testing strategy but may also reflect the younger demographics of hcws. this is suggested by the median age of hcws, 42 years old (range 17 to 85 years), compared to 45 years old (range 1 month to 107 years) for all lac a c c e p t e d m a n u s c r i p t 8 cases. an additional limitation of the data stems from the fact that interviewers were unable to interview approximately 40% of covid-19 cases; thus, there may be additional hcw cases which have not been identified. furthermore, many hospitalized cases were unable to be interviewed, which might have impacted our low hospitalization rate for hcws. in response to this data, lac dph was able to better focus resources. teams were developed to work with distinct settings and provide guidance relevant to their specific needs. for example, as outpatient facilities fully re-opened following safer at home orders ending in may, lac dph dedicated a team to provide overall outpatient guidance, as well as specific recommendations for specialized settings, such as dentists, dialysis, outpatient surgery centers, etc. healthcare facilities and their workers are a vulnerable population during the covid-19 pandemic. not only are hcws at risk of becoming ill themselves, but they also risk passing the infection to their patients, co-workers, families, and staff or patients at other locations if they work in multiple healthcare facilities. healthcare facilities continue to face obstacles, most notably in ppe supply and staffing shortages. hcw infections declined following implementation of universal masking and more aggressive symptoms screening in facilities, suggesting that a stable supply of ppe and symptom checks can best protect not only hcw but also the populations they serve. these data may also provide lessons on how best to protect the community going forward as communities reopen and consider guidance on masking and symptom checking. there are no conflicts of interest or funding sources to disclose. m a n u s c r i p t 14 licensed & certified healthcare facilities facilitycounts/facilitycounts?iframesizedtowindow=true&:embed=y&:showappbanner=f alse&:display_count=no&:showvizhome=no characteristics of health care personnel with covid-19 -united states clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan covid-2019) infection among health care workers and implications for prevention measures in a tertiary hospital in wuhan, china the impact of novel coronavirus sars-cov-2 among healthcare workers in hospitals: an aerial overview characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from a c c e p t e d m a n u s c r i p t 9 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 16 a c c e p t e d m a n u s c r i p t 18 figure 1 key: cord-329323-1cquorhs authors: ko, jean y; danielson, melissa l; town, machell; derado, gordana; greenlund, kurt j; daily kirley, pam; alden, nisha b; yousey-hindes, kimberly; anderson, evan j; ryan, patricia a; kim, sue; lynfield, ruth; torres, salina m; barney, grant r; bennett, nancy m; sutton, melissa; talbot, h keipp; hill, mary; hall, aron j; fry, alicia m; garg, shikha; kim, lindsay title: risk factors for covid-19-associated hospitalization: covid-19-associated hospitalization surveillance network and behavioral risk factor surveillance system date: 2020-09-18 journal: clin infect dis doi: 10.1093/cid/ciaa1419 sha: doc_id: 329323 cord_uid: 1cquorhs background: data on risk factors for covid-19-associated hospitalization are needed to guide prevention efforts and clinical care. we sought to identify factors independently associated with covid-19-associated hospitalizations methods: u.s. community-dwelling adults (≥18 years) hospitalized with laboratory-confirmed covid-19 during march 1–june 23, 2020 were identified from the covid-19-associated hospitalization surveillance network (covid-net), a multi-state surveillance system. to calculate hospitalization rates by age, sex, and race/ethnicity strata, covid-net data served as the numerator and behavioral risk factor surveillance system estimates served as the population denominator for characteristics of interest. underlying medical conditions examined included hypertension, coronary artery disease, history of stroke, diabetes, obesity [bmi ≥30 kg/m (2)], severe obesity [bmi≥40 kg/m (2)], chronic kidney disease, asthma, and chronic obstructive pulmonary disease. generalized poisson regression models were used to calculate adjusted rate ratios (arr) for hospitalization results: among 5,416 adults, hospitalization rates were higher among those with ≥3 underlying conditions (versus without)(arr: 5.0; 95%ci: 3.9, 6.3), severe obesity (arr:4.4; 95%ci: 3.4, 5.7), chronic kidney disease (arr:4.0; 95%ci: 3.0, 5.2), diabetes (arr:3.2; 95%ci: 2.5, 4.1), obesity (arr:2.9; 95%ci: 2.3, 3.5), hypertension (arr:2.8; 95%ci: 2.3, 3.4), and asthma (arr:1.4; 95%ci: 1.1, 1.7), after adjusting for age, sex, and race/ethnicity. adjusting for the presence of an individual underlying medical condition, higher hospitalization rates were observed for adults aged ≥65, 45-64 (versus 18-44 years), males (versus females), and non-hispanic black and other race/ethnicities (versus non-hispanic whites) conclusion: our findings elucidate groups with higher hospitalization risk that may benefit from targeted preventive and therapeutic interventions as of june 26, 2020, >9 million cases of coronavirus disease 2019 , the disease caused by sars-cov-2, have been reported worldwide (1); over 2 million cases, including >120,000 deaths, have been reported in the united states (2) . older age and underlying medical conditions are prevalent among cases (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . preliminary estimates indicate approximately 30% of u.s. laboratory-confirmed covid-19 cases were among adults aged ≥65 years (7, 8) , and about one third had underlying medical conditions (9) . among u.s. hospitalized cases, diabetes mellitus (8, 9, 10, 11, 12, 13, 14) , hypertension (10, 11, 12, 13, 14) , cardiovascular disease (8, 9, 10, 14) obesity (10, 11, 13, 14) , and chronic lung disease (8, 9, 10) were common. however, the risk of hospitalization imparted by underlying medical conditions is not clear; many of these conditions (e.g., obesity (15) , hypertension (16) , and diabetes (17) are also prevalent in the general u.s. similarly, the risk of hospitalization related to sex and race/ethnicity is unclear. an estimated 60% of new york patients hospitalized for covid-19 were male (11) ; however, other studies have found the male-female distribution among covid-19 hospitalizations was similar to the general u.s. population (50%) (10, 18) . non-hispanic black adults comprised a greater proportion of hospitalized covid-19 cases compared to the community population in 14 states (10) and to overall hospitalizations in georgia (18). to better understand the independent association of age, sex, race/ethnicity, and underlying medical conditions with covid-19-associated hospitalization relative to the nonhospitalized community-dwelling population, we calculated rate ratios for adults with and without select underlying medical conditions, adjusted for age, sex, and race/ethnicity, using data from the coronavirus disease 2019-associated hospitalization surveillance network a c c e p t e d m a n u s c r i p t 6 (covid-net) and the behavioral risk factor surveillance system (brfss), two large multi-state surveillance systems. covid-net is an all age population-based surveillance system of laboratoryconfirmed covid-19-associated hospitalizations (10, 19) . patients must have a positive sars-cov-2 test no more than 14 days before admission or during hospitalization; be a resident of the pre-identified surveillance catchment area; and be admitted to a hospital where residents of the surveillance catchment area receive care. trained surveillance officers use a standard case report form to abstract additional data, such as patient demographics, underlying medical conditions, clinical course, and outcomes data, from medical charts. community-dwelling adults (≥18 years) were identified from 70 counties in 12 states (california, colorado, connecticut, georgia, maryland, michigan, minnesota, new mexico, new york, oregon, tennessee, and utah) participating in covid-net; herein referred to as the covid-net catchment area. as of june 23, 2020, 5,715 adult covid-19-associated hospitalizations were eligible for inclusion in our analysis; 5,416 adults had underlying medical condition data and were included as hospitalized cases for this study (figure 1 ). the brfss is a nationwide cross-sectional telephone survey that collects state-based data on health-related risk behaviors, chronic health conditions, and use of preventive services from more than 400,000 community-dwelling adults (≥18 years) annually (20) . brfss data were used to calculate weighted population estimates of community-dwelling adults with and without individual underlying medical conditions by age, sex, and race/ethnicity strata for the covid-net catchment area and served as the analytic a c c e p t e d m a n u s c r i p t 7 denominators. responses from adults residing in the covid-net catchment area were weighted using the brfss iterative proportional fitting method, which incorporates a number of factors to improve the degree and extent to which the weighted brfss sample reflects the sociodemographic make-up of the covid-net catchment area (20) . weights also account for survey design, probability of selection, nonresponse bias, and non-coverage error (20) . in covid-net, underlying medical conditions were ascertained if the condition (hypertension; history of myocardial infarction, coronary artery disease, coronary artery bypass grafting; stroke; diabetes mellitus; chronic kidney disease; asthma; chronic obstructive pulmonary disease [copd] ) was present in the patient's medical chart that detailed their covid-19-associated hospitalization. in brfss, underlying medical conditions were based on self-report to the question: -has a doctor, nurse, or other health professional ever told you that you had… ‖ (high blood pressure; heart attack also called myocardial infarction, angina or coronary heart disease; stroke; diabetes, chronic kidney disease; asthma; copd, emphysema, or chronic bronchitis). for brfss, 2018 data was used for each underlying medical condition except for hypertension, where 2017 was the most recent year of available data. histories of myocardial infarction, coronary artery disease, and coronary artery bypass grafting (only available in covid-net) were categorized as coronary artery disease. in brfss, adults who self-reported having high blood pressure and answered -yes‖ to the subsequent question -are you currently taking medication for your high blood pressure?‖ were categorized as having hypertension. in covid-net, body mass index (bmi) was calculated using height and weight listed in medical charts; if these data were not available, recorded bmi was used. in brfss, self-a c c e p t e d m a n u s c r i p t 8 reported height and weight were used to calculate bmi. bmi was categorized as obese (≥30 kg/m 2 ) or severely obese (≥40 kg/m 2 ). for both covid-net and brfss data, we created an -any condition‖ variable and -number of conditions‖ variable (0; 1; 2; 3+). hypertension was not included in the -any condition‖ or -number of conditions‖ variables because covid-net catchment estimates for hypertension were derived from 2017 brfss estimates and could not be integrated with the other 2018 estimates of underlying medical conditions. although hypertension is not included in these composite variables, in 2017, 14% of adults with treated hypertension also had at least one other underlying medical condition examined in this analysis. additional details are available in supplemental table 1 . the following categories were defined for age (18-44; 45-64; ≥65 years), sex (male; female), and race/ethnicity (non-hispanic white; non-hispanic black; other). other race/ethnicity groups were aggregated due to small cell sizes once these data were stratified by age, sex and underlying medical conditions. demographic characteristics were tabulated among hospitalized covid-19 cases overall and by underlying medical condition. the percentage of adults with select underlying medical conditions was calculated among covid-19-associated hospitalizations and residents of the covid-net catchment area. to understand if the prevalence of underlying medical conditions in the covid-net catchment area was different from national estimates, nationwide brfss weighted data were compared to estimates obtained for the covid-net catchment area. hospitalization rates by underlying medical condition and age, sex, race/ethnicity strata were calculated. for each rate, the numerator was the number of hospitalized adults from covid-net in the strata of interest; the denominator was the estimated number of adults from the community in the strata of interest, derived from brfss data for the covid-m a n u s c r i p t 9 net catchment area. generalized poisson regression models with a scaled deviance term to account for overdispersion were used to calculate unadjusted and adjusted rate ratios and 95% confidence intervals (cis) associated with hospitalization. multivariable models included an individual underlying medical condition, age, sex, and race/ethnicity. model goodness of fit was assessed by evaluating standardized deviance residual plots. rate ratios with 95% cis that excluded 1 were considered statistically significant. we also assessed the prevalence of co-occurring conditions in hospitalized cases (supplemental table 2 ); however, due to the analytic design of this study and small cell counts of brfss estimates from the covid-net catchment area, we were unable to account for combinations of underlying medical conditions in our adjusted models. sas v.9.4 (sas institute, cary, nc) was used for analyses. no personal identifiers were included in either covid-net or brfss data submitted to cdc. this analysis was exempt from cdc's institutional review board, as it was considered part of public health surveillance and emergency response. participating sites obtained approval for covid-net surveillance from their respective state and local irbs, as required. of 5,416 community-dwelling adults with covid-19-associated hospitalization, 30% were aged 18-44 years, 40% were aged 45-64 years and 31% were aged 65+ years; 53% were male; 34% were non-hispanic white, 32% were non-hispanic black and 34% were of other races/ethnicities (table 1) . overall, 55% had obesity, 49% had hypertension, 33% had diabetes, 16% had severe obesity, 13% had asthma, 12% had chronic kidney disease, 9% had a history of coronary artery disease, 6% had copd, and 4% had a history of stroke. excluding hypertension, 73% of hospitalized cases had at least one underlying medical a c c e p t e d m a n u s c r i p t 10 condition. co-occurring underlying medical conditions were common among hospitalized cases (supplemental table 2 ). among hospitalized cases, the prevalence of underlying medical conditions was greatest among adults aged 65+ years except for obesity, severe obesity, and asthma (table 1 ). the prevalence of obesity (63%) and severe obesity (25%) was greatest among adults aged 18-44 years. males and females had similar prevalences of history of stroke, diabetes, and copd. the prevalence of underlying medical conditions was highest among non-hispanic black adults, except for coronary artery disease and copd. the overall prevalence of selected underlying medical conditions was greater among hospitalized cases compared to the covid-net catchment area population ( figure 2 ). covid-net catchment area estimates were similar or slightly lower than nationwide estimates: hypertension (21% vs. 25%), coronary artery disease (5% vs. 7% ), history of stroke (3% vs. 3%), diabetes (9% vs. 11%), obesity (28% vs. 31%), severe obesity (4% vs 5%), chronic kidney disease (2% vs. 3%), asthma (10% vs. 9%), and copd (5% vs. 7%). unadjusted rate ratios for covid-19-associated hospitalizations of adults 45-64 years of age and 65 years and older, versus 18-44 years, were 2.0 (95%ci: 1.8, 2.1) and 2.7 (95%ci: 2.5, 2.9), respectively ( table 2 ). the unadjusted rate ratio for hospitalization comparing males to females was 1.2 (95%ci: 1.1, 1.3) and for non-hispanic black to non-hispanic white adults was 3.9 (95%ci: 3.7, 4.2). adults with, versus without, specified underlying medical conditions had higher hospitalization rates; unadjusted rate ratios ranged from 1.2 (95%ci: 0.4, 3.8) for copd to 5.3 (95%ci: 2.4, 12.1) for chronic kidney disease. the rate ratios for most underlying medical conditions attenuated after adjustment for age, sex, and race/ethnicity; rate ratios for severe obesity and asthma remained stable ( table 2 ). the adjusted rate ratios (arr) for hospitalization by underlying medical condition were as whites had higher hospitalization rates. these associations were similar in models adjusting for any condition ( table 2) . in a separate model, after adjustment for age, sex, and race/ethnicity, rate ratios for hospitalization increased with the number of underlying conditions, with the greatest rate ratio for adults with 3+ conditions (arr: 5.0; 95%ci:3.9, 6.3) compared to those with no underlying conditions (table 3) . in this study utilizing two large multi-state surveillance systems to compare hospitalized cases with the community at risk, we found that increasing age, male sex, non-hispanic black race/ethnicity, and select underlying medical conditions were associated with a significantly greater risk for covid-19-associated hospitalization relative to the nonhospitalized community-dwelling adult population. among the underlying medical conditions studied, the magnitude of risk was greatest for severe obesity, chronic kidney a c c e p t e d m a n u s c r i p t 12 disease, diabetes, obesity, and hypertension; each of these conditions was independently associated with approximately 3 or more times the risk of hospitalization after accounting for age, sex, and race/ethnicity. our study extends the literature by quantifying the independent association of underlying medical conditions with hospitalization relative to the community population at risk. the magnitude of risk for covid-19-associated hospitalization was lower for coronary artery disease, stroke, asthma, and copd than for other medical conditions (e.g., hypertension) in our analysis. our prevalence estimates of asthma and/or copd (18%) was similar to a study among adults who tested positive for sars-cov-2 (15%), which found that asthma or copd was not independently associated with risk for hospitalization (21) . however, among hospitalized patients, coronary artery disease and copd have both been found to be associated with intensive care unit admission, need for mechanical ventilation (22, 23) and mortality (22, 23, 24) . several hypotheses are under evaluation regarding the linkage between specific underlying conditions and covid-19. obesity might predispose persons to severe covid-19 due to inflammation, altered physiology, and immune dysfunction (25) . diabetes mellitus also imparts a chronic low-grade inflammatory state (26) and predisposes persons to infections in general (27) . impaired t-cell function and increased interleukin-6 are specific factors that increase risk and severity of sars-cov-2 infection among persons with diabetes (27) . finally, the interaction of sars-cov-2 and the renin-angiotensin aldosterone system might contribute to the overrepresentation of hypertension among severe covid-19 patients (28) . we found that ages 45-64 years and 65+ years were independently associated with increased risk of hospitalization compared to ages 18-44 years after accounting for underlying medical conditions, sex, and race/ethnicity. further, the magnitude of risk for a c c e p t e d m a n u s c r i p t 13 hospitalization was greatest among adults 65 years and older, similar to other studies (21, 29) . it is important to note that the additional risk of age groups >45 years is relative to a younger adult age group (18-44 years) and should not be interpreted as absolute risk. males were 30% more likely to be hospitalized than females after accounting for age, race/ethnicity, and underlying medical conditions, similar to another study (21) . nonbiological factors may lead to a greater proportion of males being hospitalized (e.g., increased exposure or delays in care seeking). biological factors could include immune function suppression by testosterone compared to estrogen (30) or lower expression of angiotensinconverting enzyme 2, a receptor that allows entry of sars-cov-2 into host cells, due to estrogen, potentially inhibiting severe clinical progression in females compared to males (31) . over-representation of non-hispanic black adults among hospitalized covid-19 patients has been hypothesized to be due, in part, to the higher prevalence of underlying medical conditions (10, 29) such as hypertension, obesity, diabetes, and chronic kidney disease among the non-hispanic black population (15, 16, 17, 32) . while these conditions contributed to the total risk, we found that after accounting for underlying medical conditions, age, and sex, non-hispanic black adults had four times greater risk of hospitalization than non-hispanic white adults. additionally, the magnitude of risk was similar across underlying medical conditions (arr range: 4.0 to 4.7), suggesting that non-hispanic black adults experience excess risk regardless of select underlying medical conditions. this association was also observed when controlling for the presence of any condition or the number of conditions. black race was similarly associated with 3 times the risk of hospitalization in an atlanta-based study (33). it has been suggested that non-hispanic black adults might have increased risk of hospitalization due to increased exposures (e.g., related to occupation or housing) that could lead to increased incidence or more severe illness; differences in health care access or utilization; or systemic social inequities, including a c c e p t e d m a n u s c r i p t 14 racism and discrimination (34, 35, 36) . however, we were unable to assess these factors with our data. these factors may also explain similar findings of increased risk for hospitalization among other race/ethnicities compared with non-hispanic white persons. overall, these results have implications for clinical practice, as they identify high-risk patients who require closer monitoring and management of their chronic conditions during the ongoing covid-19 pandemic. while specific underlying medical conditions imparted higher risk of hospitalization, we were unable to account for the duration of each condition or the degree to which each condition was controlled (e.g., glycemic control in diabetic patients). nevertheless, clinicians might prioritize more aggressive control of underlying conditions with available treatments and encourage their patients to remain engaged in care for management of their chronic conditions while practicing preventive measures, such as wearing a cloth face covering and social distancing. these groups may also benefit from targeted preventative and therapeutic interventions. this study had several limitations. first, this analysis was based on clinical data available as of june 23, 2020 from covid-net, a surveillance system designed first to provide hospitalization rates. clinical data on underlying medical conditions was reliant on medical chart abstraction; charts from approximately 60% of the total hospitalized cases have yet to be abstracted. thus, included cases represent a convenience sample of hospitalizations with underlying medical conditions, which may have resulted in biased estimates of risk. however, bi-weekly updates of this analysis over a 2-month period with the most recently available covid-net data (i.e., additional chart abstractions) suggested consistent estimates of the frequency and distribution of underlying conditions and resulting rate ratios. second, this analysis did not include institutionalized adults. third, estimates of risk are restricted to the covid-net catchment area; the interpretation of rate ratios as risk in this analysis a c c e p t e d m a n u s c r i p t 15 assumes that risk of sars-cov-2 infection was consistent across all groups. fourth, after stratification by age, race/ethnicity, sex, and underlying conditions, each strata from the covid-net catchment population became small. thus, we were unable to assess the association of more granular race/ethnicity categories or co-occurring underlying health conditions on risk of covid-19-associated hospitalization; further investigation on both aspects is important. fifth, covid-net likely under-ascertains covid-19 cases as testing for sars-cov-2 was performed at treating health care providers' discretion and was subject to clinician bias as well as variability in testing practices and capabilities across providers and facilities. however, this probably had minimal impact on our findings as hospitalized individuals are more likely to be tested than those in the community. finally, we used brfss to obtain estimates for underlying medical conditions in the covid-net catchment area. as the ascertainment of underlying medical conditions was different across the two data systems (self-report vs. medical chart abstraction), we may have introduced bias in the rate ratio estimation. self-reported diabetes (37) and hypertension (38) have high correlation with medical examination estimates. self-report has been found to underestimate prevalence of chronic kidney disease (32) and obesity (38) ; thus, our rate ratios for these conditions may be overestimated. this analysis quantifies associations of age, sex, race/ethnicity, and underlying medical conditions with risk of covid-19 hospitalization relative to the non-hospitalized community-dwelling population. these data may aid clinicians in identifying individuals at higher risk for hospitalization who may require more vigilant care and monitoring, and groups that may benefit from targeted preventive and therapeutic interventions. emerging infections program, georgia department of health, veterans affairs medical center cdc division of population health us department of health and human services, cdc; 2020 clinical characteristics of coronavirus 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associated with worse in-hospital outcomes, and higher in-hospital epidemiology, clinical course, and outcomes of critically ill adults with covid-19 in new york city: a prospective cohort study clinical features and outcomes of 105 hospitalized patients with covid-19 in prevalence of obesity and severe obesity among adults: united states prevalence of self-reported hypertension and antihypertensive medication use among adults -united states characteristics and clinical outcomes of adult patients hospitalized with covid-19 -georgia risk factors for intensive care unit admission and in-hospital mortality among hospitalized adults identified through the u.s. coronavirus disease 2019 (covid-19)-associated hospitalization surveillance network (covid-net) us department of health and human services factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in new york city: prospective cohort study chronic obstructive pulmonary disease is associated with severe coronavirus disease 2019 (covid-19) severity and mortality associated with copd and smoking in patients with covid-19: a rapid systematic review and meta-analysis autopsy findings and venous thromboembolism in patients with covid-19 covid-19 and obesity: exploring biologic vulnerabilities, structural disparities, and weight stigma covid-19 in people with diabetes: understanding the reasons for worse outcomes diabetes in covid-19: prevalence, pathophysciology, prognosis and practical considerations covid-19 and hypertension: what we know and don't know. american college of cardiology hospitalization and mortality among black patients and white patients with covid-19 sex and gender differences in health: what the covid-19 pandemic can teach us sex-specific sars-cov-2 mortality: among hormone-modulated ace2 expression, risk of venous thromboembolism and hypovitaminosis d chronic kidney disease in the united states covid-19 and african americans sharpening the global focus on ethnicity and race in the time of covid-19 validity and reliability of self-reported diabetes in the atherosclerosis risk in communities study comparison of examination-based and self-reported risk factors for cardiovascular disease rate ratios; ci: confidence interval; copd: chronic obstructive pulmonary disease; covid-net: coronavirus disease 2019-associated hospitalization surveillance network; rr: rate ratio *each adjusted model for underlying medical condition includes the select underlying medical condition, age, sex, and race/ethnicity underlying medical condition excludes hypertension to align with 2018 brfss community estimates of underlying medical conditions; the most recent year of available brfss data for hypertension was a c c e p t e d m a n u s c r i p t 19 a c c e p t e d m a n u s c r i p t 20 mortality, in a cohort of patients with covid-19 in the bronx, new york [published online ahead of print, 2020 may 16] . metabolism. 2020;108:154262.a c c e p t e d m a n u s c r i p t key: cord-322650-q8inhgtr authors: fung, yin-wan wendy; lau, lok ting; wong, freda pui-fan; choi, kin-wing; chau, tai-nin; lai, sik-to; wang, chen g; dillon, natalie; yu, albert cheung-hoi title: use of clinical criteria and molecular diagnosis to more effectively monitor patients recovering after severe acute respiratory syndrome coronavirus infection date: 2004-08-15 journal: clin infect dis doi: 10.1086/422887 sha: doc_id: 322650 cord_uid: q8inhgtr nan 0.135 mg/l), with a maximum concentration 6 h after injection. these values are in the low range, compared with previously reported ranges [1, 3] . in those 2 reports, information concerning liver function and the concomitant drugs received are not available. these low concentrations are similar to the mics of the aspergillus species and can explain the slow evolution of the abscess. three main explanations can be invoked for these low values. pharmacokinetic interactions do not explain the low concentrations. with regard to the drugs the patient received, the interaction between tacrolimus and voriconazole leads to a higher concentration of tacrolimus and not to a lower concentration of voriconazole [4] . we did not find obvious interactions between voriconazole and prazosine, paroxetine, nicarpidine, or metopimazine. we can assess that the liver graft was not involved in these low concentrations of voriconazole, because it exhibited normal function. the sampling procedure can perhaps explain the low results. the csf specimen was collected during 2 h through the intraventricular catheter. therefore, the in vitro instability of voriconazole and/ or adsorption of voriconazole on the lines and collection materials of the intraventricular derivation are able to decrease measured csf concentrations. furthermore, we noticed in the previously reported data from lutsar et al. [1] that, among samples that yielded the 7 lowest concentrations found (which were similar to ours), 4 samples were obtained through an intraventricular catheter. a nonhomogeneous distribution of voriconazole resulting from compression of the fourth ventricle could also explain the lower concentrations in ventricles, compared with the concentration expected from a lumbar puncture. in conclusion, voriconazole is highly active against aspergillus species, but additional studies are needed to confirm that our low drug concentrations result from the method of sampling and not from poor efficacy of this molecule in the csf. in early 2003, a novel severe acute respiratory syndrome (sars) coronavirus (cov) [1] spread around the world; ultimately, more than 8000 patients in 32 countries contracted sars, many of whom died. although gold standard methods, such as viral culture, can help diagnose sars, these methods are by no means as efficient and rapid as pcr-based diagnostic tests. the speed and sensitivity of molecular diagnostic tests for sars is often considerably greater than than that of serological and viral culture methods [2] . our reported enhanced real-time pcr (ert) method [3, 4] is у100-fold more sensitive than conventional real-time pcr. the higher sensitivity of this method may reveal potential sars cov carriers who have sars cov levels that are undetectable by other methods, and the sensitivity of the ert method may be particularly important for ensuring that patients who have had sars are not infectious before discharge from the hospital [5] . in collaboration with princess margaret hospital (pmh; hong kong), samples obtained from 3 patients during recovery after sars were analyzed (table 1). six to nine weeks after the onset of infection, sars cov could still be detected by ert in certain samples (table 1), indicating that, although clinical signs and symptoms had subsided and a host immune response had been mounted, viral clearance was not complete. patient 1 was transferred on 17 june 2003 to the wong tai sin hospital (wtsh; hong kong), which was converted into a specialized center for convalescent care of patients with sars during the epidemic, but he was returned to pmh because of recurrent pneumothorax, indicated by chest radiography on 18 june. the ert method clearly demonstrated the presence of sars cov in all samples obtained from the patient on 16 june (table 1) , which was 1 day before his transfer to wtsh. the possible relapse of infection in patient 1 after his transfer to another hospital indeed raises the question of how patients with sars who have pcr results negative for sars cov should be handled [5] . standardization of clinical criteria and pcr-based methods should be emphasized to ensure accurate diagnosis of sars after hospital admission and prior to hospital discharge. more studies will be necessary to determine the infectivity status of patients who have ert results positive for sars cov. the data suggest that medical professionals should verify whether residual viral particles present in recovering patients remain infectious and whether they may constitute the source of possible future outbreaks of infection. because sars is a newly emerging disease that causes serious consequences, many countries have formulated contingency plans for possible future sars outbreaks. one of the containment activities currently undertaken by the world health organization to prevent sars from repeatedly becoming a widely established threat is to develop a robust and reliable diagnostic test [6] , which will probably rely on pcr-based technology. use of a highly sensitive method, such as the ert method [3, 4] and a similar method that was reported recently [7] , will be the first step toward more accurate screening of suspected sars carriers and will minimize the occurrence of false-negative cases. patients with false-positive cases can always be quarantined while awaiting further reconfirmation of infection. but patients with false-negative cases could be discharged into the community and pose a dangerous sars threat to the public [8] . therefore, stringent clinical criteria and use of the ert method might effectively monitor patients recovering after sars. voriconazole concentrations in the cerebrospinal fluid and brain tissue of guinea pigs and immunocompromised patients optimal testing conditions for determining mics and minimum fungicidal concentrations of new and established antifungal agents for uncommon molds: nccls collaborative study successful treatment of cerebral aspergillosis with a novel triazole (voriconazole) in a patient with acute leukaemia voriconazole inhibition of the metabolism of tacrolimus in a liver transplant recipient and in human liver microsomes service de maladies infectieuses a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome (sars): laboratory diagnostic tests a real-time pcr for sars-coronavirus incorporatingtarget gene pre-amplification boosting the sensitivity of real-time pcr sars detection by simultaneous reverse transcription and target gene pre-amplification severe acute respiratory syndrome: relapse? hospital infection? severe acute respiratory syndrome (sars): status of the outbreak and lessons for the immediate future sensitive and quantitative detection of severe acute re august) • correspondence spiratory syndrome coronavirus infection by real-time nested polymerase chain reaction the difficulties of testing for sars ᮊ 2004 by the infectious diseases society of america. all rights reserved e. denes, 1 n. pichon, 2 m. debette-gratien, 3 b. bouteille, 4 key: cord-340956-1t3o24u5 authors: borkenhagen, laura k; wang, guo-lin; simmons, ryan a; bi, zhen-qiang; lu, bing; wang, xian-jun; wang, chuang-xin; chen, shan-hui; song, shao-xia; li, min; zhao, teng; wu, meng-na; park, lawrence p; cao, wu-chun; ma, mai-juan; gray, gregory c title: high risk of influenza virus infection among swine workers: examining a dynamic cohort in china date: 2019-09-01 journal: clin infect dis doi: 10.1093/cid/ciz865 sha: doc_id: 340956 cord_uid: 1t3o24u5 background: china is thought to be a hotspot for zoonotic influenza virus emergence, yet there have been few prospective studies examining the occupational risks of such infections. methods: we present the first 2 years of data collected from a 5-year, prospective, cohort study of swine-exposed and -unexposed participants at 6 swine farms in china. we conducted serological and virological surveillance to examine evidence for swine influenza a virus infection in humans. results: of the 658 participants (521 swine-exposed and 137 swine-unexposed), 207 (31.5%) seroconverted against at least 1 swine influenza virus subtype (swine h1n1 or h3n2). swine-exposed participants’ microneutralization titers, especially those enrolled at confined animal feeding operations (cafos), were higher against the swine h1n1 virus than were other participants at 12 and 24 months. despite elevated titers, among the 187 study subjects for whom we had complete follow-up, participants working at swine cafos had significantly greater odds of seroconverting against both the swine h1n1 (odds ratio [or] 19.16, 95% confidence interval [ci] 3.55–358.65) and swine h3n2 (or 2.97, 95% ci 1.16–8.01) viruses, compared to unexposed and non-cafo swine workers with less intense swine exposure. conclusions: while some of the observed increased risk against swine viruses may have been explained by exposure to human influenza strains, study data suggest that even with elevated preexisting antibodies, swine-exposed workers were at high risk of infection with enzootic swine influenza a viruses. since the 1950s, china has led the world in swine farming. currently, china produces nearly half of the world's pork supply (approximately 45 million head in 2018) [1, 2] and the number of pigs continues to grow as china moves from small-scale swine farms to industrial farming practices [3] . these large, industrial swine farms, commonly known as confined animal feeding operations (cafos), are thought to be sites for novel pathogen emergence and transmission. in recent years, swine pathogen epizootics in china have included porcine reproductive and respiratory syndrome virus [4] , porcine epidemic diarrhea virus [5] , swine acute diarrhea syndrome coronavirus [6] , and, most recently, a 2018-19 massive outbreak of african swine fever [2] . beyond these swine-only pathogens, swine zoonotic pathogens threaten to harm not only the animals, but also the people exposed to them. in swine cafos, the continual introduction of naive animals, through onsite farrowing or the frequent introduction of new pigs, sustains the pathogen prevalences within a large farm by providing opportunities for pathogens to move from 1 barn to another. in particular, this is true for influenza a viruses (iavs). the sustained circulation of iavs and often poor biosecurity provides great opportunity for swine iavs to reassort or recombine with avian or human iavs, which can result in novel progeny-virus zoonotic transmission events [7, 8] . previous such events have underlined the importance of pigs in the generation of novel iavs, including the pandemic outbreak in 2009 [9] . still, there are relatively few robust, prospective studies examining the risk of zoonotic transmission of swine iavs to swine workers [10] . even fewer studies have captured the incidences of asymptomatic swine influenza virus (siv) infections among humans through routine surveillance [11, 12] . in this 5-year (2015-2019) prospective cohort study of swine workers in china, we are employing virological and immunological surveillance to identify patterns of iav emergence and transmission. the primary aim of our study is to identify the demographic and occupational risk factors for incident siv infections in humans through influenza-like illness (ili) surveillance and serological evidence of exposure to sivs. secondarily, we seek to examine the use of serological and mucosal immunities as biomarkers for protection against siv infections in humans. results from participant enrollment and year 1 of ili surveillance have been previously reported [7, 8] . here, we augment these results with data from the subsequent 24 months of follow-up. as previously reported [7, 8] , 299 swine-exposed participants ("exposed") were enrolled at the beginning in march of 2015 at 9 swine cafos; 97 small, house-holding swine farms; 6 abattoirs, 1 veterinary station; and 1 animal market in shandong and jiangsu provinces in china. we also enrolled 100 nonswine-exposed participants ("unexposed") in these regions in 2015. participants were asked to complete an enrollment survey with questions pertaining to demographics, health status, type of work performed, animal exposure, and personal protective equipment use. participants were also asked to permit the collection of a 5 ml blood draw. annual follow-up visits took place in march of 2016 (12 months) and march of 2017 (24 months) to collect survey data and blood samples. replacements for participants lost to follow-up were enrolled as necessary to maintain a similar sample size. throughout the 24-month study period, participants were asked to contact research staff within 24 hours of developing an ili event (acute onset of a respiratory illness with a measured oral temperature ≥37.8°c and a sore throat or cough for >4 hours) and were also contacted weekly by study staff to monitor for such events. if a participant reported an ili event, they were asked to permit the collection of a nasal swab and nasal wash, as well as a 5 ml blood draw, within 2 days of the report (acute). a second blood draw was collected more than 60 days after the illness (convalescent). nasal washes were performed by irrigating 1 nostril with saline while the participant's head was tilted back to a 70° angle; they were then coached to tilt forward as the return fluid was collected, and the procedure was repeated with the second nostril. we employed influenza a virus molecular detection, microneutralization (mn), and immunoglobin a (iga) assays, as described in our previous study [7] . to assess the possibility of neutralizing-antibody cross-reactivity against circulating human influenza strains, the same mn assay procedure was performed using sera drawn at 12 and 24 months from a random subset of 105 participants (77 exposed and 28 unexposed). a detailed description is in the supplementary information. we used standard descriptive, bivariate, and multivariable risk factor analyses to compare various subgroups of participants. the primary outcomes were seroconversion against the swine h1n1 and h3n2 viruses. our primary modeling framework was a complete case analysis, based on all individuals with full follow-ups through 24 months, using a multivariable logistic regression model to estimate the association between various demographic and occupational risk factors and seroconversion against each virus. generalized estimating equations adjusted for the correlation of participants within a swine facility and within participants over time. inverse probability weighting (ipw) was used to adjust the complete case analysis for possible biases arising from losses to follow-up. a variable selection procedure was applied to find a parsimonious model. see the supplementary information for additional details. the industrialization of pork production in china has led to significant consolidation and the shut down of millions of farms since 2002 [2] ; in 2017, 2 of the 6 farms enrolled in this study were closed, and have since been replaced ( figure 1 ). replacements for participants lost to follow-up were enrolled to maintain a similar sample size. of the 658 participants enrolled at any time point (521 exposed and 137 unexposed), 187 (28.4%) were followed for 24 months (116 exposed and 71 unexposed), 137 (20.8%) were followed for 12 months (118 exposed and 19 unexposed), and 334 (50.8%) were observed at a single time point (287 exposed and 47 unexposed; figure 2 ). the complete case analysis was based on the 187 participants (116 exposed and 71 unexposed) with follow-ups through 24 months. detailed descriptions of the cohort characteristics and changes over time can be found in the supplementary information. considering sera collected at 0, 12, and 24 months, 207 (31.5%) of the 658 enrolled participants (open cohort) seroconverted against at least 1 iav subtype, with a total of 271 seroconversion events ( table 2) . of these events, 32 (28.6%) occurred in individuals enrolled from cafos, 113 (27.6%) in individuals enrolled from non-cafo swine exposure sites (ie, small, house-holding swine farms; abattoirs; a veterinary station; and an animal market), and 62 (45.3%) occurred in swineunexposed individuals. there were 167 seroconversion events (61.6%) against swine h1n1, 97 (35.8%) against swine h3n2, 4 (1.5%) against avian h9n2, 2 (0.7%) against avian h5n6, and 1 (0.4%) against avian h7n9. there were no observed seroconversions against avian h5n1. the multivariable model results for h1n1 and h3n2 seroconversion in the complete case cohort are shown in table 4 . variable selection revealed several baseline characteristics significantly associated with seroconversion; see the supplementary information for a full list of risk factors considered. participants with elevated (≥20) baseline h1n1 mn titers had lower odds of seroconversion (odds ratio [or] 0.18, 95% confidence interval [ci] .08-.37), as did participants who had taken medication in the prior 30 days (or 0.45, 95% ci .20-.98) or who reported a respiratory infection in their household in the prior 12 months (or 0.44, 95% ci .22-.89; table 4 ). there was an association between higher h1n1-specific iga titers at baseline and future seroconversion (or 1.83 for an increase of 1 standard deviation in h1n1specific iga, 95% ci 1.08-4.49). similar to as in h1n1 titers, individuals with elevated baseline h3n2 mn titers had lower odds of seroconverting against h3n2 (or 0.28, 95% ci .11-.68). seroconversion against h3n2 was also found to be positively associated with a participant's report of an outbreak of illness among animals at work in the prior 30 days (or 2.15, 95% ci 1.41-10.76; table 4 ). after including ipw, only the enrollment site type and baseline mn titer maintained statistically significant associations with seroconversion against h1n1, though the point estimates for all risk factors remained similar. for h3n2, ipw did not significantly alter the point estimates or cis for the included risk factors. detailed multivariable modeling results from the open cohort are available in the supplementary information. in the random sample of 105 participants additionally tested with mn assays against seasonal human h1n1 and h3n2 viruses, 29 (27.6%) seroconverted against swine h1n1, 22 (20.9%) seroconverted against human h1n1, 17 (16.2%) seroconverted against swine h3n2, and 16 (15.2%) seroconverted against human h3n2. examining possible cross-reactivity, the odds of participants who seroconverted against human h1n1 also seroconverting against swine h1n1 were 0.72 (95% ci .24-2.18). similarly, the odds of participants who seroconverted against human h3n2 also seroconverting against swine h3n2 were 4.25 (95% ci 1.29-14.02). in the open cohort, the swine-exposed participants were less likely than unexposed participants to seroconvert against sivs; adjusted ors for seroconversion against swine h1n1 and swine h3n2 were 0.72 (95% ci .55-.94) and 0.49 (95% ci .36-.65), respectively ( table 2 ). this effect was also observed for seroconversion against either or both sivs (or 0.47, 95% ci .32-.69), after adjusting for differences in follow-ups using the number of follow-up visits as a proxy for person-time at risk (or 0.54, 95% ci .41-.70). stratification of the exposed group into those exposed and not exposed to a cafo revealed no significant differences in seroconversion against the swine h1n1 virus for the 3 exposure groups. however, the geometric mean of mn titers against swine h1n1 at 12 and 24 months were the same or higher among participants enrolled at cafo facilities, compared both to those enrolled at non-cafo swine facilities and to unexposed participants (table 3) . conversely, higher odds of seroconversion against swine h3n2 virus were observed among the unexposed (or 2.27, 95% ci 1.43-3.60) and cafo-exposed (or 1.98, 95% ci 1.11-3.42), when compared to the non-cafo swine workers; the unexposed participants had higher geometric mean mn titers, compared to the other 2 groups, for all time points (table 3) . there was no correlation between years of swine exposure and seroconversion against swine h1n1 at enrollment, and no differences in this relationship by enrollment site type. a negative correlation was observed between years of swine exposure and seroconversion against swine h3n2 after enrollment (spearman ρ = −0.11, 95% ci −.20 to −.02). among cafo swine workers, the spearman correlation for seroconversion against swine h3n2 was −0.01 (95% ci −.20 to .18); however, among non-cafo swine workers, the spearman correlation for the same outcome was −0.14 (95% ci −.24 to −.04). follow-up n=400 figure 2 . diagram of enrollments, replacements, and losses to follow-up for this longitudinal study. participants exposed and unexposed (unexp.) to swine were followed for influenza-like illness and sampled every 12 months for 24 months. in the complete case cohort, cafo swine workers had significantly greater odds of seroconverting against swine h1n1, compared to either non-cafo swine workers (or 20.11, 95% ci 3.62-380.34) or unexposed participants (or 17.89, 95% ci 3.12-342.01; table 4 ). cafo swine workers had higher odds (though not statistically significant) of seroconverting against h3n2 than unexposed participants (or 2.16, 95% ci .78-6.20) and significantly higher odds of seroconverting against h3n2 than non-cafo swine workers (or 3.90, 95% ci 1.41-10.76; table 4 ). further, cafo swine workers had higher odds of seroconverting against h1n1 (or 19.16, 95% ci 3.55-358.65) and h3n2 (or 2.97, 95% ci 1. 16-8.01 ) when compared to both unexposed participants and non-cafo swine workers with less intense swine exposure. between 12 and 24 months of follow-up, 15 ili events were identified among 10 exposed and 5 unexposed participants out of the open cohort, for incidence proportions of 0.019 and 0.036, respectively (table 1) . of these 15 participants, 13 were enrolled at baseline and followed through 24 months, 1 was lost to follow-up before their 24-month visit, and 1 was enrolled in 2016 and thus only had 12 months of available follow-up. among the 10 exposed participants, 1 worked at a swine cafo, 4 worked at house-holding swine farms, and 5 worked at abattoirs. the average number of pigs housed per day among the enrollment sites where these 10 participants were enrolled was 618, compared to a lower average of 553 pigs among the enrollment sites of the 511 exposed participants who did not have recorded ilis in this time period. all but 1 of these events occurred between 21 december 2016 and 5 january 2017. molecular evidence of iav was detected from either a nasal wash or swab specimen in 4 participants (3 unexposed, 1 exposed). there was 1 exposed participant who seroconverted against swine h1n1, 3 (1 exposed and 2 unexposed) who seroconverted against swine h3n2, and 2 exposed participants who seroconverted against both swine h1n1 and h3n2 viruses. in this report, we present the first 24 months of data from a 5-year prospective, cohort study of iav among participants exposed and unexposed to swine in china. while we were able to replace the participants lost to follow-up for a consistent sample size for the first 12 months, by 24 months, we were unable to maintain the original target sample size. in general, older and less educated participants were more often lost to follow-up. the characteristics of replacement participants were different for exposed and unexposed groups over time. in both follow-up visits at 12 and 24 months, new, unexposed participants were more likely to be female and more likely to have reported at least 1 respiratory tract infection in the past 12 months. this influx of participants with a history of respiratory tract infection and possible exposure to iav, as well as disproportionate follow-ups between the exposed and unexposed groups, may be partially responsible for the elevated titers and higher number of seroconversions observed in the unexposed group. thus, we considered the open cohort in several ways to explore relationships between the follow-up time and the history and extent of exposure. in addition to comparing the exposed and unexposed groups, an adjustment for person-time showed little change in the results; we suspect there were too few observation times (up to 3) for a meaningful effect to be observed with this adjustment. though the number of reported ili events (n = 15) was too low to perform statistical analyses, more ili events were observed among the swine-exposed than -unexposed participants during the study period. these exposed participants worked at enrollment sites with higher average numbers of pigs housed per day, compared to exposed participants who did not experience an ili. along this line, we further stratified the exposed group into cafo and non-cafo workers, assuming that individuals (1) swine-exposed and enrolled at cafo (exposed to cafo swine); (2) swine-exposed and enrolled at sites other than cafo (not exposed to cafo); and (3) not swine-exposed (unexposed). non-cafo sites included house-holding swine farms, abattoirs, a veterinary station, and an animal market. samples were collected at 0 (baseline), 12, and 24 months, with additional acute and convalescent sera samples collected at individually reported influenza-like illness events between follow-up visits. seroconversion against each virus was defined as a 4-fold rise in titer, relative to any previously gathered sample, and 1 titer value ≥40. predicted probabilities and ors were calculated from a univariate logistic regression model using empirical (sandwich) covariance estimates to adjust for repeated measurements within individuals over time. abbreviations: σ, standard deviation; cafo, confined animal feeding operations; ci, confidence interval; h1n1 or h2n2, swine influenza virus subtype; or, odds ratio; ref., referent group. data are stratified by swine-exposed (exposed) and non-swine exposed (unexposed) participants. sera samples were collected from 658 exposed (n = 521) and working at cafos have more intense exposure to large populations of pigs. here, we observed conflicting results, with no significant association with seroconversion against swine h1n1 by enrollment site type and increased odds of seroconversion against swine h3n2 among unexposed participants. we hypothesized that some of this effect for swine h3n2 might have been due to cross-reactivity with this human subtype, and this was supported by the subset of sera samples we tested that demonstrated higher odds of seroconversion against human h3n2 if the participant had also seroconverted against swine h3n2, a trend that was not observed for h1n1. finally, the complete case analysis we conducted among 187 participants followed through 24 months demonstrated results more consistent with previous literature [7, [13] [14] [15] [16] . the results obtained from the complete case analysis aligned with those from the first year of this study [7] , as well as results of a similar cross-sectional study of swine workers in southern china [16] that demonstrated elevated antibody titers for sivs among swine-exposed participants. despite these elevated titers, the longitudinal data still showed that cafo swine workers had a significantly higher risk of seroconverting against swine h1n1, compared to non-cafo swine workers (or 20.11, 95% ci 3.62-380.34) and unexposed participants (or 17.89, 95% ci 3.12-342.01). this is similar to results from a longitudinal study of swine workers in iowa, which found that swine-exposed individuals (or 54.9, 95% ci 13.0-232.6)-and even their nonexposed spouses (or 28.2, 95% ci 6.1-130.1)had a higher risk of seroconverting against swine h1n1 virus than other, nonexposed individuals [13] . while exposed participants were not found to be at a higher risk of seroconversion against swine h3n2 virus, the ors trended in that direction, and we present evidence that suggests some of this effect may be due to cross-reactivity with the seasonal h3n2 virus in humans. although complete case analyses are known to be biased when the data are not missing completely at random, there are several reasons our analysis is justified here. first, our primary outcome measure, by definition, required a minimum of 2 follow-up measurements; therefore, participants observed only at enrollment could not contribute to an analysis of seroconversion. participants observed at only 2 time points are similarly sera samples were collected from 116 participants who were exposed to swine and 71 who were not exposed to swine (unexposed) in the shandong and jiangsu provinces of china between march 2015 and december 2017. swine-exposed participants were either enrolled at cafo facilities (exposed to cafo swine) or at non-cafo facilities (not exposed to cafo swine), including house-holding swine farms, abattoirs, a veterinary station, and an animal market. samples were collected at each of 3 time points (0, 12, and 24 months), with additional acute and convalescent sera samples collected at individually reported influenza-like illness events between follow-up visits. seroconversion against each virus was defined as a 4-fold rise in titer, relative to any previously gathered sample, and 1 titer value ≥40. a multivariable logistic regression using empirical (sandwich) covariance estimates was used to adjust for repeated measures over time. abbreviations: cafo, confined animal feeding operations; ci, confidence interval; h1n1 or h2n2, swine influenza virus subtype; iga, immunoglobin a; mn, microneutralization; or, odds ratio; ref., referent group; sd, standard deviation. less likely to have a seroconversion event, since individuals with 3 time points have more opportunities to seroconvert. further, we observed systematic differences in demographicand exposure-related characteristics between participants who dropped out, participants who remained in the study, and participants recruited as replacement subjects at subsequent time points. overcoming these issues would require strict assumptions about the missing data mechanisms (eg, multiple imputation), which we did not feel were tenable. thus, we opted for a pragmatic approach, reporting the complete case analyses as our primary models, with sensitivity analyses using ipw. even with considerable losses to follow up in the original cohort, compelling analyses were obtained from the 187 study subjects with complete data. among these participants, the most intensively swine-exposed participants (cafo swine workers) had higher titers against swine h1n1 virus and yet higher odds of seroconversion during follow-up to both swine h1n1 and the swine h3n2 viruses. while some of this risk of seroconversion against swine h3n2 could likely be explained by cross-reactivity against human h3n2 iav, these data support the premise that persons with intense occupational exposure to pigs are at high risk (ors ranging from 17-20, depending upon the occupational comparison group) of infection with enzootic swine viruses. it seems logical, then, that more efforts should be made to conduct surveillance for novel iavs, which may first emerge at cafo human-swine interfaces. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. china's pork imports rise along with production costs united states department of agriculture foreign agricultural services united states department of agriculture foreign agricultural services. peoples republic of china livestock and products annual: multiple outbreaks of african swine fever create uncertainty for the world's largest pork producer origin of highly pathogenic porcine reproductive and respiratory syndrome virus origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china evidence for cross-species influenza a virus transmission within swine farms, china: a one health, prospective cohort study prospective surveillance for influenza a virus in chinese swine farms the role of swine in the generation of novel influenza viruses influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than a (h5n1) weighing serological evidence of human exposure to animal influenza viruses− a literature review review of influenza a virus in swine worldwide: a call for increased surveillance and research swine workers and swine influenza virus infections are swine workers in the united states at increased risk of infection with zoonotic influenza virus? serologic evidence of h1 swine influenza virus infection in swine farm residents and employees serological evidence and risk factors for swine influenza infections among chinese swine workers in guangdong province acknowledgments. the authors thank the professionals from the licheng district center for disease control and prevention (cdc), jinan, china; the shandong provincial cdc, junan, china; and the wuxi cdc, wuxi, china, who greatly helped in engaging the swine farms and in the collection of samples. they thank the farm managers and the study participants for their much-appreciated cooperation.disclaimer. the contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the funding sources listed. potential conflicts of interest. all authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-354265-udt2spoe authors: gersh, felice; lavie, carl j; o’keefe, james h title: menopause status and covid-19 date: 2020-09-23 journal: clin infect dis doi: 10.1093/cid/ciaa1447 sha: doc_id: 354265 cord_uid: udt2spoe nan a c c e p t e d m a n u s c r i p t dear editor, we greatly appreciate the publication of this important research article, for its exploration of the connection of estradiol levels and menopausal status with outcomes from infections with sars-cov-2 in women. (1) this study has been greatly needed from the inception of the covid-19 pandemic. sadly, unlike this research, most published data lacks stratification of women into pre and postmenopausal categories, making the determinations made in this article an impossibility. (2) a pervasive lack of understanding of the myriad effects that estradiol plays throughout the female body has resulted in the exclusion of this critical information being considered in much research and in the clinical care of women. we advocate for the use of physiologically dosed human-identical transdermal estradiol as hormone replacement, combined with human-identical cyclic progesterone, in appropriate recently menopausal women. our recommendations are based on a significant body of preclinical and clinical data. (3) this study's findings of a distinctly protective effect of estradiol in women with functioning ovaries is in complete alignment with our position and with science. (4) estradiol has receptors on all innate and adaptive immune cells and is a key player in the immune response, which includes both pro-inflammatory and anti-inflammatory functions. (5) estradiol (e2) is a modulator of the renin-angiotensin-aldosterone system, a major force in the instigation of the inflammatory response and in the resolution of inflammation. (6) e2 plays a major role in regulating lipid mediators and peptides involved in the processes needed for an optimal immune response, improving the likelihood of a successful outcome in the fight against an infectious agent such as sars-cov-2. (7) the use of hormone replacement therapy gains further support from this excellent study. the harmful impact of ovarian senescence affects all organ systems, inclusive of the cardiovascular system, the neurological system, the gut, the musculoskeletal system, the genitourinary system, and now in the age of the covid-19 pandemic, its vital role with the immune system is clear. (8) given the potential for serious negative effects ensuing from a state of estradiol deficiency, heightened by the covid-19 pandemic, not only should appropriate postmenopausal women be considered for hormone replacement therapy, but women being treated with aromatase inhibitors and estrogen receptor antagonists should be counseled on the risks and benefits of those drugs, personalized in each case, in light of the findings of this study. none of the authors has any potential conflicts of interest. potential influence of menstrual status and sex hormones on female sars-cov-2 infection: a cross-sectional study from multicentre in wuhan data secrecy is crippling attempts to slow covid-19's spread in u.s., epidemiologists warn back to the future: hormone replacement therapy as part of a prevention strategy for women and the onset of menopause menopause and hormone replacement therapy in the 21 st century sex hormones and immune dimorphism mind over cytokines: crosstalk and regulation between the neuroendocrine and immune systems 17-beta estradiol enhances prostaglandin e2 production in human u937-derived macrophages loww of ovarian hormones and accelerated somatic and mental aging a c c e p t e d m a n u s c r i p t key: cord-352899-bt2xg0ha authors: van kerkhove, maria d.; peiris, malik j. s.; malik, mamunur rahman; ben embarek, peter title: interpreting results from environmental contamination studies of middle east respiratory syndrome coronavirus date: 2016-10-15 journal: clin infect dis doi: 10.1093/cid/ciw478 sha: doc_id: 352899 cord_uid: bt2xg0ha nan to the editor-middle east respiratory syndrome coronavirus (mers-cov) continues to pose a threat to human health among populations in contact with infected dromedary camels and globally through the travel of persons who acquired infection from areas with enzootic dromedary infection. transmission of mers-cov is categorized as zoonotic, with repeated introductions into the human population after contact with infected dromedaries, resulting in limited human-to-human transmission, notably in healthcare settings [1] the mers-cov outbreak in south korea in 2015, resulting in 186 cases and 38 deaths [2] , reminds us that the introduction of a single case into an unsuspecting healthcare system can trigger a very large outbreak with serious public health and socioeconomic consequences. although we have known for quite some time that nosocomial transmission is responsible for more than half of reported mers-cov cases worldwide [1] , we know little about how transmission occurs in healthcare settings. we have suspected, and our involvement in several missions to affected countries in the kingdom of saudi arabia, jordan, and south korea has confirmed, that delayed recognition and slow isolation of infected patients, extended stays in overcrowded emergency rooms, and suboptimal adherence to infection prevention and control procedures have been responsible for secondary cases in these settings [3] , but the role of environmental contamination has received relatively little attention. researchers in korea have recently published studies [4, 5] evaluating environmental and/or air contamination by mers-cov and should be commended for their efforts to evaluate the hospital outbreaks in their country. taken together, these results from kim et al [4] and others [5] [6] suggest that mers-cov can persist on the surfaces in contaminated environments, such as patient rooms and equipment, and underscore the critical importance of adequate and thorough disinfection of patient rooms during and after their treatment in healthcare settings. although the findings from kim et al [4] are very interesting and raise questions about the potential presence of mers-cov in the air, they provide no evidence of airborne transmission. some concerns have been expressed that the claimed "virus isolation" did not yield virus isolates but rather was based on reverse-transcription polymerase chain reaction detection of infected cells [7] . kim et al [4] did go further to demonstrate passage of virusinfected cells leading to detectable viral antigen detection, which is better evidence of replicating virus. we are aware of a number of other environmental studies conducted in hospitals that have found surface contamination but have not been able to isolate virus from air samples. these studies have not yet been published in the peer-reviewed literature. it is important that these and any negative results are also published, or else we will be left with a skewed selection of positive findings. the partial inconsistencies in results between studies will need further examination. in particular, it is important that future studies include "negative controls" with comparable sampling and testing strategies applied in settings where patients with mers were not housed. to fully understand the possible role of environmental contamination, including the possible detection of mers-cov in the air, additional studies must be conducted to see whether these results can be replicatedfor example, in hospitals in the middle east where patients with mers-cov are treated, to evaluate virus persistence in hospital environments. a more complete understanding of the results of environmental and air contamination studies will have important implications for the application of infection prevention and control measures [8] currently used in hospitals treating patients with mers-cov, possibly leading to more detailed recommendations. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. maria d. van kerkhove, 1,2 malik j. s. peiris, 3 mamunur rahman malik, 4 and peter ben embarek 2 middle east respiratory syndrome coronavirus (mers-cov): current situation 3 years after the virus was first identified preliminary epidemiological assessment of mers-cov outbreak in south korea notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers outbreak units environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions environmental contamination and viral shedding in mers patients middle east respiratory syndrome coronavirus (mers-cov) key: cord-345045-nlui9d6e authors: zahn, matthew; adalja, amesh a; auwaerter, paul g; edelson, paul j; hansen, gail r; hynes, noreen a; jezek, amanda; macarthur, rodger d; manabe, yukari c; mcgoodwin, colin; duchin, jeffrey s title: infectious diseases physicians: improving and protecting the public’s health: why equitable compensation is critical date: 2019-07-15 journal: clin infect dis doi: 10.1093/cid/ciy888 sha: doc_id: 345045 cord_uid: nlui9d6e infectious diseases (id) physicians play a crucial role in public health in a variety of settings. unfortunately, much of this work is undercompensated despite the proven efficacy of public health interventions such as hospital acquired infection prevention, antimicrobial stewardship, disease surveillance, and outbreak response. the lack of compensation makes it difficult to attract the best and the brightest to the field of id, threatening the future of the id workforce. here, we examine compensation data for id physicians compared to their value in population and public health settings and suggest policy recommendations to address the pay disparities that exist between cognitive and procedural specialties that prevent more medical students and residents from entering the field. all id physicians should take an active role in promoting the value of the subspecialty to policymakers and influencers as well as trainees. in recent decades, emerging and reemerging infectious diseases (id) have caused outbreaks with national and international implications and have underscored the critical need for id expertise. id physicians do more than protect the health of their patients. due to the unique communicable nature of id, id physicians provide a population-level service by helping to secure the health of the community. however, as id threats to public health continue to emerge, the number of young physicians applying for id subspecialty training continues to wane. id physicians lead public health responses in their healthcare facilities and communities and at federal and global levels. in the last 2 decades, id specialists have played vital roles in responding to emerging diseases and epidemics including west nile virus (1999), severe acute respiratory syndrome (sars; 2003), h1n1 pandemic influenza (2009), middle east respiratory syndrome (mers; 2012 and ongoing), ebola virus (2014-2016 and ongoing), and zika virus (2016 and ongoing). while these outbreaks garner a great deal of media visibility, id physicians also routinely detect, prevent, or mitigate community outbreaks of vaccine-preventable diseases, foodborne illness, and healthcare-associated infections. the work performed by id doctors in the united states is substantially undercompensated. id physician salaries average around $100 000 a year less than other subspecialties [1, 2] . young physicians, who generally complete training with substantial educational debt, can be driven from considering the field [1] . developing a robust id workforce of the future requires a strategy to attract quality physicians and keep them engaged on the frontline of patient care, research, and public health. despite their documented value, id physicians' services are undervalued by payers compared to primary care and procedure-based specialties. a graduating trainee finishing an internal medicine residency can work immediately as a hospitalist at a higher mean salary than an id physician who will take 2 to 4 additional years of fellowship training. such variances in compensation for direct patient care and insufficient pay for value in other roles such as infection control creates a significant disincentive to pursuing a career in id. the 2017 medscape physician compensation report reveals that the median salary of a specialty physician to be approximately $316 000 per year [1] . the infectious diseases society of america's (idsa) 2017 compensation survey showed that the average id physician earns about $215 000 a year. full-time public health physicians averaged $30 000 per year less [2]. the average medical student leaves school with approximately $200 000 in debt [1] . this financial disincentive is a significant contributor to the 21.6% decline in the number of applicants to id fellowship training programs over the 5-year period ending in 2016 [3] . only 42.3% of these programs filled through the match, down 20% over the same period [3] . while the 2017-2018 match saw an improvement in the number of positions filled, the match rate was still significantly below that of other specialties, many of which customarily fill at or near 100% [4] . it is essential that id physicians promote the value this subspecialty brings to public health in order to affect the policy changes necessary to secure the future of the field. here, we provide concrete examples of id physicians' unique contributions to public health, which can be used to educate policymakers and influencers at the federal, state, and local levels and to advocate for needed investments to sustain the field. id specialty preparation consists of training first in general adult internal medicine or pediatrics followed by 2 to 4 years of training in id. the id subspecialty fellowship includes integrated training to provide patient care and ensure population health. the clinical training includes diagnosis, management, and treatment of patients with id; expertise in techniques for preventing healthcare-associated infections and antimicrobial resistance; prevention strategies; and research approaches to address id-related questions. in 2015, 8515 id physicians were practicing in the united states [5] , often combining clinical care with work as educators, epidemiologists, public health leaders, antimicrobial stewardship or infection prevention and control directors, researchers, administrators, and policymakers. multiple studies have demonstrated the cost-effectiveness and patient benefit of id physician care for hospitalized patients with id [6] [7] [8] [9] . further, the work of id physicians provides broader public protection against infectious threats through community and healthcare facility-based infection control and prevention activities, surveillance, outbreak response, and other public health activities. the id workforce plays a critical role in managing infections such as human immunodeficiency virus (hiv), tuberculosis, and viral hepatitis, which can cause community outbreaks if not promptly diagnosed and treated. the public health workforce, including id physicians, has been shrinking over the last decade [10, 11] . these workforce losses pose a significant barrier to carrying out routine public health activities and responding to public health threats. id physicians employed by federal agencies and state health departments have the community as their patient. along with id physicians in other practice settings, they provide leadership and subject matter expertise to enable a wide variety of community-based interventions. for example, id management and prevention. public health strategies to contain the spread of hiv rely on ensuring that people living with hiv-aids have access to id-driven clinical care. by achieving sustained virologic suppression, people stay healthy and reduce the risk of community transmission [12] . the 2015 hiv and hepatitis c virus (hcv) outbreaks linked to injection drug use in rural scott county, indiana, serve as a stark reminder of the risk for rapid disease transmission in communities that lack robust outbreak prevention resources and activities [13] . in 2017 there was an outbreak of hepatitis a virus in the homeless population of san diego, california, that required significant work from the public health community to resolve [14] . in march 2017, a national academies of medicine panel declared the elimination of hepatitis b and hcv in the united states by 2030 to be a feasible goal. a sufficient workforce with the expertise to treat patients with hcv will be critical to reaching this goal [15] . as an example of outbreak detection and response to emerging infections, the indiana state health department recognized the first identified case of mers in the united states in a traveler to that state. id specialists at the department helped develop and implement protocols to ensure appropriate care, prevent the spread of infection, and avert an outbreak. id physicians working inside or outside of public health often provide guidance to help allocate community vaccine resources and other preventive services. for example, during measles outbreaks in california in 2015 and minnesota in 2017, id physicians within the state and local health departments helped lead vaccination campaigns to halt the spread of the disease [16, 17] . even in the absence of a major outbreak, patients in hospitals and healthy individuals in communities are potentially exposed to increasingly drug-resistant and difficult-to-treat pathogens, necessitating id physician leadership to protect the population by limiting the spread of infectious threats [18] . id public health physicians act as a trusted resource, working through the media, to help educate the public about communicable diseases and prevention strategies. current topics of concern include multidrug-resistant bacteria, zika virus outbreaks, the 2017 hepatitis a outbreak in san diego, and mycobacterium chimera infections in patients treated with heart-lung bypass machines. infection prevention and control activities in healthcare settings involve a range of interventions. methods include providing oversight of programs that conduct surveillance and identify risks, providing education regarding the use of appropriate isolation procedures and personal protective equipment, developing policies to respond to novel infections, and ensuring that the healthcare environment and medical devices are properly cleaned and maintained. studies have shown that development and implementation of these activities by id physicians have resulted in improved outcomes and reductions in hospital-acquired infections in a variety of healthcare settings [19] . prevention of healthcare-associated infections leads to savings of approximately $1000 to more than $40 000 per patient depending on the specific infection in a variety of healthcare settings [20] . examples of id physician leadership in infection control and prevention and healthcare epidemiology are described here. during the west africa ebola crisis in 2014 and 2015, id physicians served as leaders of special biocontainment units at the national institutes of health, university of nebraska, emory university, and bellevue hospital. these units and their staff developed novel, sophisticated infection prevention strategies to care for patients with potentially deadly infections. they reassured medical providers, political leaders, and our nation's public by providing safe, state-of-the-art care. in seattle, washington, an id physician reported an outbreak of deadly, multidrug-resistant carbapenem-resistant enterobacteriaceae infections were attributed to contaminated, faulty duodenoscopes. this investigation helped lead to changes in guidance on endoscope reprocessing and safety [21, 22] . an id physician at vanderbilt university identified contaminated steroids as the source of a fungal meningitis outbreak that ultimately caused more than 700 infections and 46 deaths across 20 states before the cause was established [23, 24] . antimicrobial resistance is one of the most urgent public health threats of our time. infections caused by antimicrobial-resistant organisms kill more than 23 000 people and result in $20 billion in unnecessary healthcare costs each year in the united states [18] . newer antimicrobials will be precious resources, and id physician-driven expertise will be vital to ensure that they are being used appropriately. a recent clinical infectious diseases article highlighted the unique skill set and training id physicians have to lead antimicrobial stewardship programs (asps) in order to ensure that correct antimicrobials are used judiciously [25] . the centers for disease control and prevention (cdc) have acknowledged the impact and importance of asps in their release of the core elements of hospital antibiotic stewardship programs in 2014 [26] , and the joint commission released their antimicrobial stewardship standard for healthcare settings in 2017. in 2014, legislation was passed in california that mandated that every acute care hospital in the state establish a physician-supervised asp [27] . adoption of a federal government requirement that other states follow suit would help to ensure that all patients can benefit from the positive impact of asps. asps will become increasingly vital in preventing a post-antibiotic era where common infections become untreatable due to resistant organisms. id physicians need to take an active role in ensuring their facilities have adequate asps that meet the standards laid out by the cdc and joint commission. well-run asps not only help preserve the efficacy of antimicrobials but also improve patient outcomes. in fact, studies have indicated that asps generated several hundred thousand dollars a year in cost savings while also reducing rates of clostridium difficile infections [28, 29] . it is vital that the government continue to support the implementation of id-led asps in all healthcare settings to slow the development of resistance and to keep our current antimicrobials useful for as long as possible. in response to the 2001 anthrax bioterrorism attacks and the looming threat of pandemic influenza, hospitals began extensive preparations for natural and human-made bioemergencies. these activities substantially increased with the appearance of the sars, mers-coronavirus, and ebola virus epidemics. id physicians provided scientific and clinical expertise that shaped prevention, control, detection, and treatment efforts. natural disasters also carry an increased risk of outbreaks of unusual, serious infections, as seen in connection with the hurricanes and flooding in texas, florida, puerto rico, and the us virgin islands, which led to more than 70 reported cases of leptospirosis and other waterborne and vectorborne diseases [30] . id physicians also played a leading role in infection prevention at large-scale shelters for people who had been displaced by storms and flooding. hospital and healthcare system emergency preparedness requires an intimate knowledge of hospital infection control procedures and capacities, regional collaboration with other facilities, planning with public health authorities, and development of communication strategies within the hospital community, with public health departments, and with the general public. a review of recent international id outbreaks emphasizes the global dimensions of public health protection-the sars epidemic originated in hong kong, the ebola virus outbreak started in west africa, and the zika virus epidemic spread from brazil throughout south america and the caribbean. a bacterial plasmid that conferred resistance to colistin, often the last antimicrobial line of defense against gram-negative organisms, emerged from bacteria that were colonizing domestic animal populations in china and is now seen in patients on 6 continents. older epidemics, of course, continue; tuberculosis and hiv-aids remain scourges in many countries. emerging and reemerging infections with global consequences have reemphasized efforts to address global health security. the global sars epidemic illustrated how increased international travel and trade introduce new risks for the rapid worldwide spread of new infectious pathogens. the world health organization international health regulations (ihr), issued in 2005, were officially adopted by 196 countries [31] . id physicians remain crucial to ongoing ihr implementation efforts, including disease outbreak recognition, in-country training, and collaborative partnership in epidemic disease control, diagnostics, and preparedness. with prescient timing, the cdc' s global health security agenda (ghsa) was launched just months before the ebola crisis surfaced in west africa. the ghsa aims to "accelerate progress toward a world safe and secure from infectious disease threats; to promote global health security as an international priority and to spur progress toward full implementation of who ihr 2005" [32] . the goals fall into 3 major themes: prevention, detection, and response. id physicians provide critical support to all 3 arms. id training and expertise establish the necessary foundations for the prevention goal to address issues such as antimicrobial resistance, biosafety and biosecurity, and immunization needs. the detection goal promotes innovation in the area of accurate, real-time, cost-effective id diagnostics. greater use of diagnostic tests in countries currently lacking such resources is critical for containing emerging and reemerging infections, preventing outbreaks, and halting the development of antimicrobial resistance. the response goal would establish emergency operations centers that link public health with other rapid-response agencies, all of which draw on id physician expertise. multiple studies have demonstrated that consulting an id physician improves treatment outcomes and lowers patient care costs. this benefit has been documented for id consultation in general [33] , in specific patient populations such as intensive care unit patients [34] , and for specific illnesses such as staphylococcal bloodstream infections and other multidrug-resistant organism infections [9, 35] . id physicians also improve outcomes and reduce costs through antimicrobial stewardship and infection prevention [8, 36] . in addition, many complex procedures and treatments (such as bone marrow and solid organ transplantation) could not be safely conducted without input from id specialists. the idsa has implemented several initiatives in an effort to reinvigorate the pipeline of id applicants. these include mentorship programs; scholarships for medical students, residents, and fellows interested in id; research and clinical career meetings; and medical school id interest groups. however, other potential options should be considered in order to properly compensate id physicians for the public health benefit of their work. recommendations for federal, state, local, and institutional policymakers include the following: establish loan repayment opportunities for id physicians who work in public service (eg, local, state, or federal public health departments). significant medical school debt can drive new physicians away from the id specialty and public health. targeted loan repayment opportunities would allow more physicians to pursue these critical career paths. in july 2018, in a move supported by idsa, the us house of representative's energy and commerce committee passed legislation that would authorize the cdc to provide loan repayment for those who serve in the epidemic intelligence services (eis). more than 85% of eis officers continue serving in public health roles, and this proposal would significantly help make this career path more financially feasible for physicians. such loan forgiveness opportunities could also be broadened to support id physicians who work in public health at the federal, state, or local level and face similar challenges around compensation and medical student debt. establish financial compensation for id physicians who work in public service (eg, local, state, and federal public health agencies) or who do id work that provides broader public health benefits (infection control and antimicrobial stewardship). physicians who perform infection control and antimicrobial stewardship work should be compensated for these activities. as part of hospital accreditation, the centers for medicare and medicaid services (cms) mandates that every participating facility have an active infection control program [37] . states could attach an accreditation process to such mandates that would formally document id clinicians who oversee hospital infection control and antimicrobial stewardship committees, with loan forgiveness or other financial incentives attached to this accreditation. ensure that id physician compensation reflects public health value added. as a cognitive specialty, many of the clinical services id physicians provide are billed under evaluation and management codes, which have not been reevaluated in more than 20 years. the cms should update these codes to reflect the increasingly complex care provided in inpatient and outpatient settings. public and private payers, healthcare systems, and hospitals should also provide appropriate compensation and protected time for nonclinical services that are crucial to public health, including infection prevention and control, antimicrobial stewardship, and bioemergency preparedness and response. fully fund local, state, federal, and global public health agencies and build a competent workforce. without proper workforce funding, including funding of id physician positions, health departments will not be able to hire and retain the expert workforce necessary to detect, prevent, and respond to public health threats. appropriate compensation for id service in all of these forms will have significant positive effects on individual patient care, nosocomial infections, and community public health, ensuring that the future id workforce has enough of an incentive to pursue this vital field. medscape physician compensation survey results and data, specialties matching service, 2016 appointment year. national resident matching program appointment year. national resident matching program association of american medical colleges 2016 physician specialty data book employing infectious disease physicians affects clinical and economic outcomes in regional hospitals: evidence from a population-based study the value of an infectious diseases specialist the value of infectious diseases specialists: non-patient care activities infectious diseases consultation reduces 30-day and 1-year all-cause mortality for multidrug-resistant organism infections a mismatch between the educational pipeline and public health workforce: can it be reconciled? enumeration of the governmental public health workforce partner study group. sexual activity without condoms and risk of hiv transmission in serodifferent couples when the hivpositive partner is using suppressive antiretroviral therapy hiv infection linked to injection use of oxymorphone in indiana a national strategy for the elimination of hepatitis b and c: phase two report measles outbreak-california antibiotic resistance threats in the united states the value that infectious diseases physicians bring to the healthcare system health care-associated infections: a meta-analysis of costs and financial impact on the us health care system endoscopic retrograde cholangiopancreatography-associated ampc escherichia coli outbreak infections associated with reprocessed duodenoscopes doctor isolates cause in nationwide meningitis outbreak. vanderbilt magazine multistate outbreak of fungal meningitis case count infectious diseases society of america, pediatric infectious diseases society, and the society for healthcare epidemiology of america. infectious diseases physicians: leading the way in antimicrobial stewardship core elements of hospital antibiotic stewardship programs california senate bill no. 1311 clinical and economic outcomes of a prospective antimicrobial stewardship program an evaluation of the impact of antibiotic stewardship on reducing the use of high-risk antibiotics and its effect on the incidence of clostridium difficile infection in hospital settings world health organization. international health regulations. available at: www the global health security agenda infectious diseases specialty intervention is associated with decreased mortality and lower healthcare costs impact of regular collaboration between infectious diseases and critical care practitioners on antimicrobial utilization and patient outcome the value of infectious diseases consultation in staphylococcus aureus bacteremia the value that infectious diseases physicians bring to the healthcare system conditions of participation for hospitals potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-331465-humpwwk2 authors: canaday, david h; gravenstein, stefan title: on setting expectations for a sars-cov-2 vaccine date: 2020-06-04 journal: clin infect dis doi: 10.1093/cid/ciaa726 sha: doc_id: 331465 cord_uid: humpwwk2 the global coronavirus pandemic is unlike any other since 1918. a century of dramatic medical advances has produced a public expectation that the medical field will rapidly provide solutions to restore normalcy. in under 6 months, since sars-cov-2 was identified, the massive international effort to develop a sars-cov-2 vaccine has generated more than 140 vaccines in different stages of development with 9 already recruiting into clinical trials posted on clinicaltrials.gov. the long-term strategy to handle covid-19 will almost certainly rely on vaccines. but, what type of protection can we realistically expect to achieve from vaccines and when? m a n u s c r i p t protection by vaccines against respiratory viral pathogens can help set our expectations for a future sars-cov-2 vaccine. influenza has similarities, in that it also causes respiratory illness and is an rna virus. the seasonal inactivated influenza vaccine has a modest protective efficacy of 10-60% even in years when the vaccine matches the circulating strains [1]. vaccines to prevent streptococcal pneumonia, albeit a bacterial target, have similar efficacy reported at 45-64% efficacy [2, 3] . furthermore, contemporary respiratory syncytial virus (rsv) vaccine trials have yet to demonstrate more than modest protection [4] . thus, we should expect a modest protective efficacy from vaccines against respiratory pathology in the 60% to 70% range at best and potentially far worse in persons with diminished vaccine response who disproportionately count among the highest risk populations. also, unlike a vaccine targeting sars-cov-2, each of these vaccines operates in a milieu of at least some previous immunity to boost from. therefore, the expectation that a sars-cov-2 vaccine can develop this level of protection from an immune naive state, especially in the setting of immunizing elderly individuals whose naive b and t cells are substantially diminished, needs to be set with caution. influenza and pneumococcal vaccines still offer substantial clinical benefit. but, in the context of covid-19, what should we expect from a vaccine that in older and other high risk groups will likely experience a 40% or higher failure rate? will the vaccine nevertheless help mitigate disease and prevent hospitalization, mechanical ventilation, or even transmission in those who still get infected? using the influenza comparison, those who develop influenza despite vaccination still experience reduced morbidity [5] . current understanding is that influenza antibody titers help prevent infection but cell mediated immunity (cmi) is essential for recovery. data from several influenza studies suggest that increased cmi, specifically including both cd4+ and cd8+ t cells, helps mitigate influenza severity in older adults when infected despite vaccination [6] [7] [8] . an optimal vaccine would protect both by preventing infection and mitigating disease from infection that eludes antibody-derived protection. vaccine constructs from oxford and moderna have received considerable attention. the former uses the chadox1 construct, a replication-deficient adenovirus platform, which can elicit cd4+ and cd8+ t cell responses and demonstrated antibody responses in a phase 1 m. tuberculosis vaccine trial [9] . the latter is an mrna construct against sars-cov-2 which elicited neutralizing antibody in trial volunteers (phase 1); however, in a published phase 1 clinical trial using the same construct technology with 2 influenza viruses they also showed significant antibody responses, but did not demonstrate detectable t cell responses [10] . we should expect all of the existing clinical trial candidates to have incomplete effectiveness, and we need to establish whether those that ineffectively recruit cmi have inferior disease mitigation when covid-19 develops despite vaccination. the breathtaking speed of the widespread vaccine development for covid-19 has leapfrogged the fundamental immunologic work needed for a fuller understanding of the critical components of immunity. the pandemic crisis has necessitated this strategy. since we are starting without meaningful immunity, generating a robust humoral and cmi response will likely be an iterative process that will benefit from ongoing vaccine development. the most effective vaccines against respiratory-acquired pathogens are all live attenuated virus vaccines (mmr, varicella, and smallpox), while none are primary respiratory pathogens. logically, none of the current clinical trials use a live attenuated vaccine, as we simply do not know enough about sars-cov-2 virology to safely put forward such a candidate. a live attenuated vaccine m a n u s c r i p t could be optimal were it also proven to be safe. clearly, we must work in parallel, testing vaccine candidates while advancing very robust basic science efforts. we must better understand the immunology to anticipate what is ahead and optimize our current pandemic response while we build the infrastructure that can serve us for the next pandemic crisis. potential conflicts of interest s.g. and d.c. report investigator initiated grants to the university to study influenza vaccines and consulting of flu vaccines from seqirus, sanofi-pasteur, and pfizer. s.g. also reports dmc member payments from janssen and merck. past seasons vaccine effectiveness estimates polysaccharide conjugate vaccine against pneumococcal pneumonia in adults effectiveness of the 23-valent pneumococcal polysaccharide vaccine pneumococcal disease in the elderly: systematic review and meta-analysis the respiratory syncytial virus vaccine landscape: lessons from the graveyard and promising candidates influenza vaccination modifies disease severity among community-dwelling adults hospitalized with influenza t cell responses are better correlates of vaccine protection in the elderly preexisting influenza-specific cd4(+) t cells correlate with disease protection against influenza challenge in humans cytotoxic t-cell immunity to influenza a phase i trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, chadox1 85a prime -mva85a boost in healthy uk adults mrna vaccines against h10n8 and h7n9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials a c c e p t e d m a n u s c r i p t key: cord-340678-2e2s1gof authors: skowronski, danuta m; zou, macy; clarke, quinten; chambers, catharine; dickinson, james a; sabaiduc, suzana; olsha, romy; gubbay, jonathan b; drews, steven j; charest, hugues; winter, anne-luise; jassem, agatha; murti, michelle; krajden, mel; de serres, gaston title: influenza vaccine does not increase the risk of coronavirus or other non-influenza respiratory viruses: retrospective analysis from canada, 2010-11 to 2016-17 date: 2020-05-22 journal: clin infect dis doi: 10.1093/cid/ciaa626 sha: doc_id: 340678 cord_uid: 2e2s1gof influenza vaccine effectiveness against influenza and non-influenza respiratory viruses (nirv) was assessed by test-negative design using historic datasets of the community-based canadian sentinel practitioner surveillance network (spsn), spanning 2010-11 to 2016-17. vaccine significantly reduced the risk of influenza illness by >40% with no effect on coronaviruses or other nirv risk. influenza vaccine effectiveness (ve) is commonly estimated through the test-negative design (tnd), an observational method that compares the odds of vaccination among influenza test-positive cases to influenza test-negative controls through the odds ratio (or), with ve derived as (1-or)x100%. the core prerequisite for valid ve estimation by tnd is that vaccine has no effect on alternate etiologies of the same clinical syndrome included in the control group. comparison of per-protocol and tnd analyses of several large randomized-controlled trial (rct) datasets involving >6000 participants has verified this prerequisite for influenza ve estimation, with the or for influenza vaccine effect against non-influenza causes of influenza-like illness (ili) approximating 1.0 (ve approximating zero) [1] . if, however, influenza infection induces immunity that is cross-protective against noninfluenza respiratory viruses (nirv)(e.g. through non-specific innate immunity), then vaccination that effectively prevents influenza may indirectly result in greater nirv risk among vaccinated compared to unvaccinated individuals. cowling et.al. hypothesized such vaccine interference with infection-induced immunity to explain a significant four-fold increased nirv risk among 69 children randomized to receive the 2008-09 influenza vaccine compared to 46 children receiving placebo [2] . that small rct, however, included just 23 nirv cases and was under-powered to show ve against influenza, as required by the interference hypothesis [2] . conversely, in tnd analysis of six study seasons (2004[5, 6] . three other coronaviruses have been associated with more severe illness including sars-cov, mers-cov, and more recently sars-cov-2, the latter emerging in late 2019 and responsible for the ongoing pandemic of coronavirus disease 2019 (covid-19) [5, 6] . wolff's findings for seasonal coronaviruses, coincidentally published in january 2020, have triggered concern that influenza vaccination may detrimentally affect covid-19 risk [4] . here, we use historic datasets of the community-based canadian sentinel practitioner surveillance network (spsn) to assess the association between influenza vaccine and nirv risk, notably seasonal coronaviruses. we retrospectively applied tnd analysis to canadian spsn influenza ve study specimens collected during the 2010-11 to 2016-17 seasons[7], when specimens were tested for both influenza and nirv. specimens were included if collected november-april from consenting patients ≥1-year-old who presented within 7days of ili onset to a sentinel practitioner in the provinces of alberta, british columbia, ontario or quebec. ili was m a n u s c r i p t defined by fever and cough plus ≥1 of arthralgia, myalgia, prostration or sore throat. fever was not required for adults ≥65-years-old after 2010-11. specimens were tested for influenza and nirv at provincial public health reference laboratories by reverse-transcriptase-polymerase-chain-reaction (rrt-pcr) and/or commercial multiplex rt-pcr assays(supplementary_material_1). ontario panels did not include the hku1 coronavirus. during seasons for which ontario (2015-16) and alberta (2015-16/2016-17) did not perform multi-plex testing they were excluded from influenza and nirv analyses. participants who self-reported influenza vaccination≥2weeks before ili onset were considered vaccinated. participants with unknown timing or self-reporting vaccination<2weeks before ili onset were excluded; the latter were also explored as unvaccinated (per wolff) [4] . ors compared influenza vaccination rates among influenza and nirv test-positive cases relative to test-negative, pan-negative and nirv-positive controls. influenza test-positive specimens were excluded from nirv analyses. nirv cases were assessed in combination and separately grouped as coronaviruses, entero-/rhinoviruses(ev/rv), hmpv, parainfluenza, and rsv. coxsackie-/echovirus, adenovirus, and bocavirus estimates are not presented owing to limited detection but are included in combined nirv analyses. co-infections across nirv groupings were included among controls but not cases; in sensitivity analyses cases also included co-infections. all models adjusted for age, province, specimen-collection interval, calendar-time, and season; participants missing information for any of these covariates were excluded. comorbidity and sex were also assessed in sensitivity analyses but had no confounding effect. a c c e p t e d m a n u s c r i p t in this seven-season analysis by the canadian spsn, influenza vaccine was protective against medically-attended ili due to influenza viruses, significantly reducing the risk by >40%. conversely, influenza vaccine had no effect on non-influenza causes of ili, with the likelihood of vaccination among nirv cases relative to test-negative controls approaching unity. in particular, influenza vaccine did not affect seasonal coronavirus risk. our findings provide reassurance against the speculation that influenza vaccine may negatively affect covid-19 risk. addressing such speculation is important to maintain influenza vaccine coverage through the ongoing covid-19 pandemic. in assessing wolff's paper we identified a major methodological problem to account for his unexpected findings [4] . in combined nirv analysis, relative to pan-negative controls, wolff adjusted for age and excluded specimens that tested influenza-positive. in that analysis, shown in his table 3 , the or approached unity indicating no vaccine effect as expected. conversely, in unadjusted analysis of individual nirv outcomes (e.g. coronaviruses) wolff retained influenza test-positive specimens in nirv test-negative control groups, thereby violating the core prerequisite for valid tnd analysis. in the context of effective influenza vaccine, influenza cases would have lower likelihood of vaccination; as such, their inclusion would systematically reduce the proportion vaccinated in the control group and thereby inflate ors comparing vaccine exposure between nirv cases and controls. we illustrate the impact of this bias in supplementary_material_3, where we have re-analyzed wolff's data as well as our own, comparing influenza vaccine effect against nirv when influenza testpositive specimens are properly excluded (as per tnd prerequisite) or improperly included (as per wolff [4] ) within the control group. in both data sets and for all nirv, ors for influenza vaccination are biased higher when influenza cases are erroneously included in the control group. a c c e p t e d m a n u s c r i p t as for any observational design, random variation, bias and confounding may influence tnd findings. our seven-season analysis was based on substantial sample size, standardized ili testing indication, and multi-variate analysis to address those concerns; whereas, wolff relied upon a single season, general laboratory submissions, and univariate analysis, despite evidence in his dataset for confounding by age. the importance of adjustment for age and other potential confounders is reinforced by our analyses in which several unadjusted but no adjusted ors significantly differed from one(table_1;supplementary_table_s2a). vaccine status was self-reported in our study but recorded before specimen testing, minimizing differential misclassification. assays varied by province and season. two spsn provinces did not conduct nirv testing during 1-2 of the study seasons and hku1 was omitted from the coronavirus panel of one province all seasons. however, hku1 comprised a small proportion of coronavirus detections in other spsn provinces (15%;53/349) and findings were robust across nirv outcomes and in sensitivity analyses addressing variation in provincial contribution (not displayed). finally, although we did not find evidence for vaccine interference, population surveillance signals elsewhere suggesting cross-pathogen immunological interactions still warrant immuno-epidemiological investigation [3, 8, 9] . in conclusion, our findings provide reassurance that protective influenza vaccination does not negatively affect nirv risk, including coronaviruses. valid tnd estimates require that etiologies against which vaccine is effective are specifically excluded from the testnegative control group, and this applies also when exploring vaccine effects on non-vaccine target pathogens. these methodological insights have important implications for other tnd applications, including future evaluations of influenza vaccine effects against covid-19, and vice-versa when sars-cov-2 vaccines become available. a c c e p t e d m a n u s c r i p t the authors gratefully acknowledge the contribution of sentinel sites whose regular submission of specimens and data provide the basis of our analyses. we also wish to acknowledge those who provided administrative and coordination support and those who provided laboratory and technical support in each participating province. the views expressed herein do not necessarily represent the view of the public health agency of canada. funders had no role in data analysis or interpretation or in the decision to publish the test-negative design : validity, accuracy and precision of vaccine efficacy estimates compared to the gold standard of randomised placebo-controlled clinical trials increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine influenza vaccination is not associated with detection of noninfluenza respiratory viruses in seasonal studies of influenza vaccine effectiveness. clinical infectious diseases influenza vaccination and respiratory virus interference among department of defense personnel during the 2017-2018 influenza season. vaccine coronavirus infections in children including covid-19. an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection and association of antibody responses with severity of disease m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-339859-anatn295 authors: paret, michal; lighter, jennifer; pellett madan, rebecca; raabe, vanessa n; shust, gail f; ratner, adam j title: sars-cov-2 infection (covid-19) in febrile infants without respiratory distress date: 2020-04-17 journal: clin infect dis doi: 10.1093/cid/ciaa452 sha: doc_id: 339859 cord_uid: anatn295 we report two cases of sars-cov-2 infection (covid-19) in infants presenting with fever in the absence of respiratory distress who required hospitalization for evaluation of possible invasive bacterial infections. the diagnoses resulted from routine isolation and real-time rt-pcr-based testing for sars-cov-2 for febrile infants in an outbreak setting. m a n u s c r i p t 3 sars-cov-2 infection (covid-19) has rapidly emerged as a worldwide cause of severe respiratory disease in adults. [1] early reports indicate that the course of disease is generally milder in children, but fatal cases have been described. [2] [3] [4] even in the setting of asymptomatic or mildly symptomatic infection, children may represent a source of sars-cov-2 spread in community or hospital settings, so understanding the spectrum of covid-19 illness in infants, particularly regarding conditions that result in hospitalization, is crucial to establishment of effective infection control interventions. [5] in the first months of life, infants presenting with fever frequently undergo diagnostic evaluations for invasive bacterial disease, even in the absence of other clinical signs. such evaluations generally include cultures of blood, urine, and, in some cases, cerebrospinal fluid (csf), followed by observation and empiric antibiotic therapy in a hospital setting. [6] a significant percentage of febrile infants have infections with respiratory viruses, including respiratory syncytial virus, enteroviruses, and influenza. [7] these viral infections may occur in the presence or absence of invasive bacterial infections. over a oneweek period in late march 2020 corresponding to a time of widespread community transmission of sars-cov-2 in new york city, we encountered two febrile infants presenting with minimal or no respiratory symptoms who were found to have sars-cov-2 infection without other etiologies despite thorough evaluations. case 1: a 25 day-old full-term male infant was brought to the pediatric emergency department because of fever (38.5°c (101.3°f) on arrival) and irritability. he had had no cough, tachypnea, rhinorrhea, or respiratory distress. there was no change in feeding, and no vomiting or diarrhea was reported. the child had not travelled. both parents were symptomatic with sore throat and subjective fever in the prior two days but had not sought medical attention for themselves. there were no other ill contacts and no known a c c e p t e d m a n u s c r i p t 4 contacts with sars-cov-2 infection. on examination, the patient was alert and active and was noted to have an erythematous, papular facial rash. the remainder of the physical examination was within normal limits. samples of blood, urine, and csf were obtained for analysis. vital signs and pertinent laboratory findings appear in the a real-time rt-pcr assay performed at the new york state department of health detected sars-cov-2 rna in the patient's np sample. empiric therapy with parenteral ampicillin and cefepime was started on admission and continued until the blood, urine, and csf cultures were negative for >48 hours. no antiviral medications were given. the patient was discharged to home in stable condition with infection precautions consistent with centers for disease control and prevention guidelines. [8] case 2: a 56 day-old male infant born at 35 weeks gestation who had had an uneventful perinatal course presented to the hospital with a temperature of 38.2°c (100.8°f) taken rectally at home. the child had no respiratory or gastrointestinal symptoms and had normal oral intake and activity level. his mother, father, and siblings were all well, and there were no other known ill contacts and no history of travel. in the emergency department, the child was febrile but well appearing, with a normal physical examination. vital signs and laboratory findings appear in the table. blood and urine cultures were done, but sampling of the csf was not performed, nor was radiographic imaging. the biofire respiratory pcr panel performed on an np sample was negative, and the np sars-cov-2 real-time rt-pcr (cobas, roche) a c c e p t e d m a n u s c r i p t 5 was positive. the child was treated empirically with parenteral ceftriaxone until the results of blood and urine cultures were negative for >36 hours. no antiviral medication was used, and the patient was discharged in stable condition with infection control guidance. these two cases present a common problem in pediatric medicinethe febrile infantwith an important twist, diagnosis of sars-cov-2 infection during an explosive community-based outbreak in new york city. the epidemic coronaviruses, including mers-cov, sars-cov, and sars-cov-2, have the potential for spread within healthcare settings, making case identification and prompt isolation crucial to protecting patients, physicians, and staff. [9] [10] [11] in the context of an ongoing outbreak, we encouraged routine testing of febrile infants for sars-cov-2, even in the absence of respiratory symptoms. for both cases presented above, because sars-cov-2 testing was sent, and contact/droplet/eye shield precautions were instituted in the emergency department, with n95 masks used during np swab collection because of the potential for aerosol generation. in addition, family members were required to wear surgical masks and upon discharge received instructions for home isolation. we did not find non-sars-cov-2 etiologies for fever in these two infants, and fever without localizing signs has been reported in older children with sars-cov-2. [5] lymphopenia has been described in adult patients with sars-cov-2 infection, and we noted a low absolute lymphocyte count in patient 2. [11] testing for sars-cov-2 in non-np samples was not performed in either child, and both infants had a benign clinical course. however, the need to routinely hospitalize febrile infants for workup of potential invasive bacterial disease may serve as an characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention epidemiology of covid-19 among children in china detection of covid-19 in children in early cdc covid-19 response team. coronavirus disease 2019 in children -united states characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding a prediction model to identify febrile infants </=60 days at low risk of invasive bacterial infection the risk of serious bacterial infection in febrile infants 0-90 days of life with a respiratory viral infection discontinuation of transmission-based precautions and disposition of patients with covid-19 in healthcare settings (interim guidance) hospital outbreak of middle east respiratory syndrome coronavirus sars in healthcare facilities clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china the authors are grateful to the members of the department of pediatrics at nyu grossman school of medicine, nyu langone health/hassenfeld children's hospital, and bellevue hospital center for their dedication to outstanding pediatric care under the extreme conditions of the sars-cov-2 outbreak. the authors received support from the department of pediatrics at nyu grossman school of medicine.there was no external funding for this work. a.j.r. has served as a consultant to pfizer outside the scope of this work. all other authors report no conflicts. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-351314-atsuh8e2 authors: bryson-cahn, chloe; duchin, jeffrey; makarewicz, vanessa a; kay, meagan; rietberg, krista; napolitano, nathanael; kamangu, carole; dellit, timothy h; lynch, john b title: a novel approach for a novel pathogen: using a home assessment team to evaluate patients for 2019 novel coronavirus (sars-cov-2) date: 2020-03-12 journal: clin infect dis doi: 10.1093/cid/ciaa256 sha: doc_id: 351314 cord_uid: atsuh8e2 thousands of people in the united states have required testing for sars-cov-2. evaluation for a special pathogen is resource intensive. we report an innovative approach to home assessment that, in collaboration with public health, enables safe evaluation and specimen collection outside the healthcare setting, avoiding unnecessary exposures and resource utilization. the 2019 novel coronavirus (sars-cov-2), identified as the cause of an outbreak of respiratory infection in wuhan, china, is rapidly spreading around the globe. the first case in the united states was identified on january 20 th , 2020. 1 as of march 6, 2020, 164 additional cases in the united states have been diagnosed, though thousands of people have been tested for sars-cov-2. 2 we anticipate many more will require testing in the future. the transmission dynamics of sars-cov-2 remain under study, and conservative infection control processes and intensive contact tracing are required for confirmed or suspected cases of infection. public health agencies are charged to monitor persons with sars-cov-2 exposures and perform assessments if symptoms develop. depending on the outcome of these assessments, testing for sars-cov-2 might be recommended. safe evaluation of persons for suspected infection with a special pathogen (including sars-cov-2) in the traditional healthcare environment is costly and resource intensive. it requires specialized rooms, use of personal protective equipment (ppe), monitored donning and doffing, logistically-complicated patient transportation from the community to the healthcare facility and back (typically through emergency medical services), and appropriate decontamination of transport and hospital environments. additionally, evaluating a patient for emerging pathogens in a healthcare setting runs the risk of exposing patients and staff to infection. in a clinically stable patient with mild symptoms, alternative methods for special pathogen evaluation/testing are vital to preserve healthcare resources and prevent unnecessary exposures. patients with symptoms of illness and possible exposure to sars-cov-2 are identified either by public health or through a call to any part of the university of washington healthcare system, with calls routed to the ipc team responsible for hat activities. if the patient meets center for disease control and prevention (cdc) criteria for testing, 3 public health determines appropriateness of a hat visit and sars-cov-2 testing in the field. once public health determines that a hat visit is indicated, a hat member calls the patient to confirm that they are safe for a visit (breathing comfortably, able to eat and drink, mobilizing around their home), discuss details of the visit, determine locations for donning/doffing, and to register the patient within our system to allow for medical record documentation. each hat is made-up of 1 physician, 1 nurse, 1 or more trained ppe observers, and a site-commander, and uses one or two vehicles to transport personnel, ppe, and testing materials. the physician and nurse enter the patient's dwelling in appropriate ppe recommended by the cdc, using standard, contact, and airborne precautions with eye protection. 4 the patient is evaluated by the physician, who gathers a focused history and all involved hat members complete a daily log including temperature and respiratory and gastrointestinal symptom reporting through employee health for 14 days or until sars-cov-2 testing returns negative from the index visit. to date, hat has successfully conducted 15 community-based assessment visits, including in single-family homes and commercial properties where patients were isolated. all patients were deemed clinically stable and appropriate to remain out of the hospital; none have required subsequent hospitalization. approximate time from dispatch to return is 3 hours on average; including 10 minutes for donning, 30 minutes for face-to-face patient care, and 30 minutes for doffing/waste procedures. to date, 2 patients have tested positive for sars-cov-2. other evaluated patients have tested positive for human coronavirus, rhinovirus, parainfluenza, or had negative results. patients have reported that evaluation in this setting is highly acceptable. we noted no instances of contamination of healthcare worker ppe with visible blood or other body fluids and no instances of breech of ppe doffing protocol. . no hat members have developed symptoms requiring evaluation. to our knowledge, this is the first hospital team-based covid-19 assessment program in the united states to evaluate patients outside of the traditional healthcare setting. this model benefits both the public health and clinical healthcare systems by increasing safety and efficiency while reducing the costs and complexity of sars-cov-2 testing for patients who do not require emergency evaluation or hospitalization. given concerns with healthcare exposures and healthcare-associated outbreaks with sars-cov-2, as well as historical experiences with sars-cov and mers-cov transmission in healthcare settings, minimizing the exposure risk to clinics, emergency departments, and hospitals is crucial. 5, 6 as healthcare workers are known to be at risk of contamination during doffing of ppe, even when caring for patients with routine respiratory infections, 7 the importance of well monitored appropriate ppe doffing for special pathogens cannot be over-emphasized. given the novel nature of this pathogen, that the exact mechanisms of transmission have yet to be defined and unclear morbidity and mortality, this is an important yet often overlooked step in keeping our healthcare workers safe. following initiation of our protocols, the cdc has released new guidance for covid-19 assessment in residential settings 8 with waste management recommendations that differ from ours. for future novel pathogen assessments prior to release of national guidance, especially for pathogens with a greater risk of transmission, our protocol regarding secure ppe waste control remains pertinent. the hat program is a scalable, cost-saving model that cuts-down on resources required to isolate and care for patients. importantly, this model can likely be used for other novel outbreaks and potentially in unconventional settings like ships, planes, and airports that can be served by scaling-up hat size to allow large scale screening of individuals. our hospital has a long history of collaborating closely with public health on routinely occurring and emerging infectious disease responses. the use of home assessment teams in the setting of novel infectious disease epidemics demonstrates the value of integrated and coordinated public health and healthcare systems and corresponding benefits to our patients and the public. first case of 2019 novel coronavirus in the united states novel coronavirus (2019-ncov) in the u centers for disease control and prevention. evaluating and reporting persons under investigation (pui centers for disease control and prevention. interim infection prevention and control recommendations for patients with confirmed 2019 novel coronavirus (2019-ncov) or persons under investigation for 2019-ncov in healthcare settings sars transmission among hospital workers in hong kong clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china respiratory viruses on personal protective equipment and bodies of healthcare workers interim guidance for public health personnel evaluating persons under investigation (puis) and asymptomatic close contacts of confirmed cases at their home or non-home residential settings we thank the leadership of harborview medical center, uw medicine, and washington state department of health for supporting this project. dr. lynch reports funding for the program from washington state department of health, during the conduct of the study.none of the authors has potential conflicts to disclose. key: cord-331978-y4uo7o8g authors: maxwell, daniel n; perl, trish m; cutrell, james b title: “the art of war” in the era of coronavirus disease 2019 (covid-19) date: 2020-03-04 journal: clin infect dis doi: 10.1093/cid/ciaa229 sha: doc_id: 331978 cord_uid: y4uo7o8g nan novel coronavirus disease (covid-19) caused by the virus sars-cov-2, began in wuhan, china, and has spread worldwide, with over 101,700 cases and 3,461 deaths in more than 75 countries. with rapidly increasing cases and local community transmission in multiple countries outside of china, including the united states, the outbreak has entered a new phase, which requires a shift in primary battle strategy from a focus on containment in china to international mitigation. what will be required to fight this novel virus as it travels the globe? the metaphor of war is often used in the infectious diseases field, with its interspecies fight for survival. military strategies can be applied to outbreak management, and advice from one of the oldest and bestknown military sages -china's own sun tzu discusses the importance of preparation in the art of war. sun notes that victory is achieved before any fighting begins and that those headed toward failure look for victory only after the battle has already begun 1 . so again, how do we prepare to fight covid-19? as cases of covid-19 explode internationally, a strategic shift is required away from primarily containment, keeping the virus "out there", to home-based mitigation and public health responses. the task of healthcare systems is no longer screening and treating small numbers of infected returning travelers in highly specialized units with expert teams. now, the task is bearing the burden of identifying, isolating, triaging and managing the rising number of cases, necessitating total engagement of the medical community, public health sector, governments and society as a whole. for the medical and public health communities, this enormous task requires approaches that are both rapidly scalable and sustainable. we need to use existing teams and resources efficiently and to build capacity where it is lacking. two reports in this issue of clinical infectious diseases shed light on possible steps forward. first, we must learn from our own and others' battles. marchand-senecal et. al. 2 report on the successful management of the first hospitalized case of covid-19 in canada. they draw on and highlight lessons from the 2003 sars experience in toronto. notably, while they utilized airborne, contact, and droplet precautions in a negative pressure room, no advanced personal protective equipment (ppe) such as powered air-purifying respirators (paprs) was used. the rationale was simple. prior research demonstrates that using unfamiliar or increasingly complex ppe increases the risk of selfcontamination 3 . consequently, training for healthcare workers (hcw) focused on ensuring proper donning and doffing techniques with familiar, well-rehearsed ppe procedures. as sun tzu noted, "if in training soldiers' commands are habitually enforced, the army will be well-disciplined." 1 the authors also highlight improvements in infection prevention and control (ipc) infrastructure, administrative controls, and public health coordination compared to their 2003 sars experience. standard staffing models rather than a dedicated covid-19 team were used safely. strategies that focus on maintaining the workforce by requiring sufficient training for all staff offer potential for more sustainable, scalable hcw capacity in these extraordinary settings. still, these authors note the paucity of evidence-based guidance for initial triage and discharge timing decisions in hospitalized covid-19 patients. second, we must train the way we intend to fight. as illustrated by the canadian report, the allure and novelty of ppe "maximalism" should be avoided in favor of proven strategies that hcw have practiced and conduct with a high-degree of fidelity without self-contamination. regarding triage, bryson-cahn 4 and colleagues in washington state present a novel framework for home screening and evaluation of persons under investigation (puis) based on prior preparation for ebola community screening in 2014. their experience describes nine community-based assessment visits during which teams screened puis in a variety of community settings after the ipc team determined a home assessment was appropriate. detailed protocols are given for how a hcw team, with appropriate training and required supplies, can safely perform a focused assessment and collection of screening samples outside the healthcare setting. this approach avoids unnecessary exposures and resource utilization for those who otherwise are safe to remain at home. their explicit protocols provide a framework for other healthcare and public health systems to weigh along with cost-effectiveness and scalability. both papers highlight the power of collaborative partnerships and communication between public health and healthcare facilities required in these events. finally, we must identify our weaknesses and vulnerabilities the "enemy" can exploit. as sun tzu exhorted, "carefully compare the opposing army with your own, so that you may know where strength is superabundant and where it is deficient." 1 we want to highlight four critical vulnerabilities at present within the united states context but with global applicability. first, a paramount vulnerability that must be rapidly addressed is the limited diagnostic testing capacity for sars-cov-2 in the clinical arena. at this stage where screening must expand from narrow geographic-based criteria to syndromic surveillance, rapid and validated testing at scale must be available to help inform clinicians and public health officials for isolation, triage and care of patients. fortunately, fda emergency use authorization regulatory requirements have been relaxed to allow more laboratory developed tests to come online even as the cdc races to expand testing capacity in the public health sector. these efforts must be given utmost priority to define the scope of current community transmission and to allow proactive, rather than reactionary, public health responses. second, aggressive supply chain management during periods of increased demand is critical. public panic and fear can create or exacerbate real supply shortages, especially in an era of social media and just-in-time supply chain management. the world health organization and others have issued helpful guidance on the rational use of ppe for covid-19, aimed at optimizing hcw safety while mitigating disruptions in the global ppe supply chain. 5 rapid scalability in the supply of pharmaceuticals and ppe must be considered a public health imperative. moreover, preventing rushes on the public market through measured risk communication with the public can help safeguard needed supplies. finally, we must consider strategies to decrease less urgent use of ppe and identify situations where we can use different types of protection, where elements of ppe can be reused, or where the use of ppe is not supported by evidence-based practice. third, efforts to build and leverage margin and flexibility within healthcare staff capacity must be prioritized. marchand-senecal et. al. point out that specialized, dedicated teams in an outbreak, while attractive, could be quickly overwhelmed as cases increase. moreover, longer shifts and increased work intensity may lead to hcw fatigue and lapses in ppe techniques, driving nosocomial transmission, a painful reminder from the battle with sars. initial reports indicate about 4% of chinese hcw caring for covid-19 patients were infected, with 15% classified as severe or critical disease. 6 transmission to hcws, a feature seen with sars and mers, is devastating as it simultaneously diverts resources, depletes hcw capacity, saps morale, and drives public fear. to mitigate this, healthcare systems experiencing a surge in cases should consider all measures to liberate resources and staff, including telemedicine triage, drive-thru testing, and preparations to reschedule elective medical care. fourth, and finally, our national and global commitments to funding for public health and epidemic preparedness must be expanded and sustained. rather than the current "boom and bust" funding roller-coaster responsive to the latest outbreak, governments must provide expanded, stable funding levels to improve disease surveillance and response and to build technical capacity for rapid deployment of diagnostics, vaccine development, and clinical trials of pharmaceuticals for this outbreak and the next. 7 the folly of short-sighted cuts to public health and research funding is manifest in the significant costs associated with a lack of preparedness and threatens global health and security. as the battle against covid-19 ramps up worldwide, it is imperative that the entire global community join together in solidarity, apply the hard-fought lessons of this and prior epidemics, and move rapidly to implement proven public health and ipc principles to turn the tide against this foe. quoting sun tzu one final time, "he who knows these things, and in fighting puts his knowledge into practice, will win his battles." none of the authors has any conflicts of interest. the art of war, circa 6 th century b.c.e, translation by lionel giles diagnosis and management of first case of covid-19 in canada: lessons applied from sars common behaviors and faults when doffing personal protective equipment for patients with serious communicable diseases clinical infectious diseases 5. who. rational use of personal protective equipment for coronavirus disease 2019 (covid-19): interim guidance characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention responding to covid-19-a once-in-a-century pandemic? key: cord-352837-a29d5dkv authors: hirsch, hans h title: spatiotemporal virus surveillance for severe acute respiratory infections in resource-limited settings: how deep need we go? date: 2019-04-01 journal: clin infect dis doi: 10.1093/cid/ciy663 sha: doc_id: 352837 cord_uid: a29d5dkv nan following the discovery of molecular cloning, nucleic acid sequencing, and polymerase chain reaction, the wonderland of molecular biology has opened its doors again to virtually unlimited possibilities, as next-generation/deep sequencing or, better, massive parallel sequencing allows for the comprehensive analysis of all genomic nucleic acids of any origin in any given sample in real time. initially applied to the bacterial microbiome, deep sequencing has similarly detected a multitude of viral genome sequences, and changed hitherto common views on persisting viral infections [1] , putting forward the concept of the "virome" in general, and of the "human viromes" in particular [2] . while the numeric summary of viral genome sequences is mind-blowing, even after careful annotation and curation through refined bioinformatic tools, the utility of massive parallel sequencing has been prominently demonstrated in epidemiologic cluster analysis, for example, leading to the identification of a novel arenavirus as the cause of donor-derived death of 3 solid organ transplant recipients [3] , or of a novel human polyomavirus causing lethal multiorgan failure in a pancreas transplant recipient [4] . given the technical and bioinformatic advances as well as the declining laboratory costs, the application of deep sequencing to identify etiologic agents in clinical samples has been approached in different pathologies, including those caused by community-acquired respiratory viruses (carvs). despite the general enthusiasm for metagenomics, it remains important to realize that there are caveats. in fact, multiple technical and conceptual aspects influence the results of deep sequencing, may cause significant differences in the read-outs, and hence bias their interpretation. conceptually, it is clear that the human virome is different in different organ sites and body location (eg, the gastrointestinal, urogenital, or respiratory tract) and is subject to dynamic changes of the viral constituents, not only as a result of physiological changes (eg, dietary, hormonal, aging), but also due to complex pathological alterations such as trauma, inflammation, and immune responses [5] , exemplified in inflammatory bowel disease or transplantation [6, 7] . technically, nucleic acid extraction is presumed to be representative across all different agents, whereas in fact different sample preparations, buffers, purification, single fragment partitioning, and sequence analytic procedures may introduce an as-yet little-understood bias, which can lead to underrepresentation of certain viral genomes-for example, human coronavirus compared with paramyxovirus genomes (h. h. hirsch, unpublished observation). thus, every step of the wet and dry laboratory procedure is dependent on the methodology, and may yield potentially different results [8] . independent confirmatory testing has become an essential part of the presumably hypothesis-free deep sequencing [9] . these aspects are important when discussing the results of massive parallel sequencing to either rule in or rule out the involvement of a pathogen in a given pathology. with these caveats in mind, and given the significant global burden of viral respiratory tract disease in the very young and the very old [10] [11] [12] oropharyngeal (np/op) samples of sari cases were identified through a national surveillance study conducted by the uganda virus research institute from 2010 through 2015. the virome present in influenza virus-negative np/op samples was analyzed by deep sequencing enriching for viral sequences. the epidemiologic data were collected by sentinel hospitals in 9 different geographic areas, and hence focused on sari (eg, pediatric and adult patients admitted to hospital for severe influenza-like illness), thereby combining feasibility and clinical significance. the numbers are impressive, as 3921 sari cases were identified, 335 (8.5%) of which were influenza-positive np/op samples. of the remaining 3586 cases of influenza-negative sari, 2901 had spatial and temporal data available to pinpoint 9 clusters involving 301 (10.4%) influenza-negative sari cases. notably, the demographic characteristics of the ugandan sari cases indicated that mostly young children were enrolled, 80% of whom were <1 year of age, both within and outside of clusters. although coughing and shortness of breath were leading features of sari cases of both clustered and nonclustered cases, fever with temperatures >38°c and stridor were significantly more frequent in cluster cases than in noncluster cases. in two-thirds of the sari cluster cases, a virus-capturing technique and massive parallel sequencing using the hiseq illumina system yielded informative genome sequences in 82%, identifying human rhinovirus in 34.5%, human paramyxoviruses consisting of respiratory syncytial virus in 17.9%, and parainfluenza viruses in 11.7%, metapneumovirus in 6.9%, followed by coronaviruses (5.5%), adenoviruses (4.1%), and coxsackie virus (5.5%) and other enteroviruses (4.8%). these numbers compare well with results from resource-rich settings testing symptomatic children and adults using multiplex nucleic acid testing (nat) [13] [14] [15] , whereby the detection rate in children was around 70% and significantly higher than the 30% found in adults [13] , and where human rhinoviruses/enteroviruses together accounted for >45% of the detected carvs [13] . while this suggests that, by and large, a significant part of the pathogens could have been detected by multiplex nat for carvs, the deep sequencing approach of this study revealed a number of remarkable surprises, which may help to close the gap on at least 30%-50% of the carvnegative multiplex nat results [16] . cytomegalovirus (cmv) was identified in 26.9% of the sari clusters, and may either reflect reactivation as a bystander of significant inflammatory states including infections with other including viral pathogens [17] [18] [19] , or may in fact reflect the manifestation of cmv primary infections as sari, a hypothesis viable in view of the high seroprevalence in developing countries [20] and the very young age of 80% of the sari cluster patients described here. similarly, the discovery modus of deep sequencing identified more somewhat expected pathogens in that age group, such as human herpesvirus 6 and parvovirus b19, for which similar arguments could be made. by the same token, it is surprising that other viruses were not reported, some of which had been proposed to be associated with respiratory symptoms or febrile tonsillitis such as bk virus and other polyomaviruses [21] . similarly, the complete absence of the apparently ubiquitous anelloviruses, which have received much attention in studies of respiratory samples from patients with inflammatory conditions or transplantation, is notable [7, [22] [23] [24] . however, no samples from the lower respiratory tract were available for this analysis. the discovery potential of deep sequencing of this us-ugandan study is, however, highlighted impressively by the identification of picobirnavirus in 2 cases, as well as measles, as the cause of sari clusters in 18 cases, of which 13 belonged to genotype b3, which is endemic in eastern africa. remarkably, 5 cases were caused by genotype b4, and could be traced back to an unvaccinated tourist from england and an outbreak in manchester, united kingdom, in 2011. thus, global travel and outbreak have been linked. even though undervaccination may play an important role in the ugandan setting, it is clear that measles outbreaks have been a problem in resource-rich settings such as in switzerland, where 105 cases have occurred in the year 2017 (incidence of 12.5 per million inhabitants), causing outbreaks among mostly unvaccinated individuals, despite the current 2-dose vaccination coverage of 85% and 93% of 2-and 16-year-old children, respectively [25] . of note, measles virus also belongs to the human paramyxovirus family and is transmitted via respiratory fluids, but was detected alone as well as together with other viral pathogens in the present study by cummings et al. taken together, this report from resource-limiting settings is also of relevance for resource-rich countries and raises the question about how to best expand current first-or second-line testing for respiratory viral pathogens including cmv, parvovirus b19, and measles, and how to move to more deep sequencing virome analysis and comprehensive metagenomics in the near future. the spatiotemporal cluster analysis, combined with viral metagenomics presented in this study, is of interest also for other settings, and raises the question of how and when this is can be best delivered in real time. such efforts are under way for influenza virus vaccine prediction [26] , the pacemaker of respiratory viral research, and will be of utility for characterizing vaccine failure and antiviral drug resistance. the average ugandan cluster consisted of 30 cases within a 13-km radius and 38 days, and viruses were detected in only some of the patients. not unexpectedly, certain factors were associated with the ugandan sari clusters, such as human immunodeficiency virus (hiv) infection, urban location, higher population density (eg, 431.5 vs 250 persons/km 2 ), or higher median annual rainfall of 1293 vs 1150 mm/year. these differences, though statistically significant, need to be put in perspective, as the study was anchored in sentinel hospitals, typically located in urban areas where hiv status is more likely to be known, whereas sari cases in rural areas may not have been equally able to seek hospital admission. given the >10-fold higher average population density of boston (5000 persons/km 2 ), or basel (7500 persons/km 2 ), one might suspect that the associations in the ugandan setting may have been a surrogate for other factors such as general hygiene standards, other medical conditions, and socioeconomic factors in certain suburban quarters. similarly, the differences in annual rainfall of <10% need to be balanced against larger seasonal changes in short-term higher rainfall, which may be more important for case clustering and should be addressed in future studies. despite a critical appreciation, this collaborative us-ugandan study combining epidemiology with state-of-the-art modeling and virology not only sets standards for resource-rich industrial settings, but provides next to hiv, ebola, and malaria another proof-of-concept of feasibility and impact of innovative joint projects and partnerships between advanced research groups and dedicated institutions in resource-limited countries. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. redefining chronic viral infection translational aspects of the human respiratory virome a new arenavirus in a cluster of fatal transplant-associated diseases identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy the human gut virome: inter-individual variation and dynamic response to diet intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation temporal response of the human virome to immunosuppression and antiviral therapy full-length haplotype reconstruction to infer the structure of heterogeneous virus populations evaluation of unbiased next-generation sequencing of rna (rna-seq) as a diagnostic method in influenza virus-positive respiratory samples global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis mortality associated with influenza and respiratory syncytial virus in the united states global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study comparing viral respiratory tract infections in symptomatic children and adults: multiplex nat from 2010-2015 comparing luminex nxtag-respiratory pathogen panel and respifinder-22 for multiplex detection of respiratory pathogens community-acquired respiratory paramyxovirus infection after allogeneic hematopoietic cell transplantation: a single-center experience comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h1n1: what else is out there? high incidence of active cytomegalovirus infection among septic patients sepsis and cytomegalovirus: foes or conspirators? viral impact on long-term kidney graft function human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments bk virus: opportunity makes a pathogen reactivation of multiple viruses in patients with sepsis torque teno virus in children who underwent orthotopic liver transplantation: new insights about a common pathogen viral infection in acute exacerbation of idiopathic pulmonary fibrosis the evolution of seasonal influenza viruses key: cord-348178-6bjimde4 authors: li, ling; gu, jiang; shi, xicheng; gong, encong; li, xingwang; shao, hongquan; shi, xueying; jiang, huijun; gao, xiaoqiang; cheng, daiyun; guo, lizhu; wang, hao; shi, xiaohong; wang, peizhi; zhang, qianying; shen, bing title: biosafety level 3 laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects date: 2005-09-15 journal: clin infect dis doi: 10.1086/432720 sha: doc_id: 348178 cord_uid: 6bjimde4 background. during the outbreak of the emergent severe acute respiratory syndrome (sars) infection, >30% of the ∼8000 infected persons were health care workers. the highly infectious nature of sars coronavirus (sars-cov) compelled our pathologists to consider biosafety issues in the autopsy room and for tissue processing procedures. methods. a specially designed biosafety level 3 (bsl-3) autopsy laboratory was constructed and divided into a clean area, a semicontaminated area, a contaminated area, and 2 buffer zones. high-efficiency particulate air filters were placed in the air supply and exhaust systems. laminar air flow was from the clean areas to the less clean areas. the negative pressures of the contaminated, semicontaminated, and clean areas were approximately -50 pa, -25 pa, and -5 pa, respectively. personal protective equipment, including gas mask, impermeable protective clothing, and 3 layers of gloves worn during autopsies; the equipment was decontaminated before it was allowed to exit the facility. strict bsl-3 practices were followed. results. when a given concentration of particulate sarin simulant was introduced into the contaminated area, it could not be detected in either the semicontaminated area or clean area, and particles >0.3 μm in size were not detected in the exhaust air. a total of 16 complete postmortem examinations for probable and suspected sars were performed during a 2-month period. of these, 7 reported confirmed cases of sars. none of the 23 pathologists and technicians who participated in these autopsies was infected with sars-cov. conclusions. our experience suggests that bsl-3 laboratory operating principles should be among the special requirements for performing autopsies of contaminated bodies and that they can safeguard the clinicians and the environment involved in these procedures. in november of 2002, severe acute respiratory syndrome (sars), which is caused by a novel sars coronavirus (sars-cov), originated in the guangdong province of china. during the subsequent 6 months, it quickly spread to 33 countries worldwide. beijing was one of the most strongly affected areas. during this new epidemic, which had a mortality rate of 10%-15%, complete autopsy became necessary to investigate the cause, pathogenesis, and pathological changes of the syndrome [1] . according to the guidelines of world health organization and the centers for disease control and prevention (cdc) in the united states, sars-cov fulfills the criteria for a biohazard group 3 pathogen. therefore, autopsies of persons who died of sars must be performed in a biosafety level 3 (bsl-3) laboratory [2] [3] [4] [5] . however, although the monograph published by the cdc made recommendations for the performance of such autopsies, the information it provided was insufficiently detailed [6] . complete autopsy of patients who have had sars or who probably had sars is a very high-risk procedure. autopsy-transmitted infections may occur through percutaneous injury, exposure to infectious aerosols produced by drills, gross contamination with blood, and escape of gas from hollow organs [7] [8] [9] . a bsl-3 autopsy laboratory for sars was established in beijing ditan hospital (which was designated the sars hospital during the outbreak of sars in china) in may 2003. sixteen complete autopsies were performed on patients with clinically confirmed or suspected sars in the subsequent 2-month period. seven of the cases were later confirmed to be sars infections. none of the 23 pathologists and technicians involved in these autopsies became infected with sars-cov. in this article, we describe our experience with the construction of the bsl-3 autopsy laboratory and with its operating principles. laboratory construction. the bsl-3 autopsy laboratory was physically separated from other parts of the building. it was divided into 5 sections (a clean area, a semicontaminated area, a contaminated area, and 2 buffer zones), and each section was segregated by an airtight door. the mortuary was connected to buffer zone ii (figure 1). the contaminated area served as the autopsy room, with a dissection table located in the center. there was no tap water or sewage system in this room. autopsy instrument cabinets, specimen-fixation containers, liquid nitrogen tanks, a deep freezer, and remote control cameras were installed at the periphery of the room. specimen collection, dissection, and fixation were all performed exclusively on the dissection table. buffer zone i separated the contaminated area from the semicontaminated area. it consisted of a chemical decontamination unit and an air decontamination unit, in which the persons decontaminated their personal protective equipment (ppe) before leaving for the semicontaminated area. the semicontaminated area connected buffer zone i with the clean area. the monitoring station of our laboratory was installed in this area because of space limitations (after the sars outbreak ended, it was moved out). the laboratory director and his assistant (in full ppe) monitored the complete autopsy process with use of a remote control video camera, a color monitor, and an intercom. air-cleaning equipment (research institute of chemical defense) was used to kill airborne pathogens continuously with plasma (an electrically neutral, highly ionized gas composed of ions, electrons, and neutral particles), catalysis, oxidation, and filtration. three 760-l tanks, each of which contained 0.5% peracetic acid, were used for ppe decontamination and were placed near the exit of the semicontaminated area. a transmission cabin was designed to transport clean articles into the contaminated area when needed, but this was not actually used during the autopsies. an antidraft valve was installed in the wall to maintain the air-pressure gradient. buffer zone ii was designed to connect the mortuary with the contaminated area and was used for transportation of potentially infectious materials, such as corpses, specimens, and waste products. it was divided into an outer part and an inner part. an autoclave was installed in the wall between the outer part and the contaminated area. an exit led straight outside from buffer zone ii. ventilation systems. the 2 ventilation systems of the bsl-3 autopsy laboratory were kept physically separated from the air supply system for other parts of the building. one system controlled the airflow for the mortuary and buffer zone ii, which was separated from the other parts of the laboratory. laminar airflow was from clean areas to less clean areas. the average rate of the airflow in the laboratory was ∼2000 m 3 /h. negative pressures in the clean area, the semicontaminated area, and the contaminated area were kept at ϫ5 pa, ϫ25 pa, and ϫ50 pa, respectively. negative pressure in the mortuary was approximately the same as that for the contaminated area. the rate of airflow and the pressure in each area were monitored by a central automatic control system in the clean area. highefficiency particulate air (hepa) filters (ricd) were placed in the supply and exhaust air systems; the filters were capable of trapping 99.9999% of 0.3-mm particles (figure 1). in the contaminated area, downdraft table ventilation was used to decrease exposure to aerosolized pathogens. the hepafiltered air entered from the ceiling, flowed down over the autopsy table, with airflow splitting ∼6-18 cm from the operating surface, and escaped through the outlets at the bottom of the wall. aerosol particles generated at the work surface were immediately captured by the downward airflow, establishing an air-screen between the corpse on the table and the investigators (figure 2). ppe. to protect the eyes, skin, and mucous membranes of investigators, use of full ppe was mandatory in the clean area. the ppe included gas mask, impermeable protective clothing, and 2 layers of surgical gloves plus an additional outer pair of rubber gloves (ricd) (figure 3). the filters on the gas mask consisted of hepa and gas filters. they protected against gas, chemical vapors, and particles. these filters were disposable and had a maximum life of 4-6 h. the gas mask formed an air-proof seal around the face. all inhaled and exhaled air passed through the mask. the impermeable protective clothing was made of a specific type of rubber and wholly enclosed the investigator, except for the face. the air-proof space around the body was tested each time before investigators entered the contaminated area. the overlap and the tight adhesion between the mask and clothing eliminated air leakage entirely. the ppe was decontaminated in 3 steps after an autopsy was completed. first, investigators wiped the blood remaining on the surface of ppe with 0.5% peracetic acid, especially in the areas of hands, arms, and thorax. the ppe was then washed with high-pressure 0.5% peracetic acid for 5 min within the enclosed liquid decontamination unit in buffer zone i and was dried with sterilized towel. finally, the ppe was sprayed with multiple-direction cold plasma (as from p4) for 5 min in the air-decontamination unit of buffer zone i. the impermeable protective clothing and gas mask were removed, each in turn, for further decontamination in the semicontaminated area. after removal of the gas mask, the investigators held their breath in the semicontaminated area until they entered into the clean area, then showered in the clean area before putting on street clothing and exiting the outside. the efficiency of decontamination was evaluated by a sarin simulant test, as described below. effluent and waste disposal. solid waste products included the corpses, dressings, and other discarded material. waste products were taken to a crematory designated for sars cases under government authority; specially trained staff transported the materials in ambulances used only for this purpose. all staff involved in the transportation wore full ppe. before transportation, the waste products were placed in 3 layers of fully sealed, leak-proof, heavy-duty plastic bags with appropriate labeling. liquid waste products from the corpses were decontaminated with neutralbuffered formaldehyde for 1-2 weeks and were then released into the sewage disposal system of beijing ditan hospital. beijing ditan hospital's infectious diseases sewage system also has its own primary sewage decontamination system. decontamination and maintenance of the laboratory. because the gross spillage of potentially infectious materials during autopsy was inevitable, we decontaminated the area after each autopsy. the air was sterilized with uv light for 1 h, surfaces and the floor were decontaminated with 0.5% peracetic acid containing chlorine, 3000 mg/l, and the same disinfectant was distributed by aerosols at a concentration of 20 ml/m 3 . two additional lower-power fans in the ventilation system maintained a persistent, mild negative pressure gradient in our laboratory during working breaks. staff training and administration. twenty-three pathologists and technicians who participated in autopsies underwent intensive biosafety training. before any autopsies were performed, comprehensive biosafety training concerning the facility designs, proper use of ppe, and appropriate procedures and administrative rules was performed in our department. each group (which consisted of 3 pathologists and 1 technician) rehearsed twice under the direction of the biosafety specialists. after the ventilation system in the newly constructed bsl-3 autopsy laboratory had been running for 10 min, negative pressure in the contaminated area approached ϫ40 pa (the lowest value in the predefined range), and the negative pressure gradient from the clean area to the less clean area reached ∼20 pa. at this time, a sarin simulant aerosol of 0.3-mm particles at 4 mg/l was generated and spread by a special device in the contaminated area. one staff member wearing full ppe stayed in this room for 5 min and then went into the clean area through buffer zone i and the semicontaminated area after undergoing normal decontamination procedures. during the process, the protective function of the ventilation system was tested every 5-10 min for 40 min. the negative pressures in the contaminated area, the semicontaminated area, and the clean area were within the predetermined ranges of ϫ40 to ϫ50 pa, ϫ20 to ϫ25 pa, and ϫ5 pa, respectively. the pressure gradient from the clean area to less clean areas was ∼20 pa, as expected (table 1). the concentration of sarin simulant in the contaminated area decreased from 10 ϫ2 ppm to 0 ppm, and sarin was undetectable in the clean area and the semicontaminated area (table 2). particles of 10.3 mm in diameter were undetectable in the exhaust air. laboratory application. sixteen complete postmortem examinations were performed on the cadavers of persons with clinically diagnosed or suspected sars in the newly constructed bsl-3 autopsy laboratory from may to july 2003. seven cases in this study were confirmed to be sars-cov infection on the basis of identification of the pathogen in these patients by in situ hybridization, rt-pcr, or electron microscopy. gross examination of each organ collected from these 16 patients was performed, and photographs were taken with a digital camera. large numbers of specimens were collected from each system, including nasal mucosa, conjunctiva, brain, spinal cord, bone marrow, blood, urine, feces, ascites, and pleural fluid. for each case, 3 series of specimens were obtained. the first series for light microscopy were fixed in neutral formaldehyde for at least 2 weeks. the second series for electron microscopy were fixed in cold 3%-5% glutaraldehyde for 3-5 days before further processing. the third series were stored in the deep freezer and in liquid nitrogen for further research. comprehensive videotape recorded the process of each autopsy and has subsequently been used for further biosafety training. twenty-three pathologists and technicians participated in these autopsies. in accordance with government policy, each cohort of 3 pathologists and 1 technician was kept in isolation for 2 weeks after completing an autopsy. during this period of time, body temperatures were taken twice per day, and the serum test for sars-cov was performed as well. none of these personnel demonstrated any evidence of sars infection. the dangers of transmission of infectious diseases in the autopsy room have long been appreciated [10] . however, specific autopsy biosafety issues may not have been seriously taken into consideration by pathologists in china until the outbreak of sars. most autopsy facilities in china are located in older premises. they often share ventilation with other spaces in the building, and many do not meet the design criteria for bsl-2. furthermore, many pathologists in china have been slow in complying with precautions promulgated to prevent the transmission of both bloodborne and aerosolized pathogens [11, 12] . in fact, 17 autopsies of patients with sars or suspected sars were performed under such inadequate conditions [13] [14] [15] [16] [17] . sars-cov fulfills the criteria of biohazard group 3. the main modes of transmission are through droplet spread and close/ direct contact, but situations conducive to aerosol generation appeared to be associated with higher risk, and there are still many factors concerning the mode of transmission and the environmental risk that need to be clarified [18] . studies have shown that sars-cov can be found in sputum, tears, blood, urine, feces, and sweat glands. the virus can be shed in the feces for 30 days, and it has been shown to survive on hard surfaces for 124 h [19, 20] . therefore, autopsies involving sars autopsies may be considered more dangerous than are autopsies involving more common biohazard group 3 organisms, such as mycobacterium tuberculosis. autopsies involving sars should be performed in at least a bsl-3 laboratory. to ensure the safety of the staff and the environment, a bsl-3 autopsy laboratory was constructed in beijing ditan hospital in may 2003. after 16 complete autopsies were performed, none of the 23 involved pathologists and technicians showed evi-dence of sars infection. since the last autopsy was performed, our bsl-3 autopsy laboratory has been maintained, and it has now become the autopsy center for the sars research laboratory network system and emergency infectious diseases in china. autopsies involving 2 persons with suspected sars, 1 person with aids, and 1 person with gas gangrene have been safely performed in this facility during the 2 years following its initial use for sars cases. the biosafety of our bsl-3 autopsy laboratory has been ensured in 4 ways: through the design of the facility, use of ppe, decontamination, and administrative regulation. first, use of a bsl-3 ventilation system is the prerequisite for sars autopsies. second, because ppe is the first barrier of protection, we invested in the maximum protection available, including gas masks, impermeable protective clothing, and 3 layers of gloves. because the use of oscillating saws, spray, and aspirators hoses may generate infectious aerosols, and because dissection of lungs can also create aerosols and droplets [8, 9] , respiratory protection for sars autopsies is especially important. the combined filter system of the gas masks used in our facility provided the highest level of protection available to the investigators. although the cdc recommended that n-100 particulate respirators or powered air-purifying respirators (paprs) with hepa filters should be used if aerosols may be generated [6, 21] , paprs were not available in beijing during the sars outbreak; thus, the gas mask became the practical choice for use in our facility. however, we noticed that the gas masks made the investigators feel uncomfortable. therefore, paprs would be the better choice in the future. the 3 layers of gloves made the investigators work more cumbersome and prone to mistakes, but this was nevertheless necessary for secure protection. third, a 3-step procedure for decontamination of ppe was strictly followed. this was indispensable for the safety of the investigators, laboratory, and environment. last but not least, we strictly adhered to administration controls, including rigorous biosafety training, supervision, and operational procedures. our experiences suggest that monitoring personal behaviors, together with teaching of biosafety and correct attitudes to those involved in the autopsies, best ensure the safety of autopsy performance. it is noteworthy that safety administration may be neglected until something goes wrong. for example, 2 sars cases that appeared at the end of 2003 were laboratory acquired [22, 23] . the small sars outbreak in late march and mid-april 2004 in china also originated from contamination of 2 researchers at the national institute of virology in china's center for disease control [24] . these incidences indicated that safety precautions and the personal attitudes of staff members are what really counts, provided that stringent engineering and ppe standards are applied. on the basis of our experience, compared with the experience at the facilities at which the 3 accidents occurred, we feel that conscientiousness about biosafety cannot be overly stressed. although the following was not the focus of this article, we would like to briefly share some observations that we made during this work. when confronting the potential threats of novel emerging infectious diseases and bioterrorist events, we need a better-prepared national bsl-3 laboratory network, including an autopsy laboratory, research laboratory, and mobile autopsy facilities. such facilities could be capable of response to public health events. as a result of the imbalance in economic development in china and of china's vast territory, and considering the costs of remolding old facilities and of new construction, a mobile autopsy facility constructed to operate at bsl 3 or 4 would be beneficial in providing autopsy support to regions with inadequate facilities when they are confronted with contagious cases. such a laboratory could be set up in a specially designed bus with 2 sections, including a diagnostic laboratory and an autopsy room equipped with an autonomous water supply and sewage system, a heating system, and necessary medical equipment, allowing one to perform autopsies of patients with infectious cases in rural conditions [25] . in conclusion, our experience of 16 complete postmortem examinations for probable and suspected sars suggests that application of bsl-3 laboratory operating principles to the special requirements for contaminated autopsies can safeguard the investigators and the environment for performance of sars autopsies. world health organization. world health organization issues emergency travel advisory geneva: world health organization centers for disease control, and prevention and national institutes of health world health organization. who post-outbreak biosafety guidelines for handling of sars-cov specimens and cultures interim laboratory biosafety guidelines for handling and processing specimens associated with severe acute respiratory syndrome (sars) department of health and human services safe handling of human remains of severe acute respiratory syndrome (sars) patients: interim domestic guidance protection of laboratory workers from instrument biohazards and infectious disease transmitted by blood, body fluids, and tissue; approved guideline. approved standard m-29a-17 characteristics of aerosols generated during autopsy procedures and their potential role as carriers of infectious agents aerosols in the mortuary association of clinical pathologists' surveys of infection in british clinical laboratories, 1970-1989 construction of autopsy room and personnel protection of sars autopsy staff draw inspiration from sars pathology morphological study of severe acute respiratory syndrome (sars) clinical pathology and pathogenesis of severe acute respiratory syndrome expression of the monoclonal antibody against nucleocapsid antigen of sars-associated coronavirus in autopsy tissues from sars patients pathological and ultramicrostructural changes of tissues in a patient with severe acute respiratory syndrome cases of severe acute respiratory syndrome the epidemiology of the outbreak of severe acute respiratory syndrome (sars) in hongkong-what we do know and what we don't listening to sars: lessons for infection control organ distribution of severe acute respiratory syndrome (sars) associated cornnavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities laboratory-acquired severe acute respiratory syndrome severe acute respiratory syndrome-taiwan sars: china dumps cdc head, probes lab mobile laboratory for autopsies and histological studies we would like to express grateful thanks to dr. michael a. mcnutt for editing the manuscript.financial support. this study was supported by 2 grants: one from the ministry of science and technology, peoples republic of china (entitled "autopsies, sample collection, and pathogenesis of sars"), and another from beijing science and technology research committee, peoples republic of china (entitled "collaborative investigation of the immunopathology of sars between china and england"). the project title was "investigation of the etiology and pathogenesis of sars" (h030230100130).potential conflicts of interest. all authors: no conflicts. key: cord-341838-lkz8ro90 authors: gervasoni, cristina; meraviglia, paola; riva, agostino; giacomelli, andrea; oreni, letizia; minisci, davide; atzori, chiara; ridolfo, annalisa; cattaneo, dario title: clinical features and outcomes of hiv patients with coronavirus disease 2019 date: 2020-05-14 journal: clin infect dis doi: 10.1093/cid/ciaa579 sha: doc_id: 341838 cord_uid: lkz8ro90 little is known about the clinical outcomes of hiv patients infected with sars-cov-2. we describe 47 patients referred to our hospital between 21 february and 16 april 2020 with proven/probable covid-19, 45 (96%) of whom fully recovered and two died. as of april 20, the pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection had affected 2.5 million people all over the world, and led to more than 165,000 deaths (update available at https://coronavirus.jhu.edu/map.html). initial information from china and evidence accumulated over recent weeks has allowed the identification of some of the risk factors associated with a negative prognosis, includingaging, male gender, hypertension, diabetes mellitus, and cardiovascular, lung and/or kidney diseases. [1] [2] [3] [4] however, little is known about the impact of hiv infection on the clinical outcomes of patients infected with sars-cov-2 because, to the best of our knowledge, only case reports or small case series have so far been published. [5] [6] [7] treated people living with hiv who have a normal cd4 t cell count and suppressed viral load may not be at increased risk of serious illness, but many also have other conditions that increase their overall risk: almost half of hiv patients are males, aged >50 years, and affected by chronic cardiovascular and lung diseases. the aim of this retrospective study was to describe the clinical characteristics and outcomes of hivinfected patients with a probable/proven diagnosis of sars-cov-2 infection who have been regularly followed up by our hospital. a c c e p t e d m a n u s c r i p t 4 we searched our database for hiv patients diagnosed as having probable or proven sars-cov-2 infection between 21 february and 20 april 2020. a diagnosis of probable sars-cov-2 infection was based on the presence of fever and respiratory symptoms (cough and dyspnea), epidemiological risk factors such as relatives or close colleagues with a proven diagnosis of covid-19, and/or a chest xray or ct diagnosis of interstitial pneumonia; proven sars-cov-2 infection required a throat swab positive for viral nucleic acid. we also recorded their main demographic data, pharmacological treatments and clinical outcomes. the database of our department of infectious diseases included nearly 6,000 hiv-positive patients (74% males; mean age 52±12 years); 90% had <20 copies/ml of hiv viral load and 76% had a cd4 cell count of >500 cells/mm 3 (3% had <200 cells/mm 3 ). during the observation period, 47 hiv patients with proven or probable sars-cov-2 infection were identified. they were mainly males (76%) and had a mean age of 51±11 years. as shown in table 1 , most of the patients showed suppressed hiv viremia and acceptable immune reconstitution (cd4 cell count >350 cells/mm 3 ); three (all males) had detectable hiv viral loads of 52, 72 and 134 copies/ml. nearly 64% had at least one co-morbidity (82% of the males and 58% of the females), mainly dyslipidemia (32%), arterial hypertension (30%) and hepatitis b or hepatitis c co-infections (11%). approximately 80% of the identified patients were receiving integrase inhibitor-based antiretroviral treatment and 11% a protease inhibitor-based regimen (11%); 42% were receiving a tenofovir-based regimen ( table 2) . twenty-eight patients (>50%) tested positive for sars-cov-2, including one female asymptomatic patient who was tested because she was a healthcare provider; the remainder were not tested mainly because they lived in the high-risk provinces of bergamo and brescia (lombardy) but were isolated at home and cared for by their general practitioners. the covid-19 diagnosis of the untested patients was based on their clinical symptoms and the presence of risk factors (13% were healthcare providers; 9% had been in close contact with sars-cov-2 positive working colleagues and 23% with sars-cov-2 positive relatives). interstitial pneumonia was diagnosed by means of an x-ray in three cases, and ground-glass opacity was identified by means of ct in one. thirteen of the 28 sars-cov-2 positive patients were hospitalised. six had severe lung disease (respiratory rate ≥30 breaths/min; resting percutaneous oxygen saturation ≤93% in room air), two of whom required mechanical ventilation: one recovered and was discharged and the other (a 47-year-old overweight man without other co-morbidities) died. another a c c e p t e d m a n u s c r i p t 6 patient with cardiovascular disease and a recent diagnosis of lung cancer died during hospitalisation. for comparative purposes, the crude mortality rate of the hiv-negative covid-19 patients in our hospital (n=502, 67% males, mean age 61±16 years) is currently forty-five of the patients recovered 14±8 days after symptom onset, with no significant difference between the females and males (11±7 versus 14±9 days; p=0.338). table 2 , fewer than 50% of the patients were given potential anti-sars-cov-2 treatments, specifically hydroxychloroquine (17%), azithromycin (15%), lopinavir/ritonavir (11%); one was treated with tocilizumab and remdesivir, and one with toxicizumab alone. this report describes our experience with hiv-positive patients regularly followed by our hospital (a reference hospital for the management of hiv infection in italy) who were infected with sars-cov-2. as in the general population, the large majority of our patients were males, but their mean age was nearly 10 years lower than that observed in hiv-negative covid-19 patients. [8] [9] [10] this is in line with the recent finding of blanco et al. who described five hiv-infected patients with covid-19 from spain who were aged 29-49 years. 5 the difference in age between hiv-positive and hiv-negative patients with covid-19 may possibly be explained by the general belief that hiv infection can add nearly 10 years to the chronological age. 11, 12 this is indirectly supported by the fact that, although they are about 50 years old, the large majority of our patients have at least one co-morbidity, a picture that is more frequently observed in patients aged >60 years in the general population. the risk of severe disease in our hiv-patients compared favourably with that observed in the general population of covid-19 patients. 13 likewise, the risk of death or admission to an intensive care unit was lower than that observed in the hiv-negative patients treated at our hospital and in another cohort of hiv-negative covid-19 patients of a similar mean age. 14 it is worth noting that these positive outcomes were achieved even though fewer than 50% of the patients were treated with drugs currently considered to be potential treatments for sars-cov-2 infection, and only two received remdesivir or tocilizumab. it could be argued that antiretroviral therapy may have played a role in the positive evolution of covid-19 in our hiv patients; indeed, lopinavir/ritonavir, darunavir/ritonavir and darunavir/cobicistat were all initially suggested as candidate treatments for sars-cov-2 infection. 15 however, we have recently shown that darunavir does not prevent sars-cov-2 infection in people living with hiv or protect against worsening respiratory function, at least not at a dose of 800 mg. 16 furthermore, the findings of this study document favourable outcomes in hiv patients treated mainly with integrase inhibitors (11% protease inhibitors), which apparently indicates that antiretroviral therapy does not play a key role, a c c e p t e d m a n u s c r i p t 8 although a potentially protective effect of tenofovir cannot be ruled out given its recently reported effect against sars-cov-2 rna-dependent rna polymerase. 17 another possible explanation of the more favourable clinical outcome observed in our hiv-positive patients is that, despite their effective antiretroviral therapy, they still had deficient immune systems (albeit to different degrees) and showed some persistent immune activation: consequently, the evolution of covid-19 may be milder and not progress to a severe cytokine storm. 18 finally, as all of our hiv-negative patients required hospitalisation, they may have been more susceptible to an unfavourable outcome. the main limitation of this retrospective observational study is that it also includes hiv patients with probable but not confirmed covid-19. however, given the paucity of the data published so far, we believe that our real-life experience can provide useful information concerning the management of hiv-infected patients with sars-cov-2 infection that may eventually be used for prospective investigations. in conclusion, our findings suggest that hiv-positive patients with sars-cov-2 infection are not at greater risk of severe disease or death than hiv-negative patients. however, the observed more favourable outcomes need to be confirmed in larger cohort studies. m a n u s c r i p t 15 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study risk factors of healthcare workers with corona virus disease 2019: a retrospective cohort study in a designated hospital of wuhan in china risk factors associated with disease severity and length of hospital stay in covid-19 patients covid-19 in patients with hiv: clinical case series early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv co-infection of sars-cov-2 and hiv in a patient in wuhan city review of the clinical characteristics of coronavirus disease 2019 (covid-19) clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region do people living with hiv experience greater age advancement than their hiv-negative counterparts? basic science and pathogenesis of ageing with hiv: potential mechanisms and biomarkers clinical characteristics of coronavirus disease 2019 in china clinical features and short-term outcomes of 102 patients with corona virus disease 2019 in wuhan, china. clin infect dis. 2020 therapeutic options for the 2019 novel coronavirus darunavir does not prevent sars-cov-2 infection in hiv patients ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study inflammation, immune activation, and antiretroviral therapy in hiv a c c e p t e d m a n u s c r i p t 10 a c c e p t e d m a n u s c r i p t 13 key: cord-343827-jo61t3m0 authors: qian, qun; fan, lifang; liu, weicheng; li, jin; yue, junqiu; wang, mingwei; ke, xianliang; yin, yan; chen, quanjiao; jiang, congqing title: direct evidence of active sars-cov-2 replication in the intestine date: 2020-07-08 journal: clin infect dis doi: 10.1093/cid/ciaa925 sha: doc_id: 343827 cord_uid: jo61t3m0 background: currently, there is no direct evidence to prove the active sars-cov-2 replication in the intestinal tract and relevant pathological changes in the colon and rectum. we investigated the presence of virions and pathological changes in surgical rectal tissues of a clinically confirmed covid-19 patient with rectal adenocarcinoma. methods: here, the clinical data were collected during hospitalization and follow-up of this patient. quantitative rt-pcr was performed on the rectal tissue specimens obtained from surgical resection, succus entericus and intestinal mucosa of ileostomy, and rectal mucosa during follow-up after recovery. ultrathin sections of surgical samples were observed for sars-cov-2 virions using electron microscopy. histopathological examination was performed using hematoxylin-eosin stain. immunohistochemical analysis and immunofluorescence were carried out on rectal tissues to evaluate the distribution of sars-cov-2 antigen, and immune cell infiltrations. results: the patient had fever and cough on day 3 postoperatively, was diagnosed with covid-19 on day 7, and was discharged from the hospital on day 41. rna of sars-cov-2 was detected in surgically resected rectal specimens, but not in samples collected on 37 day after discharge. notably, coincidence with rectal tissues of surgical specimens tested nucleic acid positive for sars-cov-2, typical coronavirus virions in rectal tissue were observed under electron microscopy. moreover, abundant lymphocytes and macrophages (some are sars-cov-2 positive) infiltrating the lamina propria were found with no significant mucosal damage. conclusions: we firstly reported that direct evidence of the active sars-cov-2 replication in the patient's rectum during the incubation period, which might explain sars-cov-2 fecal-oral transmission. the current coronavirus disease identified in 2019 , caused by a novel coronavirus, has become a global public health problem [1, 2] . as of may 27, 2020, a total of 5,451,532 cases of covid-19 have been confirmed globally, including 345,752 deaths [3] . there are many reports suggesting that sars-cov-2 rna can be detected and identified in anal/rectal swabs [4, 5] and stool specimens [6, 7] . in fact, one recent small sample study found that rna was consistently detected in rectal swabs, even after viral clearance from the upper respiratory tract, indicating extended duration of viral shedding in fecal samples and raising the possibility of fecal-oral transmission of sars-cov-2 [5] . similar results were reported in another study with more cases involved, raising the possibility of prolonged presence of sars-cov-2 in stools. notably, fecal samples remained positive for sars-cov-2 rna nearly 5 weeks after the viral clearance from the upper respiratory tract in covid-19 patients [8] . considering a high degree of sequence homology between the sars-cov-2 and sars-cov, angiotensin-converting enzyme ii (ace2) has been identified as the entry receptor of sars-cov-2. since this receptor is highly expressed on the epithelial cells from the ileum and colon [9] , the intestinal tract may be a potential route for sarapatients with cancer are considered to be more susceptible to sars-cov-2 [10, 11] . one patient with rectal cancer was admitted to zhongnan hospital of wuhan university for radical operation. on postoperative day 3, the patient began to develop cough and a c c e p t e d m a n u s c r i p t fever; chest ct revealed radiologic characteristics of viral pneumonia. on postoperative day 7, the patient was confirmed to be infected with sars-cov-2. although live sars-cov-2 had been successfully isolated from the fecal sample of a laboratory confirmed sars-cov-2 patient [12] , until now, there has been no direct evidence to prove active sars-cov-2 virus replication in the intestinal tract. it remains unknown whether there are pathological changes related to sars-cov-2 infection existing in colorectal mucosa in covid-19 patients. to clarify the above questions, we performed a retrospective study to detect the presence of sars-cov-2 virions and determine the pathological changes in rectal tissues of this patient. the patient's clinical information is described in supplementary samples of rectal tissues, succus entericus and intestinal mucosa of ileostomy, and rectal mucosa were tested for sars-cov-2 nucleic acid using qrt-pcr. the qrt-pcr analyses were performed following a previously described method [13] . the qrt-pcr test kits (biogerm) were recommended by the chinese center for disease control a c c e p t e d m a n u s c r i p t and prevention. the rectal tissue obtained by resection was soaked in rnalater solution overnight, the solution was discarded and the tissue was frozen at -80°c. the rectal tissue was cut into 1 mm thick sections and fixed in 2.5% glutaraldehyde and 1% osmium tetroxide in a biosafety cabinet with level 2 protection, and subsequently dehydrated using different ascending concentrations of alcohol (30% to 100%), and immersed and embedded in epoxy resin. ultrathin sections (80-100 nm) were prepared on formvar-coated copper grids (200 mesh). the virions were observed with a tecnai g2 20 twin electron microscope (fei company) under 200 kv. immunohistochemical staining was performed on formalin-fixed and paraffinembedded tissue sections (4-μm). sections of rectal tissues were immunostained to evaluate the expression and distribution of the sars-cov-2 antigen. briefly, sections were deparaffinized with xylene and alcohol and subsequently heated in citrate buffer (ph 6.0) for antigen retrieval. after blockage with 3% bsa in pbs for 30 minutes, a rabbit antibody against rp3 np protein (kindly provided by dr. zhengli shi, wuhan institute of virology, chinese academy of science [14] ) was incubated with the sections overnight at 4°c. for immunochemistry study, the slides were subsequently figure 1 . the patient's clinical information is shown in supplementary table 1 clinical characteristics of coronavirus disease 2019 in china the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status world health organization. coronavirus disease (covid-2019) situation reports diarrhea may be underestimated: a missing link in 2019 novel coronavirus characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding the presence of sars-cov-2 rna in the feces of covid-19 patients epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore prolonged presence of sars-cov-2 viral rna in faecal samples the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes clinical features of patients infected with 2019 novel coronavirus in wuhan sars-cov-2 transmission in patients with cancer at a tertiary care hospital in wuhan, china isolation of 2019-ncov from a stool specimen of a laboratory confirmed case of the coronavirus disease 2019 (covid-19) positive rt-pcr test results in patients recovered from covid-19 a pneumonia outbreak associated with a new coronavirus of probable bat origin molecular biology, and pathogenesis of avian influenza a (h5n1) infection in humans influenza viremia and the potential for blood-borne transmission influenza viral rna detection in blood as a marker to predict disease severity in hematopoietic cell transplant recipients the fate of influenza a virus after infection of human macrophages and dendritic cells viral infection triggers rapid differentiation of monocytes into dendritic cells influenza virus a infection of human monocyte and macrophage subpopulations reveals increased susceptibility associated with cell differentiation from influenza-induced acute lung injury to annual update in intensive care and emergency medicine evidence for gastrointestinal infection of sars-cov-2 we are grateful to center for instrumental analysis and metrology, the corefacility and technical support, wuhan institute of virology, cas. we declare that we have no conflicts of interest.a c c e p t e d m a n u s c r i p t key: cord-341435-b36h69r1 authors: dawson, patrick; rabold, elizabeth m; laws, rebecca l; conners, erin e; gharpure, radhika; yin, sherry; buono, sean a; dasu, trivikram; bhattacharyya, sanjib; westergaard, ryan p; pray, ian w; ye, dongni; nabity, scott a; tate, jacqueline e; kirking, hannah l title: loss of taste and smell as distinguishing symptoms of covid-19 date: 2020-06-21 journal: clin infect dis doi: 10.1093/cid/ciaa799 sha: doc_id: 341435 cord_uid: b36h69r1 in a household study, loss of taste and/or smell was the fourth most reported symptom (26/42; 62%) among covid-19 case-patients and had the highest positive predictive value (83%; 95% ci: 55–95%) among household contacts. olfactory and taste dysfunctions should be considered for covid-19 case identification and testing prioritization. m a n u s c r i p t the global coronavirus disease 2019 (covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has prompted robust public health investigations to characterize the disease course. early published reports identified fever, cough, and shortness of breath as predominant symptoms of covid-19 (1, 2) , and these classic symptoms have been used for case identification and testing prioritization. as subsequent reports cdc, in partnership with wisconsin department of health services and local health departments, conducted a household study that included known covid-19 index cases and their household members. this investigation provided an opportunity to identify household covid-19 cases and describe their symptom profiles, including loss of taste and smell, prior to diagnosis. all activities in this investigation were part of the public health response and determined as not human subjects research. during march-april 2020, health department personnel in milwaukee county, wisconsin, assisted cdc in identifying a convenience sample of households with a laboratory-confirmed covid-19 index case ≤10 days from diagnosis who resided in the m a n u s c r i p t home at enrollment and lived with at least one other household member. informed consent was obtained for all participants. all participants completed questionnaires assessing basic demographic information, medical history, symptoms since the index case's disease onset, and other epidemiological information. a parent or legal guardian assisted in completing questionnaires for children <18 years. at enrollment, we collected nasopharyngeal (np) swabs from all participants; to fulfill a secondary objective of this investigation, we also collected nasal self-collect (nsc) swabs from index cases and symptomatic household members. the city of milwaukee health department laboratory tested swabs using the cdc sars-cov-2 real-time reverse transcription polymerase chain reaction (rt-pcr) assay (7) . we classified any participant having a sars-cov-2-positive np swab or nsc swab collected in the 10 days prior to and including enrollment as a covid-19 case. we compared the prevalence of symptoms among cases descriptively and computed bivariate analyses of demographic, medical history, and symptoms with reported loss of taste and/or smell using two-tailed pearson's chi-squared test or fisher's exact test. we calculated the positive predictive value and 95% cis of individual symptoms for covid-19 among household contacts. statistical analyses were performed using sas® version 9.4, sas institute inc., cary, north carolina. we enrolled 90 participants from 26 households, including 26 index cases and 64 household members. overall, 48 (53%) study participants were male; 69 (77%) were adults ≥18 years old (median=31 years; range: <1-90 years); 41 (46%) were black non-hispanic/latino, 37 (41%) were white non-hispanic/latino, and 12 (13%) were other races/ethnicities. preexisting table 1) ; 38 (90%) reported at least one classic covid-19 symptom. the most frequently reported symptoms were cough (81%), headache (76%), fever (subjective or measured ≥100.4°f) (64%), loss of taste and/or smell (62%), and nasal congestion (62%). among the 26 participants with covid-19 who reported loss of taste and/or smell, 24 reported loss of taste with 14/24 (58%) reporting complete loss; 18 participants reported loss of smell with 13/18 (72%) reporting complete loss. there were no significant differences in reporting loss of taste and/or smell by sex, age <18 years versus ≥18 years, race/ethnicity, presence of medical conditions, or between index and household member cases (all p>0.05). of the 26 participants reporting any loss of taste and/or smell, 9 (35%) reported it in the absence of nasal congestion. participants with covid-19 reporting loss of taste and/or smell were more likely to report headache (88% vs. 56%; p=0.03) but were no more or less likely to report any other symptom (p>0.05). no participant reported loss of taste and/or smell as the only symptom. when loss of taste and/or smell was added to the classic symptoms, 95% of participants with a c c e p t e d m a n u s c r i p t covid-19 reported at least one of loss of taste and/or smell, fever, cough, and shortness of breath. among the 64 household members of covid-19 index cases, loss of taste and/or smell was reported by 12 individuals, of whom 10 were positive for sars-cov-2. the positive predictive value (ppv) of any loss of taste and/or smell (83%, 95% ci: 55-95%) was higher than for fever (subjective or measured) and cough, two of the three classic symptoms, and equal to the third, shortness of breath (83%, 95% ci: 44-97%) ( table 1 ). the ppv for complete loss of taste and/or smell (86%, 95% ci: 49-97%) was the highest among any of the symptoms. in this household-based population of individuals with covid-19, including mildly symptomatic individuals who otherwise may not have been tested according to contemporaneous public health guidance (8), loss of taste and/or smell was reported by more than three of every five individuals with confirmed covid-19. among the 64 household contacts of covid-19 index cases, it had the highest ppv of all symptoms, matched only by shortness of breath. when compared to other symptoms, loss of taste and/or smell appeared highly predictive of sars-cov-2 infection and was more predictive than cough. nasal congestion alone is unlikely to explain the taste and smell alterations, as one-third of patients reporting loss of taste and/or smell did not report nasal congestion; other analyses have shown an even smaller proportion of covid-19 cases with concurrent nasal congestion (9) . investigations into the mechanism of covid-19-related olfactory and taste dysfunction include demonstration of olfactory nerve infection by sars-cov, the virus that causes sars, and expression of angiotensin converting enzyme 2 (ace2), the receptor involved in sars-a c c e p t e d m a n u s c r i p t cov-2 pathogenesis, on olfactory epithelial cells in animal models (5, 10) . studies in human models have shown broad expression of ace2 on the epithelial cells of the tongue and oral cavity mucosa (11, 12) . the underlying mechanism for these symptoms is an area of active investigation. this analysis is subject to limitations. all symptoms and medical histories were selfreported and limited by patient recall, health literacy, and availability of home thermometers. because prior and current symptom ascertainment and sars-cov-2 testing were conducted at enrollment, any subsequent development of symptoms or infection that could affect ppvs were not captured, and timelines for pre-enrollment symptoms are not available. however, this approach likely reduced selection biases that may be present in other reports describing the association between loss of taste and smell with sars-cov-2 infection. due to the high prevalence (25%) of covid-19 infection within this population, the ppv identified in this analysis may not be representative of all clinical encounters. however, our findings may be particularly relevant for screening individuals in close contact with known cases. finally, the sample size was small, resulting in wide overlapping confidence intervals for the reported ppvs. in this investigation, adding loss of taste and/or smell to the classic clinical criteria would have captured 95% of cases while only misidentifying two non-cases as cases, compared to 14 non-cases that would have been misidentified by the classic symptoms. in the absence of confirmatory laboratory testing, cste criteria for a probable covid-19 case now include loss of taste and/or smell in conjunction with other non-classic symptoms (3). our findings suggest that adding loss of taste and/or smell to the singular cste clinical criteria, which currently include cough, shortness of breath, and difficulty breathing, may increase the efficiency of probable covid-19 case identification. a c c e p t e d m a n u s c r i p t due to limited testing capacity, most states have prioritized testing of moderately to severely ill patients. however, as the availability of contact tracing and testing expands, testing and diagnoses will shift to include outpatients with milder illness. identifying these cases will assist in appropriate isolation recommendations and the prevention of additional spread within the community. clinicians will benefit from further characterization of the full spectrum of illness in patients and may consider using loss of taste and/or smell in their testing strategies. m a n u s c r i p t clinical characteristics of coronavirus disease 2019 in china hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan technical guidance interim-20-id-01: standardized surveillance case definition and national notification for 2019 novel coronavirus disease (covid-19) self-reported olfactory and taste disorders in patients with severe acute respiratory coronavirus 2 infection: a cross-sectional study olfactory and gustatory dysfunction in coronavirus disease 19 (covid-19) acute-onset smell and taste disorders in the context of covid-19: a pilot multicentre polymerase chain reaction based case-control study interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19) urgent update -prioritization of covid-19 testing for hospitalized patients alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection expression of the sars-cov-2 cells of the olfactory epithelium: identification of cell types and trends with age severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa a c c e p t e d m a n u s c r i p t key: cord-349566-zx9kt144 authors: de alencar, julio cesar garcia; moreira, claudia de lucena; müller, alicia dudy; chaves, cleuber esteves; fukuhara, marina akemi; da silva, elizabeth aparecida; miyamoto, maria de fátima silva; pinto, vanusa barbosa; bueno, cauê gasparotto; lazar, felippe; gomez, luz marina; menezes, maria clara saad; marchini, julio flavio meirelles; marino, lucas oliveira; brandão, rodrigo antônio; souza, heraldo possolo title: double-blind, randomized, placebo-controlled trial with n-acetylcysteine for treatment of severe acute respiratory syndrome caused by covid-19 date: 2020-09-23 journal: clin infect dis doi: 10.1093/cid/ciaa1443 sha: doc_id: 349566 cord_uid: zx9kt144 background: a local increase in angiotensin 2 after inactivation of angiotensin-converting enzyme 2 by sars-cov-2 may induce a redox imbalance in alveolar epithelium cells, causing apoptosis, increased inflammation and, consequently, impaired gas exchange. we hypothesized that n-acetylcysteine (nac) administration could restore this redox homeostasis and suppress unfavorable evolution in covid-19 patients. objective: to determine whether nac in high doses can avoid respiratory failure in patients with covid-19. methods: it was a double-blind, randomized, placebo-controlled, unicentric trial, conducted at the emergency department of hospital das clínicas, são paulo, brazil. we enrolled 135 patients with severe covid-19 (confirmed or suspected), with an oxyhemoglobin saturation of less than 94% or respiratory rate higher than 24 breaths/min. patients were randomized to receive nac 21 g (approximately 300 mg/kg) for 20 hours, or dextrose 5%. primary endpoint was the need for mechanical ventilation. secondary endpoints were time of mechanical ventilation, admission to icu, time in icu, and mortality. results: baseline characteristics were very similar in the two groups, with no significant difference in age, sex, comorbidities, medicines taken, and disease severity. also, groups were similar in laboratory tests and chest ct scan findings. sixteen patients (23.9%) in the placebo group were submitted to endotracheal intubation and mechanical ventilation, compared to 14 patients (20.6%) in the nac group (p=0.675). no difference was observed in secondary endpoints. conclusion: administration of nac in high doses did not affect the evolution of severe covid-19. severe acute respiratory infection (sari) caused by the sars-cov-2 virus may lead to lung failure and the necessity of mechanical ventilation [1] [2] [3] . the mechanisms by which the virus affect the alveolar epithelia are not entirely understood [4] ; however, it seems that, upon reaching the lower airways, the virus spike protein binds to the angiotensin-converting enzyme 2 (ace2) and utilizes it as a means to enter the alveolar cells [5] . ace2 is an enzyme that catalyzes the conversion of angiotensin ii (angii) in angiotensin 1-7 [6] , and it appears to be inactivated by the action of the virus. there is still no robust experimental confirmation for this fact; however, increased serum angii levels have been reported in patients with severe covid-19 cases [7] and enhanced expression of ace2 in adults, compared to children, has been advocated to cause the difference of disease prevalence in these age ranges [8] . physiological intracellular signaling of angii involves increased production of reactive oxygen species (ros), either through the activity of the nox enzyme [9] or mitochondria [10] . at first, these ros are used in signaling mechanisms; however, in excess, they can lead to cell apoptosis or necrosis [11] . moreover, vascular smooth muscle cells in culture exposed to angii enhance the expression of cd40, a crucial mediator of the acquired immune response [12] . this phenomenon occurs through a redox-signaling pathway, involving enhanced nox expression and hydrogen peroxide generation. in these experiments, angii-induced cd40 expression was blocked by treatment with n-acetylcysteine (nac) [12] . nac has been in clinical use as a mucolytic since the 1960s [13] . it is also currently used in acute liver failure [14] or acetaminophen poisoning [15] . its safety is well documented, and its effectiveness in lung diseases may be beyond its mucolytic action, since it may also interfere with the inflammatory response and bronchial tone [16] . nac may also replenish intracellular reduced glutathione (gsh) pools by providing cysteine, an essential precursor in gsh synthesis [17] . therefore, nac administration could restore intracellular redox signaling through enhanced activity of the reduced-oxidized glutathione pair (gsh-gssg), the primary intracellular antioxidant system [18] . a c c e p t e d m a n u s c r i p t 4 we hypothesize that the local increase in angii after inactivation of ace2 by sars-cov-2 leads to a redox imbalance in the alveolar epithelium cells, causing apoptosis, breaking of the alveolar-capillary barrier and, consequently, impaired gas exchange and respiratory failure. other authors also raised this hypothesis [19] . therefore, we designed a double-blind, placebo-controlled, randomized clinical trial to determine whether nac (in doses used to treat acute liver failure), is able to protect alveolar cells and avoid respiratory failure in patients with severe acute respiratory syndrome caused by (confirmed or suspect) covid-19. this is a double-blind, randomized, placebo-controlled trial to assess the effectiveness and safety of intravenous nac to prevent respiratory failure in patients with confirmed or suspect severe covid-19. for paracetamol poisoning [20] . twenty-one grams of nac were administered intravenously for each patient in the nac group, divided into two doses: 14 g in the first 4 hours and 7 g in the next 16 hours. nac was diluted in dextrose 5%, 500ml. therefore, the concentration for each dose was 28 mg/ml, and 14 mg/ml, respectively, and the amount of volume received for each patient was 1000 ml in 20 hours. patients in the placebo group received the same amounts of dextrose 5% in water (1.000 ml in total) intravenously. all patients received standard care, according to the institutional protocol. at the emergency department, it included oxygen supplementation, invasive ventilation, and antibiotics. all patients received the drugs at the emergency department and were later forwarded to hospital wards, icus, or discharged, according to the in-charge emergency physician discretion. all patients in this study received empirically ceftriaxone 2g / day and azithromycin 500mg / day, which was the protocol in our emergency department for severe covid-19. a c c e p t e d m a n u s c r i p t 6 routine laboratory tests were collected from all patients included in the study, including white blood cell counts, c-reactive protein, renal function, lactate dehydrogenase, electrolytes and d-dimer. we confirmed covid-19 by reverse-transcriptase polymerase chain reaction (rt-pcr) assay of nasopharyngeal swab or tracheal aspiration specimens. chest computed tomography was also obtained for all patients. lung involvement extension was classified as higher or lower than 50% of lung volume. the primary clinical endpoint was the necessity for intubation and invasive mechanical ventilation. secondary outcomes were time of invasive mechanical ventilation, admission to icu, time of icu internment, and mortality. the original total sample size was set at 140, since it would provide the trial with 80% power to detect a reduction in the need for invasive mechanical ventilation of 50%, at a twosided significance level of α=0.05. on april 30, 2020, the data and safety monitoring board reviewed results. this review was initially planned as an interim analysis. the planned enrollment of 100 patients in the trial occurred quickly, and the assessment at that point was that the trial was underpowered; thus, a decision was made to continue enrollment by investigators. primary efficacy analysis was on an intention-to-treat basis and included all the patients who had undergone randomization. because the statistical analysis plan did not include a provision for correcting for multiplicity in tests for secondary or other outcomes, results are reported as point estimates and 95% confidence intervals. during the study period, 612 individuals were assessed for eligibility, and 140 were chosen and randomized (figure 1 ). from the 140 sets prepared by the pharmacy, two bags were mishandled during administration, and three patients lost their drug packs while been transferred inside the hospital. therefore, at the end of the study, we had 67 patients in the placebo group and 68 patients in the nac group. these 135 patients were included in the final analysis (table 1) baseline characteristics were very similar between the two groups. the median age was almost identical, as well as the incidence of comorbidities. we present the two most prevalent (hypertension and diabetes), but other diseases, like cancer and autoimmune diseases, were also identical in both groups. of the 63 hypertensive patients, 30 (47.6%) used angiotensin-receptor blockers. these patients were divided equally into both groups (14 of 32 in the placebo group and 16 of 31 in the nac group). the time between the first symptoms of the disease and the protocol inclusion for patients in the nac group was seven days (iqr: 5-10), while for the placebo group was also seven days (iqr: 6-11.5). at the time of hospital admission, oxyhemoglobin saturation was equal in both groups, and all patients needed oxygen supplementation upon arrival at the emergency department. we assessed disease severity utilizing the seven-category ordinal scale, proposed by the world health organization [21] . the vast majority of patients (99.3%) were classified in categories 4 and 5, hospitalized, and about 68% requiring oxygen supplementation or non-invasive ventilation and ongoing care. the severity categories in both groups were, again, very similar. laboratory findings were also similar in both groups. we show in table 1 in summary, the placebo and the experimental groups were equal, suggesting that our randomization process was very effective. table 2) . mortality in both groups was almost equal: 10 patients in placebo (14.7%) and 9 in nac (13.4%). at the end of the study, six patients were still in icu, three in each group. these patients were not included in mortality analysis. no difference was observed time of hospitalization between the two groups. when only the 128 patients with positive rt-pcr for sar-cov-2 were included, the results were the same. no adverse effect was observed in patients who received nac. all patients tolerated the drug and the volume well. in summary, nac administration did not affect the evolution of sars caused by covid-19. the evolution of covid-19 to respiratory failure involves a complex net of inflammatory and vascular events [4] . we hypothesized that the increased availability of angii caused by the sars-cov-2 virus-induced ace2 blockade could be a crucial component in the disease pathogenesis. considering that angii signals inside the cell through a redox mechanism, we designed this clinical trial, aiming to block the intracellular action of angii and prevent alveolar cells inflammation and apoptosis. this was a double-blind, placebo-controlled, randomized trial, where 135 patients diagnosed with severe acute respiratory infection were enrolled. the baseline characteristics of the two groups were very similar in all aspects. a c c e p t e d m a n u s c r i p t regarding the outcome, there was no difference between the placebo and nac groups neither in the primary outcome (need for invasive mechanical ventilation) nor the secondary ones (mortality, icu admission, time of invasive mechanical ventilation). several papers have been published advocating that redox imbalance caused by angii could be a crucial point in sars-cov-2 pathogenesis [20, 22, 23] . our data do not support these hypotheses. it is necessary, therefore, to discuss the causes of this treatment failure in improving respiratory function in covid-19 patients. first, we have to speculate whether the drug and dosage proposed to restore redox balance in these patients were the right ones. since our objective was to correct intracellular redox imbalance, nac was a natural choice, for acting on the reduced-oxidized glutathione pair, the central intracellular antioxidant system [12, 18, 24] . moreover, nac has shown the ability to restore the intracellular redox imbalance in vitro experiments [12, 25, 26] and has been safely prescribed in patients with acute liver failure [14] and acetaminophen poisoning [15] . regarding the posology, preparing individual doses (300 mg/kg, as recommended in these studies) would be time-consuming, and we had a small window for drug administration. hence, we chose a fixed dose (21 g), an average for the recommended dose [20] . although we did not measure patients' weight, we did not include very obese patients. therefore, we believe that all patients received approximately 300 mg/kg, a dose even higher than the ones that have been used successfully in other clinical conditions [27, 28] . the second hypothesis for the failure is related to the time of disease when nac was administered. we chose patients with severe covid-19 since we believed that in patients already in respiratory failure, the inflammation could not be controlled acting on redox signaling [29] . all included patients had pulmonary infiltrates in chest ct scan and needed oxygen supplementation, which suggests they already had substantial lung involvement. we can speculate that earlier nac prescription could be more effective at the start of the symptoms; however, we know that most of these patients would recover spontaneously [30] . it would not be feasible or ethical to treat these patients for 20 hours in a hospital, knowing that most of them would not need it. finally, we cannot exclude the fact that our initial hypothesis was wrong; that is, excess angii neither leads to intracellular redox imbalance nor influences the disease pathogenesis. it is documented that there is an increase in angii in severe covid-19 patients a c c e p t e d m a n u s c r i p t 10 [7] ; however, it can be just a collateral phenomenon, and it has nothing to do with the disease pathophysiology. against this hypothesis is the fact that published data [31, 32] , and our own series of cases (submitted to publication), show a better prognosis for patients in the use of angiotensin receptor blockers. these data suggest that angii may be involved, someway, in sars-cov-2 pathogenesis. the main limitation of our study is the small number of patients enrolled. this protocol has been initially proposed as a preparatory stage before a larger multicentric trial; however, given the unambiguous negative results, the larger study was aborted. nevertheless, several opinion articles continue to propose acting on angii [33] or redox signaling [34, 35] as a viable therapeutics for covid-19. that is the importance of our work. we have shown here that the administration of nac in high doses did not affect the evolution of severe covid-19. the question remains on how to act on these signaling pathways activated by angii without impairing patients' hemodynamics and renal function. we believe that interactions between angii and inflammation should be better studied in the near future and can probably furnish new ideas for treating a c c e p t e d m a n u s c r i p t comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the features of 20 133 uk patients in hospital with covid-19 using the isaric who clinical characterisation protocol: prospective observational cohort study baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region the trinity of covid-19: immunity, inflammation and intervention sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor ace2 the janus-faced protein -from cardiovascular protection to severe acute respiratory syndrome-coronavirus and covid-19 elevation of plasma angiotensin ii level is a potential pathogenesis for the critically ill covid-19 patients nasal gene expression of angiotensin-converting enzyme 2 in children and adults redox aspects of vascular response to injury angiotensin ii-induced production of mitochondrial reactive oxygen species: potential mechanisms and relevance for cardiovascular disease activation of apoptosis signalling pathways by reactive oxygen species angiotensin ii modulates cd40 expression in vascular smooth muscle cells an improved inhaled mucolytic to treat airway muco-obstructive diseases intravenous n-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure evaluation and treatment of acetaminophen toxicity thiol-based drugs in pulmonary medicine: much more than mucolytics statpearls. treasure island (fl): statpearls publishing implications of plasma thiol redox in disease covid-19: the renin-angiotensin system imbalance hypothesis simplification of the standard three-bag intravenous acetylcysteine regimen for paracetamol poisoning results in a lower incidence of adverse drug reactions a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 the renin-angiotensin system: an integrated view of lung disease and coagulopathy in covid-19 and therapeutic implications the renin-angiotensin system -a therapeutic target in covid-19 n-acetylcysteine--a safe antidote for cysteine/glutathione deficiency n-acetyl cysteine attenuates oxidative stress and glutathione-dependent redox imbalance caused by high glucose/high palmitic acid treatment in pancreatic rin-5f cells n-acetylcysteine protects epithelial cells against the oxidative imbalance due to clostridium difficile toxins improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure efficacy of oral n-acetylcysteine in the treatment of acetaminophen overdose. analysis of the national multicenter study (1976 to 1985) pathological inflammation in patients with covid-19: a key role for monocytes and macrophages estimating clinical severity of covid-19 from the transmission dynamics in wuhan, china estimation of renin-angiotensin-aldosterone-system (raas)-inhibitor effect on covid-19 outcome: a meta-analysis use of raas inhibitors and risk of clinical deterioration in covid-19: results from an italian cohort of 133 hypertensives angiotensin receptor blockers as tentative sars-cov-2 therapeutics an anti-oxidative therapy for ameliorating cardiac injuries of critically ill covid-19-infected patients covid-19: melatonin as a potential adjuvant treatment a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 18 a c c e p t e d m a n u s c r i p t figure 1 key: cord-333943-9d93na7s authors: jeong, han eol; lee, hyesung; shin, hyun joon; choe, young june; filion, kristian b; shin, ju-young title: association between nsaids use and adverse clinical outcomes among adults hospitalized with covid-19 in south korea: a nationwide study date: 2020-07-27 journal: clin infect dis doi: 10.1093/cid/ciaa1056 sha: doc_id: 333943 cord_uid: 9d93na7s background: non-steroidal anti-inflammatory drugs (nsaids) may exacerbate covid-19 and worsen associated outcomes by upregulating the enzyme that sars-cov-2 binds to enter cells. to our knowledge, no study has examined the association between nsaid use and the risk of covid-19-related outcomes. methods: we conducted a cohort study using south korea’s nationwide healthcare database, which contains data of all subjects who received a test for covid-19 (n=69,793) as of april 8, 2020. we identified adults hospitalized with covid-19, where cohort entry was the date of hospitalization. nsaids users were those prescribed nsaids in the 7 days before and including cohort entry and non-users were those not prescribed nsaids during this period. our primary outcome was a composite of in-hospital death, intensive care unit admission, mechanical ventilation use, and sepsis; our secondary outcomes were cardiovascular complications and acute renal failure. we conducted logistic regression analysis to estimate odds ratio (or) with 95% confidence intervals (ci) using inverse probability of treatment weighting to minimize confounding. results: of 1,824 adults hospitalized with covid-19 (mean age 49.0 years; female 59%), 354 were nsaids users and 1,470 were non-users. compared with non-use, nsaids use was associated with increased risks of the primary composite outcome (or 1.54 [95% ci 1.13-2.11]) but insignificantly associated with cardiovascular complications (1.54 [0.96-2.48]) or acute renal failure (1.45 [0.49-4.14]). conclusion: while awaiting the results of confirmatory studies, we suggest nsaids be used with caution among patients with covid-19 as the harms associated with their use may outweigh their benefits in this population. coronavirus disease 2019 (covid19) , which is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is a global pandemic. [1, 2] concerns exist that the use of nonsteroidal anti-inflammatory drugs (nsaids) may exacerbate covid-19 by upregulating angiotensin-converting enzyme 2 (ace2) expressions, [3, 4] the enzyme which sars-cov-2 binds to enter cells. in addition, nsaids inhibit cyclooxygenase (cox), [5] which could be involved in the pathogenesis of viral infections to result in tissue damage. [6, 7] these concerns were based on unconfirmed anecdotal reports of four young covid-19 patients who developed serious infectious complications following nsaids use. [8] the health minister of france subsequently recommended that paracetamol (acetaminophen) be used as first-line antipyretic agents over nsaids. in contrast, the us food and drug administration, [9] european medicine agency, [10] and australia's therapeutic goods administration [11] stated that there is insufficient evidence to draw conclusions regarding this safety concern and thus, current clinical practice should not be changed until further evidence becomes available. this position is supported by a recent systematic review of randomized trials and observational studies of respiratory viral infections, which concluded that there is currently no evidence to support that nsaids are harmful with respect to covid-19. [12] despite the widespread use of nsaids, to our knowledge, there is currently no published observational study that specifically assessed the association between nsaids use and clinical outcomes among covid-19 patients. this cohort study therefore aimed to examine the association between nsaids use, compared to non-use, and worsened clinical outcomes among adults hospitalized with a c c e p t e d m a n u s c r i p t page | 5 covid-19 using south korea's nationwide healthcare database containing all covid-19 patients. we used the health insurance review and assessment service (hira) database of south korea, provided as part of the #opendata4covid19 project, a global research collaboration on covid-19 jointly conducted by ministry of health and welfare of korea and hira. [13] briefly, the south korean government released the world's first de-identified covid-19 nationwide patient data on march 27, 2020. owing to south korea's national health insurance system, which is the universal single-payer healthcare provider covering the entire korean population of 50 million, and its fee-for-service reimbursement system, the database includes information from both inpatient and outpatient settings. the hira covid-19 database contains data of all subjects who received a test for covid-19 as of april 8, 2020, linked to their administrative healthcare data from the previous 3 years (january 1, 2017 to april 8, 2020). the hira covid-19 database includes anonymized patient identifiers, sociodemographic characteristics, healthcare utilization history, diagnoses (international classification of diseases, 10 th revision; icd-10), and drug prescription information (anatomical therapeutic chemical classification codes); use of over-the-counter drugs are not collected in this database (supplementary material 1). [14] this study was approved by the institutional review board of sungkyunkwan university (skku 2020-03-012), which waived the requirement of obtaining informed consent. a c c e p t e d m a n u s c r i p t page | 6 of 69,793 individuals who received a diagnostic test for covid-19 between january 1, 2020 to april 8, 2020, 5,707 tested positive for covid-19 ( figure 1) we defined exposure using inpatient and outpatient prescription records of nsaids from the hira database, including both oral and intravenous formulations (supplementary material 2). we ascertained exposure to nsaids according to an intention-to-treat approach, in which exposure was defined in the index period of 7 days before and including cohort entry among hospitalized covid-19 patients. patients prescribed nsaids during this period were classified as nsaids users whereas those not prescribed nsaids during this period a c c e p t e d m a n u s c r i p t page | 7 were classified as non-users. to minimize any time-related biases such as immortal time, [17] follow-up was initiated from the date of cohort entry for both nsaids users and non-users. our primary outcome was a composite endpoint of in-hospital death, intensive care unit (icu) admission, mechanical ventilation use, and sepsis. our secondary outcomes were a composite endpoint of cardiovascular complications (myocardial infarction, stroke, heart failure), and acute renal failure. we defined outcomes using in-hospital icd-10 diagnostic codes and procedures using the national procedure coding system (supplementary material 2). study outcomes were measured between the cohort entry date and the earliest of the date of hospital discharge or end of study period (april 8, 2020). we assessed sociodemographic and clinical factors considered to be associated with nsaids use and risk of the outcomes of interest. for sociodemographic factors, we assessed age, sex, and health insurance type at cohort entry; age was grouped into 10-year bands. clinical variables included comorbidities and use of co-medications assessed in the year before cohort entry (supplementary material 2). we used the expanded benefit coverage codes in addition to diagnosis codes to define malignancy to minimize false positives. baseline characteristics were summarized for nsaids users and non-users using counts (proportions) or mean (standard deviation) for categorical or continuous variables, respectively. we calculated the absolute standardized difference (asd) to determine important imbalances between exposure groups, with asd ≥0.1 considered important. a c c e p t e d m a n u s c r i p t page | 8 we estimated the cumulative incidence of the primary and secondary outcomes among nsaids users versus non-users. we used three outcome models using logistic regression to estimate odds ratio (or) with 95% confidence intervals (cis) of the association of interest. the first model was unadjusted. the second model included all covariates described above. the third model, considered our primary analysis, was weighted by propensity scores (ps) using the inverse probability of treatment weight (iptw) approach. [18] the ps, or probability of receiving nsaids, was estimated using multivariable logistic regression analysis, with all confounders mentioned above included as independent variables. the c-statistic was used to determine model discrimination, with a value between 0.6 and 0.8 considered adequate to predict treatment status based on covariates included. [19] the iptw approach involves weighting the inverse probability of receiving nsaids (1/ps for nsaids, and 1/(1−ps) for non-user groups). we conducted sex-and age-stratified analyses, with age classified into three groups (<45, 45-65, ≥65 years), for the risk of the primary outcome associated with nsaids use. in addition, we stratified by route of administration (oral versus intravenous) and by history of hypertension, hyperlipidemia, or diabetes mellitus. the ps were re-calculated in all subgroup analyses using multivariable logistic regression models. a c c e p t e d m a n u s c r i p t page | 9 as there is currently no data available on how fast nsaids increase ace2 tissue expressions, we varied the exposure ascertainment window to 14 days and 30 days before and including cohort entry. patients prescribed nsaids during these periods were classified as nsaids users whereas those not prescribed nsaids were classified as non-users. follow-up was initiated from cohort entry. to examine the potential effects of confounding by indication, we compared nsaids to paracetamol as these are used for similar indications (supplementary material 2). we classified patients based on their exposure to nsaids or paracetamol in the 7 days before and including cohort entry, excluding those not exposed to one of the two drugs of interest and those who received both drugs during this exposure window. follow-up was initiated from cohort entry for both exposure groups. as outcome misclassification of sepsis from inaccuracy of coding or reverse causality between nsaids use and sepsis is possible, we repeated our main analysis by using a redefined primary outcome that was a composite endpoint of in-hospital death, icu admission, and mechanical ventilation use. a c c e p t e d m a n u s c r i p t page | 10 first, to improve comparability between exposure groups, we excluded the most extreme 1% of ps values (iptw with trimming). second, we included the estimated ps, in addition to other covariates, into our multivariable logistic regression model. third, we stratified on the ps in deciles. last, we applied standardized mortality ratio weights (smrw) (1 for nsaids, and ps/(1−ps) for non-user groups). [18] all statistical analyses were performed using the sas enterprise guide software (version 6.1). nsaids users (19%) and 1,470 non-users (81%). nsaids users were older than non-users there was no difference between the association between nsaid use and the risk of our primary composite endpoint by formulation of nsaids, sex, and histories of hypertension and hyperlipidemia ( figure 2 ). however, we found effect modification in age groups (p-for-interaction <0.0001) and history of diabetes mellitus (p-for-interaction 0.0180). findings from sensitivity analyses remained largely consistent, where all effect estimates showed positive associations between the primary outcome and nsaids users, as compared with non-users, when varying the exposure window, applying other methods involving ps, or redefining the primary outcome. when comparing to paracetamol, our sample size was greatly reduced, and there were no events that occurred in the nsaid group (cumulative incidence for nsaids users: 0.0%; paracetamol users 4.1%). results of sensitivity analyses for the secondary outcomes were also generally consistent ( figure 3 ). to the best of our knowledge, this is the first population-based cohort study to have investigated the association between nsaid use and adverse outcomes among patients with likewise, despite use of nsaids known to result in nephrotoxicity [27, 28] , our findings suggest no additional risk of acute renal failure when covid-19 patients were exposed to nsaids. the underlying pathogenic link between nsaids and covid-19 has yet to be elucidated. however, one animal study found increased ace2 expressions with nsaids (ibuprofen) [29] in various organs such as the lung, heart, and kidneys. [4, 30, 31] thus, ace2 upregulation induced by nsaids could theoretically heighten the infectivity of sars-cov-2 to worsen clinical outcomes, resulting in multiple organ failure in severe cases. other hypothetical mechanisms have also been suggested. nsaids could aggravate infections by upregulating cox-2 in activated b lymphocytes to interfere with antibody productions, [32] or by selectively inhibiting interferon-γ productions that are vital for immunity against foreign pathogens. [33] however, with inconsistent findings from animal studies and the precise biological mechanisms yet to be understood, it remains unclear as to whether these findings are readily transferable to humans. we defined exposure using an approach analogous to an intention-to-treat, with exposure assessed in the 7 days before and including the day of cohort entry (hospital admission). we used this approach to avoid time-related biases that could be introduced by assessing in-hospital nsaid use as the date of prescription was not available for ~50% of inhospital prescriptions. the length of hospital stay not only influences the probability of being exposed to nsaids while hospitalized but is also associated with worse prognosis. however, with exposure defined using pre-hospital medication use, our exposure assessment was independent of in-hospital outcomes and the duration of hospital stay. although predicting the direction resulting from this bias is difficult as occurrence of confounders during hospitalization are accounted for in this study, the use of this exposure definition is likely to a c c e p t e d m a n u s c r i p t page | 14 bias our findings towards the null. this is because, by not accounting for nsaid use during hospitalization, the observed increased risk is suggested to be a conservative estimate. our study has several strengths. to our knowledge, this is the first population-based study conducted using all hospitalized patients with covid-19 to assess the association between nsaid use and covid-19 related outcomes. moreover, we used a nationwide healthcare database of south korea that includes information on healthcare utilization of all covid-19 cases as of april 8, 2020. therefore, our findings provide real-world evidence that is highly generalizable to everyday clinical practice. with its large source population, our data source was sufficiently large to assess this clinically important issue. in addition, our findings were consistent in sensitivity analyses that extended the index period. our study also has some limitations. first, outcome misclassification is possible. however, misclassification of in-hospital death is likely to be very small, and the validity of procedure codes to define icu admission or mechanical ventilation use are also expected to be high as these codes are used for reimbursement processes by the health insurance authority. also, the positive predictive value of diagnosis codes between claims data and electronic medical records was previously reported to be 82%,[34] and we believe its validity to be greater for sepsis, myocardial infarction, stroke, heart failure, and acute renal failure as we restricted to hospitalized patients receiving close monitoring. second, our findings may have theoretically underestimated the association between nsaids users and clinical outcome due to depletion of susceptible patients,[35] as we included prevalent users of nsaids. however, our study period included the start of the covid-19 pandemic in south korea, making it unlikely that patients who were susceptible to adverse covid-19 related outcomes were excluded prior to entering our cohort. third, our results may be affected by confounding by indication given our use of an unexposed reference group. despite attempting to address this by comparing nsaids users to paracetamol users, we were unable a c c e p t e d m a n u s c r i p t page | 15 to provide meaningful results as there were no events among nsaids users upon excluding those prescribed both nsaids and paracetamol during the exposure window. this therefore suggests that the 23 exposed events from our main analysis were exposed to both drugs during the exposure window, implying that these patients were severe cases who were world health organization. coronavirus disease (covid-19) situation dashboard rapid asymptomatic transmission of covid-19 during the incubation period demonstrating strong infectivity in a cluster of youngsters aged 16-23 years outside wuhan and characteristics of young patients with covid-19: a prospective contact-tracing study are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor roles of cyclooxygenase (cox)-1 and cox-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by cox-2 hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza h5n1 infection a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses update -coronavirus: french health minister and who issue warning over taking anti-inflammatories. the local france fda advises patients on use of non-steroidal antiinflammatory drugs (nsaids) for covid-19. us food and drug administration ema gives advice on the use of non-steroidal antiinflammatories for covid-19 therapeutic goods administration. no evidence to support claims ibuprofen worsens covid-19 symptoms. therapeutic goods administration scientific brief: the use of non-steroidal antiinflammatory drugs (nsaids) in patients with covid-19. world health organization ministry of health and welfare. #opendata4covid19. available at towards actualizing the value potential of korea health insurance review and assessment (hira) data as a resource for health research: strengths, limitations, applications, and strategies for optimal use of hira data world health organization. laboratory testing for coronavirus disease in suspected human cases: interim guidance. world health organization,. 2020. 16. ministry of health and welfare rok. about covid-19: patient treatment & management immortal time bias in pharmaco-epidemiology alternative approaches for confounding adjustment in observational studies using weighting based on the propensity score: a primer for practitioners reducing bias in a propensity score matched-pair sample using greedy matching techniques agence nationale de sécurité du médicament et des produits de santé (ansm) anti-inflammatoires non stéroïdiens (ains) et complications infectieuses graves -point d'information. l'agence nationale de sécurité du médicament et des produits de santé (ansm) risks related to the use of non-steroidal anti-inflammatory drugs in community-acquired pneumonia in adult and pediatric patients non-steroidal anti-inflammatory drugs and covid-19 acute respiratory infection and use of nonsteroidal anti-inflammatory drugs on risk of acute myocardial infarction: a nationwide case-crossover study risk of stroke associated with use of nonsteroidal anti-inflammatory drugs during acute respiratory infection episode cardiovascular safety of non-aspirin non-steroidal anti-inflammatory drugs: review and position paper by the working group for cardiovascular pharmacotherapy of the european society of cardiology vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials the invisible threat of nonsteroidal anti-inflammatory drugs for kidneys non-steroidal anti-inflammatory drug induced acute kidney injury in the community dwelling general population and people with chronic kidney disease: systematic review and meta-analysis ibuprofen attenuates cardiac fibrosis in streptozotocin-induced diabetic rats angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury ibuprofen and other widely used non steroidal anti-inflammatory drugs inhibit antibody production in human cells , and co-medications (angiotensin converting enzyme inhibitors, angiotensinreceptor ii blockers, β-blockers, calcium channel blockers, diuretics, nitrates, anticoagulants, inhaled therapy for respiratory for respiratory disease, lipid lowering drugs, opioids, oral glucocorticoids, platelet inhibitors, dipeptidyl peptidase-4 inhibitors, metformin, insulin, sulfonylurea, sodium-glucose cotransporter-2 inhibitors) ‡ ipt weighted multivariable logistic regression model (main model), where the propensity score used was estimated using a multiple logistic regression model that included the following independent variables: age, sex, health insurance type, comorbidities (hypertension, hyperlipidemia, diabetes mellitus, asthma, chronic obstructive pulmonary disease, malignancy, atherosclerosis, chronic renal failure, chronic liver disease, rheumatoid arthritis, osteoarthritis, gastrointestinal conditions, peripheral arterial disease, atrial fibrillation, other arrythmia, ischemic heart disease, pneumonia, psychiatric disorders, depression, thyroid disease), and co-medications (angiotensin converting enzyme inhibitors, angiotensin-receptor ii blockers, β-blockers, calcium channel blockers, diuretics, nitrates, anticoagulants, inhaled therapy for respiratory for respiratory disease, lipid lowering drugs, opioids, oral glucocorticoids, platelet inhibitors, dipeptidyl peptidase-4 inhibitors, metformin, insulin, sulfonylurea, sodium-glucose co-transporter-2 inhibitors) (c-statistics: 0.668 for nsaids users versus non-users) ⁋ cardiovascular complications include myocardial infarction, heart failure, and stroke ⁑ inestimable due to small number of events relative to the large number of confounders key: cord-354877-n5du3bqt authors: vasoo, shawn; stevens, jane; singh, kamaljit title: rapid antigen tests for diagnosis of pandemic (swine) influenza a/h1n1 date: 2009-10-01 journal: clin infect dis doi: 10.1086/644743 sha: doc_id: 354877 cord_uid: n5du3bqt we found that the sensitivities of 3 rapid influenza antigen tests for pandemic influenza a/h1n1 virus were low to moderate: bd directigen ez flu a+b test (becton dickinson), 46.7%; binaxnow influenza a&b (inverness medical), 38.3%; and quickvue influenza a+b test (quidel), 53.3%. a patient with influenza-like illness who has a negative rapid antigen test result should undergo further testing using reverse-transcription polymerase chain reaction. influenza viruses and can cause similar symptoms, diagnosing influenza on the basis of clinical presentation alone is difficult, with reported sensitivity ranging from 38% [4] to 79% [5] . rapid influenza antigen tests (point-of-care tests) might prove useful, because they have a fast turnaround time (10-15 min) and require minimal training to perform. however, there are few data on the diagnostic accuracy of rapid influenza antigen tests for pandemic influenza a/h1n1 virus [6] . soon after the onset of the pandemic influenza a/h1n1 outbreak, we received reports of poor performance of rapid influenza antigen tests. we report our findings of the clinical accuracy of 3 commonly used rapid influenza tests for diagnosis of pandemic a/h1n1 influenza. methods. during the period from 1 may 2009 through 2 june 2009, a convenience sample of 84 positive, nonduplicate nasopharyngeal specimens were tested using 3 different rapid antigen test kits: bd directigen ez flu a+b test (becton dickinson), binaxnow influenza a&b (inverness medical), and quickvue influenza a+b test (quidel). testing was performed in accordance with the manufacturer's instructions, and each test result was interpreted by at least 2 of the study authors. nasopharyngeal specimens were collected from patients presenting with influenza-like illness using a sterile polyester nasopharyngeal swab and were transported to the microbiology laboratory in m4rt viral transport medium (remel). only specimens that were positive for respiratory viruses using the luminex xtag rvp (luminex) were included. the luminex xtag rvp is a multiplex rt-pcr assay that allows for the simultaneous detection of 17 respiratory virus types/subtypes, including metapneumovirus, coronaviruses, influenza a virus subtypes h1 and h3, influenza b virus, parainfluenza virus types 1-4, respiratory syncytial virus, adenovirus, and rhinovirus. all specimens were refrigerated, and rapid antigen tests were performed р48 h after specimen receipt. specimens included 60 nasopharyngeal samples positive for pandemic influenza a/h1n1 virus, with results confirmed using the cdc's rt-pcr assay for pandemic influenza a/h1n1 virus. control specimens included 24 nasopharyngeal specimens positive for other respiratory viruses, as follows: adenovirus ( ), coro-n p 1 navirus ( ), concurrent coronavirus and rhinovirus ( n p 3 n p ), metapneumovirus ( ), rhinovirus ( ), concurrent 1 n p 4 n p 4 rhinovirus and parainfluenza virus ( ), parainfluenza virus n p 1 ( ), and respiratory syncytial virus ( ). n p 7 n p 3 we calculated the sensitivity and specificity for each rapid antigen test. we determined the prevalence of pandemic a/ h1n1 influenza over the study period; this was used to calculate the positive predictive value (ppv) and negative predictive value (npv). patient charts were reviewed, if available, for potential factors that may have been associated with rapid antigen test result; these included patient age, duration of symptoms before presentation, inpatient or emergency department versus outpatient status, and the median number of rt-pcr fluorescence intensity (mfi) units. data analysis was performed using spss, version 16.0 (spss). the mann-whitney u test was used for comparison of continuous variables, and x 2 and mcnemar tests were used for categorical independent and paired variables, respectively. p values !.05 were considered to be statistically significant. this study was approved by the institutional review board of rush university medical center (chicago, il). results. the majority of patients with pandemic a/h1n1 influenza were children and young adults; the median age was 12.5 years (range, 7 months to 58 years). a total of 34 patients (57%) were assessed in the emergency department, and 26 patients (43%) were seen in physicians' offices. twenty-two (65%) of 34 patients evaluated in the emergency department were subsequently hospitalized. information for the duration of illness before presentation was available for 38 of 60 patients. the majority of patients (76%) presented р3 days after developing symptoms influenza-like illness (mean interval, 2.8 days; range, 1-9 days). control patients had a median age of 7 years (range, 4 months to 66 years); 14 control patients (58%) presented to the emergency department, and 10 (42%) were outpatients. the analytic performance of the 3 rapid antigen tests, compared with that of the luminex xtag rvp are presented in table 1 . the overall respective sensitivity and specificity of the bd directigen ez flu a+b test were 46.7% (28 of 60 specimens) and 100%; for the binaxnow influenza a&b test, 38.3% (23 of 60 specimens) and 100%; and for the quickvue influenza a+b test, 53.3% (32 of 60 specimens) and 100%. the quick-vue influenza a+b test was significantly more sensitive than the binaxnow influenza a&b test ( ), but there was no sig-p ! .01 nificant difference between the other rapid antigen tests. during the study period, the overall prevalence of pandemic a/h1n1 virus was 17.9% (95% confidence interval, 8.24%-27.6%) among all specimens submitted for respiratory virus rt-pcr testing. on the basis of this prevalence, the calculated ppvs were 100% for all 3 tests, and the calculated npvs were 89.6%, 88.2%, and 90.8% for bd directigen ez flu a+b test, binaxnow influenza a&b, and quickvue influenza a+b test, respectively. patient age and duration of symptoms prior to collection of the respiratory specimen did not correlate significantly with the results of rapid antigen tests ( ). a higher mfi unit with rt-pcr (which p 1 .05 correlates with higher viral load) was significantly associated with positive results for all rapid antigen tests ( ) (table 2) . p ! .01 discussion. rapid antigen tests are commonly used in many hospital laboratories, emergency departments, and doctors' offices. most influenza rapid antigen tests are lateral flow chromatographic immunoassays in which patient samples are applied to a chromatographic strip that contains monoclonal antibodies against influenza a and b viruses. on-site diagnosis of influenza by point-of-care tests has been shown to limit antibiotic prescriptions, use of blood cultures, and chest radiography and ultimately to reduce patient costs [7] . however, rapid antigen tests have demonstrated a wide range of sensitivities for detection of seasonal influenza viruses, ranging from 69%-96% for the bd directigen ez flu a+b test [8, 9] , 64.9%-73% for the binaxnow influenza a&b test [8, 10] , and 19.7%-85% for the quickvue influenza a+b test [11, 12] . we found that the 3 rapid antigen tests had low to modest sensitivities for diagnosis of pandemic influenza a/h1n1, although all tests demonstrated excellent specificity, with no cross-reactivity against other cocirculating respiratory viruses. possible reasons for the low sensitivities in our study include the fact that the sample collection technique was not standardized and that specimens were refrigerated and only tested after completion of rt-pcr, which may have led to antigen degradation. however, we think that this is unlikely, because our findings are consistent with those of a similar study by ginocchio et al [6] in which rapid antigen tests had a sensitivity of only 17.8% for pandemic influenza a/h1n1 virus, compared with the luminex xtag rvp. an alternative explanation may be related to differences in test sensitivity for detection of pandemic influenza a/h1n1 virus, compared with seasonal influenza viruses. in a study that used titered pandemic influenza a/h1n1 virus grown in cell cultures, rapid antigen tests required 1-1.5-log higher viral loads than did human seasonal virus for a positive result [13] . other limitations of our study include the fact that we employed a known convenience sample of positive specimens. because of the widespread interest regarding the clinical performance of rapid antigen tests for detection of pandemic influenza a/h1n1 virus, we used a convenience sample of positive specimens to allow for a quick yet relatively accurate assessment of these tests. in addition, all specimens were decoded, and both investigators performing the tests were blinded to the rt-pcr results. finally, because the design of our study did not permit an accurate determination of the npv and ppv, we calculated these values on the basis of the prevalence of novel influenza h1n1 during the study period. these values would clearly vary according to the prevalence of disease, and the ppv would be highest during the peak of an outbreak, with increased numbers of false-positive results when disease prevalence is low (table 1). despite modest sensitivities, rapid influenza antigen testing could prove to be useful because of the tests' speed, portability, and ease of performance. clinicians should be aware that patients presenting with an influenza-like illness and a negative rapid antigen test result should undergo further laboratory testing. however, a positive rapid antigen test result would allow presumptive diagnosis of novel influenza h1n1 virus infection and should lead to timely institution of infection control measures, treatment, and prophylaxis. finally, as we approach the 2009-2010 winter season, it is desirable for manufacturers to develop sensitive rapid antigen kits that can also differentiate seasonal influenza a viruses from pandemic influenza a/h1n1 virus in view of their different antiviral susceptibilities. novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans world health organization. influenza a(h1n1)-update 46 chicago department of public health. health alert, swine influenza update 19 accuracy of clinical diagnosis of influenza in outpatient children clinical signs and symptoms predicting influenza infection evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized prospective controlled trial performance of six influenza rapid tests in detecting human influenza in clinical specimens evaluation of the directigen flua+b test for rapid diagnosis of influenza virus type a and b infections comparison of the binax now flu a enzyme immunochromatographic assay and r-mix shell vial culture for the 2003-2004 influenza season evaluation of the quidel quickvue test for detection of influenza a and b viruses in the pediatric emergency medicine setting by use of three specimen collection methods low sensitivity of rapid diagnostic test for influenza performance of influenza rapid point-of-care tests in the detection of swine lineage a (h1n1) influenza viruses potential conflicts of interest. k.s. is on the speaker panel for wyeth. s.v. and j.s.: no conflicts. key: cord-349070-bqv03u2e authors: jiang, shih sheng; chen, tsan-chi; yang, jyh-yuan; hsiung, chao a.; su, ih-jen; liu, ying-lan; chen, po-cheng; juang, jyh-lyh title: sensitive and quantitative detection of severe acute respiratory syndrome coronavirus infection by real-time nested polymerase chain reaction date: 2004-01-15 journal: clin infect dis doi: 10.1086/380841 sha: doc_id: 349070 cord_uid: bqv03u2e a quantitative, real-time, nested polymerase chain reaction (pcr) method, combining the high sensitivity of nested pcr with time-saving real-time instrumentation, was developed for large-scale screening for severe acute coronavirus (sars) coronavirus. forty-six clinical specimens were analyzed by this method, and results were compared with those obtained by conventional, single-round, real-time reverse-transcriptase pcr (rt-pcr) performed in parallel. of the 17 positive results, 2 identified by our method were not detected by single-round, real-time rt-pcr, which suggests that real-time nested pcr has the potential for increased sensitivity, leading to earlier detection of sars. the outbreak of severe acute respiratory syndrome (sars) in southeast asia and on other continents reached serious global epidemic proportions in 130 countries [1] [2] [3] . in the absence of a vaccine or effective therapeutic drugs, the key to preventing and controlling future epidemics is to block transmission of infection through a strict quarantine policy. therefore, rapid, sensitive, and specific diagnostic methods are essential for the assessment of patients suspected of being infected and for prevention of spread to the larger community. real-time rt-pcr detection is currently favored for the de-tection of sars coronavirus (sars-cov) because of its advantages as a rapid and quantitative assay [3, 4] . unfortunately, results obtained with this method vary on the basis of which detection method is used as part of a real-time, high-throughput clinical screening protocol. in particular, because the concentration of extracted viral rna from samples obtained during the early phase of infection is exceptionally low, the aforementioned problems would usually worsen. in most of the cases, we and others have found that the single-step real time rt-pcr methods (as suggested by the world health organization [who] ; available at http://www.who.int/csr/sars/diagnostic tests/en/) could specifically detect sars-cov but were unable to proficiently detect !10 copies of virus per test, suggesting that the conventional rt-pcr assay may actually yield falsenegative results. failure of early detection of sars has meant that "super-spreaders" of sars have not been isolated and quarantined, thus highlighting the importance of a more sensitive early diagnostic method. to solve this problem of diagnostic methods, several nested pcr methods have been reported [5] [6] [7] [8] [9] to enhance both the specificity and sensitivity of the assay. however, those nested rt-pcr methods are either unfavorable for quantitative assay or require lengthy and labor-intensive procedures, making these protocols impractical for large-scale screening. in the present report, we describe an innovative 2-round, real-time pcr method for detection of sars-cov. we utilized the on-line detection method of the lightcycler (roche) instrument to optimize the first-round pcr conditions for linear amplification, and then used real-time nested pcr for sensitive and quantitative virus detection. we show that this method is rapid and highly sensitive and could be scaled up for routine screening for early diagnosis of sars-cov infection in a laboratory setting. methods. forty-six clinical throat swab specimens (1 each from 46 patients with suspected or reported sars), which had been obtained during the period of april through may 2003 by contracted hospitals of the taiwan center for disease control, were randomly selected for this study. according to the who's definition (http://www.who.int/csr/sars/casedefinition/ en/), a patient with a suspected case of sars has high fever (temperature, 138њc), the symptoms of cough or breathing difficulty, and a history of exposure to or contact with a person with suspected or probable sars. in our study, reported case patients are people who had symptoms similar to those of patients with suspected cases of sars, except that a history of contact was not clear. viral rna was extracted from 200 ml figure 1 . a, results of the real-time nested pcr with indicated starting rna copy number in each assay. छ, !1 rna copy; ⅺ, 10-fold dilution of the control rna sample used for the single-copy rna test; nc, negative control, in which input rna was replaced by deionized water. b, linear amplification by real-time nested pcr. the starting viral rna copy number was plotted against the threshold cycle number of each dilution and fitted with a linear regression model. of viral transport medium using the qiaamp viral rna mini kit (quiagen) and eluted in 50 ml of rnase-free water, which was frozen in aliquots at ϫ70њc until use. for the control experiment, viral rna was extracted from the supernatant of viral culture medium. the concentration of this control viral rna was calibrated using the realart hpa-coronavirus rt-pcr reagents kit (artus), in accordance with the manufacturer's instructions. the titer of calibrated viral rna was then adjusted to copies/ml as high-titer rna stock. 5 5.2 ϫ 10 two pairs of pcr primers made available by the who (bniouts/bnioutas and bniins/bniinas; see http://www .who.int/csr/sars/primers/en/) were used for the first round of rt-pcr and the subsequent nested pcr, respectively. the firstround pcr amplification was performed using lightcycler (roche) with 2 ml of viral rna diluted to a volume of 20 ml with pcr mix (lightcycler rna master sybr green i kit; roche) containing 2 mmol/l mn(oac)l 2 , and 0.5 mmol/l of bniouts/bnioutas primers at the following settings: 61њc for 20 min and 95њc for 30 s, followed by 25 cycles of 95њc at 1 s, 55њc for 10 s, 72њc for 8 s. the nested pcr was then conducted using 1 ml of the first-round amplicon as template diluted to a volume of 20 ml with pcr mix (lightcycler faststart dna master sybr green i kit; roche) containing 2 mmol/l mgcl 2 and 0.5 mmol/l bniins/bniinas primers, with the following settings: 95њc for 10 min, followed by 25-35 cycles of 95њc for 10 s, 56њc for 5 s, and 72њc for 5 s. after amplification, melting curve analysis at temperatures of 65њc-95њc, with a temperature transition rate of 0.1њc/s, was performed to mark out the presence of pcr-amplified product. the sizes of the product were analyzed by gel electrophoresis analysis in 2% agarose. furthermore, the sequence of nested pcr amplicon was subjected to sequence analysis with an abi 3700 auto-sequencer (abi) to confirm whether it was a sars-cov sequence. to reduce the risk of random or carry-over contamination of nested pcr [10] , sample preparation, reagent preparation, and pcr amplification were performed in different buildings or rooms with separated air-conditioning using different sets of the pipette system. all samples and reagents were transferred via filter tips to protect the pcr from aerosol contamination. for real-time nested pcr data analysis, the threshold cycle was calculated with a "fit points" algorithm using 2-points calculation, and the crossing point was then determined automatically. for quantification of the sars-cov rna load in the samples, a standard curve was generated using various dilutions of calibrated control viral rna, as mentioned previously. to confirm positive pcr results, occurrence of seroconversion was determined by elisa using available serum samples obtained during the convalescent phase of infection (128 days after illness) [11] . the sars elisa antigen was kindly provided by the us centers for disease control and prevention (at-lanta). the optimal dilution (1:1000) for the use of this antigen was determined by checkerboard titration against human serum samples obtained during the convalescent phase. the control antigen, prepared from uninfected vero e6 cells, was used to control for the specific reactivity of tested serum. the conjugates used were goat anti-human igg, iga, and igm conjugated to fluorescein isothiocyanate and horseradish peroxidase for the indirect fluorescence antibody test and elisa, respectively. approval for this study was obtained from the center for disease control, department of health, taiwan. results. using the single-step rt-pcr protocol, our firstround pcr yielded a minor amplification signal, although the nonspecific fluorescence signal background frequently occurred after 20 cycles of amplification (data not shown). in contrast, the second-round amplification by nested real-time pcr proficiently generated a signal of sars-cov dna without apparent background, compared with no detectable signal for the negative control samples ( figure 1a ). in addition, by melting curve analysis, we could clearly define an average melting temperature of 84.2њc for the nested amplicon of sars-cov, in contrast to no indication of melting temperature for the negative control (data not shown). the size (110 bps) of every nested pcr amplicon was confirmed by the agarose gel analysis, and the sequence was further validated by direct sequencing, to verify the specific detection of sars-cov. to determine the detection limit for this developed method, samples of various dilutions of control sars-cov rna were subjected to the assay. after 25 cycles of first-round amplification and 25 cycles of nested pcr amplification, our assay could detect a theoretical single copy of extracted viral rna (figure 1a), suggesting its superior sensitivity for detection of sars-cov. nonetheless, for the quantitative assay, the cycle number of the first-round rt-pcr should be controlled to !30 cycles to prevent nonlinear amplification, as reflected by saturation of the fluorescence signal. we found that, for most cases, 25 cycles was the optimized condition for the first-round pcr to produce the most adequate amplicon level as the template for subsequent nested pcr. under such conditions, the linear relationship between the copy number of input rna and the threshold cycle number of nested pcr was accurately held within the range of 10 3 -10 0 in the semilog plot ( figure 1b) . in general, our developed assay is highly sensitive and specific for detection of trace amounts of virus. to further validate this method for real clinical diagnoses, we then examined rna samples from 46 patients with sus-pected or reported sars using this method and another commercial single-round rt-pcr kit (artus) in parallel for comparison. we found that the single-round pcr detected 15 of 46 positive cases, comparison with 17 of 46 cases for our 2round, real-time pcr. of the 17 positive cases, 15 were those identified by single-round pcr, and only 2 were missed by the single-round pcr (table 1) . for those 2 possible false-negative results for the single-round pcr, we used direct sequencing analysis to confirm the positive results detected by real-time nested pcr. furthermore, we carefully avoided the possibility of carry-over contamination by following the protocols described in methods and by conducting 3 independent tests for all assays. most importantly, we also confirmed seroconversion for the convalescent-phase serum samples for these 2 cases (table 1), further supporting the result obtained by the nested real-time pcr method. it is interesting to note that virus titers for the aforementioned 2 possible false-negative cases were actually quantified and shown to contain !10 copies of viral genome in the test. of the 15 cases that yielded positive results by both assays, only 1 contained !10 copies of viral genome in the assay, indicating that the single-round pcr method may be insensitive for the low-titer virus assay. discussion. our assay combines the 2-round pcr amplification method with the real-time detection approach to identify sars-cov, thus providing an alternative, sensitive means of detection of sars. these findings reveal several advantages over the conventional real-time rt-pcr and/or nested pcr. first, the assay is simple and rapid. the conventional nested pcr requires arduous processing steps, demanding rt-pcr procedures, performance of nested pcr on regular thermocycler, and use of agarose gel analysis, tests that normally will take at least 4-5 h to complete. this dual real-time pcr method can be easily completed within 2 h using lightcycler, which includes a 45-min, 1-step rt-pcr followed by 40-min real-time nested pcr, making it an ideal routine protocol for high-throughput screening of sars-cov. second, the assay had a detection limit of !10 copies of sars-cov, to reduce the rate of false-negative results for trace virus samples. in this report, we show that the developed assay is sensitive enough to detect trace amounts of virus in the sample, suggesting that this test is an excellent alternative to the existing sars-cov assay methods, which frequently generate false-negative results for samples obtained during the early phase of infection. last, this assay allowed for quantification of sars-cov rna in a range over a 3-log span. in theory, the 2-round amplification protocol can be optimized through the on-line detection system for linear amplification of viral rna without saturation for quantitative analysis. thus, quantitative analysis for detection of a wider range of values (14-log span) should be achievable. to our knowledge, this is the first report of a quantitative nested pcr assay for virus detection. through analysis of 46 clinical cases using our developed assay in parallel with a commercial, single-round rt-pcr kit, we have demonstrated that our new method has efficacy at least equal to that of the commercial kit while possessing the potential for increased sensitivity for early detection of sars-cov infection. in practice, this innovative assay method can be easily adapted to detect the rna or dna of other pathogens. a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) single-tube two-round polymerase chain reaction using the lightcycler instrument detection of cryptococcus neoformans dna in tissue samples by nested and real-time pcr assays two-round rapidcycle rt-pcr in single closed capillaries increases the sensitivity of hcv rna detection and avoids amplicon carry-over diagnosis of bartonella endocarditis by a real-time nested pcr assay using serum detection of influenza virus from throat and pharyngeal swabs with a nested duplex light cycler rt-pcr nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented dna clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study we are grateful to dr. i-shou chang for the critical discussions of quantitative pcr, and dr. clifford mcdonald for critical reading of this manuscript. we also thank hsiang-chi li and sheng-fan wang for generous technical assistance. key: cord-350338-lcsa06gm authors: wang, kun; zuo, peiyuan; liu, yuwei; zhang, meng; zhao, xiaofang; xie, songpu; zhang, hao; chen, xinglin; liu, chengyun title: clinical and laboratory predictors of in-hospital mortality in patients with covid-19: a cohort study in wuhan, china date: 2020-05-03 journal: clin infect dis doi: 10.1093/cid/ciaa538 sha: doc_id: 350338 cord_uid: lcsa06gm background: this study aimed to develop mortality-prediction models for patients with coronavirus disease 2019 (covid-19). methods: the training cohort were consecutive patients with covid-19 in the first people’s hospital of jiangxia district in wuhan from january 7, 2020 to february 11, 2020. we selected baseline clinical and laboratory data through the stepwise akaike information criterion and ensemble xgboost model to build mortality-prediction models. we then validated these models by randomly collecting covid-19 patients in the infection department of union hospital in wuhan from january 1, 2020, to february 20, 2020. results: 296 patients with covid-19 were enrolled in the training cohort, 19 of whom died during hospitalization and 277 were discharged from the hospital. the clinical model developed with age, history of hypertension and coronary heart disease showed auc of 0.88 (95% ci, 0.80-0.95); threshold, -2.6551; sensitivity, 92.31%; specificity, 77.44% and negative predictive value (npv), 99.34%. the laboratory model developed with age, high-sensitivity c-reactive protein (hscrp), peripheral capillary oxygen saturation (spo2), neutrophil and lymphocyte count, d-dimer, aspartate aminotransferase (ast) and glomerular filtration rate (gfr) had a significantly stronger discriminatory power than the clinical model (p=0.0157), with auc of 0.98 (95% ci, 0.92-0.99); threshold, -2.998; sensitivity, 100.00%; specificity, 92.82% and npv, 100.00%. in the subsequent validation cohort (n=44), the aucs (95% ci) were 0.83 (0.68, 0.93) and 0.88 (0.75, 0.96) for clinical model and laboratory model, respectively. conclusions: we developed two predictive models for the in-hospital mortality of patients with covid-19 in wuhan and validated in patients from another center. we developed a clinical model and laboratory model for predicting the in-hospital mortality of covid-19 patients, the aucs (95% ci) were 0.88 (0.80, 0.95) and 0.98 (0.92, 0.99) in training cohort, and 0.83 (0.68, 0.93) and 0.88 (0.77, 0.95) in validation cohort, respectively. several cases of "unknown viral pneumonia" have been reported in wuhan, hubei 2 province, china since december 2019. the causative agent was revealed as a novel 3 coronavirus named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the 4 international committee on taxonomy of viruses. the disease caused by sars-cov-2 was 5 named coronavirus disease 2019 (covid-19) by the world health organization (who). 1 6 this infectious disease has rapidly spread from wuhan to other chinese regions 2 . since mid-7 march 2020, cases have been detected in most countries worldwide and community spread is mild acute respiratory infection symptoms, such as fever, dry cough, and fatigue, 13 commonly occur in the early stages of covid-19, but some patients might rapidly develop 14 acute respiratory distress syndrome, acute respiratory failure, multiple organ failure, and other 15 fatal complications. 3, 4 no specific treatment has been fully developed for covid-19; thus, 16 early identification of patients with poor prognosis may facilitate the provision of proper 17 supportive treatment in advance and reduce mortality. the participants in the training cohort were all the consecutive patients diagnosed with 28 covid-19 in the first people's hospital of jiangxia district in wuhan, a major hospital in 29 the jiangxia district. we collected data on patients hospitalized from january 7, 2020, 17:58 30 to february 11, 2020, 22:01. a total of 296 patients with final outcome (i.e. discharged or 31 dead) were enrolled in this study before february 12, 2020, 14:00. we then randomly 32 collected patients with covid-19 who had been hospitalized in the infection department of 33 union hospital in wuhan from january 1, 2020, to february 20, 2020 to form our validation 34 cohort. a flow diagram is showed in figure 1 . 35 the data of these participants were used to construct two predictive models for in-36 hospital mortality. the study protocol was approved by the medical ethics committee of the 37 first people's hospital of jiangxia district and union hospital, and was complied with the 38 declaration of helsinki. we verbally informed the patients that their data would be used 39 anonymously for medical studies and obtained their permission. written informed consent 40 was not gathered, because the data were anonymous and the study was observational. 41 previous medical history, age, cough and fever (the oral temperature>37.5 ℃, the 43 axillary temperature>37℃, or the body temperature fluctuates more than 1℃ in a day) for 44 every subject were obtained by trained nurses. the laboratory data of the first examination 45 after admission of every subject were also collected. 46 all blood and urinary samples were processed within two hours of collection. routine 47 106 range (iqr), 8.6-15.5] days, respectively. the mean and median hospital stay of the 107 survivors were 6.2 ± 5.0 and 4.9 (iqr, 2.6-10.5) days, respectively. the mean and median 108 time interval between symptom onset and admission of the non-survivors were 5.2 ± 3.7 and 109 5.0 (iqr, 3.0-7.0) days, respectively. and for the survivors were 6.8 ± 4.0 and 5.5 (iqr, 3.0-110 9.2) days, respectively. 111 baseline clinical and laboratory characteristics of study population by training and 112 validation cohort are shown in table 1 . we observed significant differences between the two 113 cohorts in age, outcome, symptoms, and clinical indicators. the patients in validation cohort 114 were remarkably older, with higher rates of diabetes and hypertension, lower spo2, and 115 worse markers of inflammation, clotting status, and liver and kidney function. 116 the comparison between the survivors and the non-survivors were shown in table 2 . the 117 mean age of the non-survivor group was remarkably higher than that of the survivor group in 118 both cohorts. medical history showed that the non-survivor group had a higher proportion of 119 basic disease. no substantial difference was observed in the sex composition and habits of 120 smoking and drinking between survivors and non-survivors. in the training cohort, non-121 survivors had remarkably lower spo2 than survivors. inflammatory cells, namely, wbc and 122 neutrophil, were considerably higher whereas lymphocyte was remarkably lower in the non-123 survivor group than in the survivor group. meanwhile, hscrp, a marker of inflammation, was 124 also substantially elevated in the non-survivor group. in terms of blood coagulation indexes, 125 the non-survivor group had higher d-dimer and thrombin time and lower activated partial 126 thromboplastin time than the survivor group. cr, bun, alt, ast, ldh, and blood ammonia 127 were remarkably higher whereas gfr and serum alb were significantly lower in the non-128 survivor group. 129 in the model-development phase, the clinical model developed according to age, history 130 of hypertension and coronary heart disease showed good discriminatory power with auc of table 4 ) 142 the roc of the two models in training and validation cohort were plotted in figure 2 . exhibited relatively good discriminatory power the and the external verification was also 151 satisfactory. we believe that this is the first study to establish models for predicting the 152 mortality of patients with covid19 . 153 the clinical model based on age, history of hypertension, and coronary heart disease had 154 achieved good predictive power. elderly people are at higher risks for chronic diseases and 155 more susceptible to infection. age might be the risk factor for worse outcomes in patients 156 with covid-19 partially because age-related immune dysfunctions result from low-grade 157 chronic inflammation according to our speculation. 5, 11 in addition, elderly patients may 158 possess other risk factors, such as comorbidities and sarcopenia. hypertension is one of the 159 most common diseases in the elderly. history of hypertension is an important risk indicator in 160 the mulbsta score, which is a viral pneumonia death warning model developed by chinese 161 scholars. 12 our results are consistent with the above research. in addition, angiotensinwith chd history and infected with sars-cov-2 has to work harder to ensure that sufficient 168 blood oxygen is provided throughout the body. the problem of increased heart burden will 169 become more prominent. reasonable precautions must be taken to prevent these patients from 170 the viral infection. 171 xgboost showed that hscrp was the most important predictor for the mortality of patients 172 with covid-19, followed by age, spo2, ast, neutrophil count, d-dimer, gfr and 173 lymphocyte count. this finding is consistent with our clinical observation. 174 a low spo2 level suggests that the patients might have a serious illness at the time of 175 admission. we found that most of the patients with covid-19 had mild acute respiratory 176 infection symptoms initially; however, the conditions of some patients would rapidly 177 exacerbate and result in multiple organ failure or even death. we suspected this exacerbation 178 was primarily due to the "cytokine storm" and consequent immunologic abnormality. 179 cytokine storm is an important cause of death in severe acute respiratory syndrome (sars), 180 middle east respiratory syndrome coronavirus, and influenza a virus subtype h1n1 181 infection. [15] [16] [17] cytokine storm also seems to be a remarkable mechanism in the present 182 outbreak of covid-19 and contributed to the death of several patients, especially young 183 patients. a recent study showed that patients requiring icu admission had higher 184 concentrations of granulocyte colony-stimulating factor, interferon-induced protein 10, 185 monocyte chemoattractant protein 1, macrophage inflammatory protein 1 alpha, and tumor 186 necrosis factor alpha than those who did not require icu admission, suggesting that cytokine 187 storm is associated with disease severity. 4 a remarkable finding of our study was that the 188 increasing level of hscrp and neutrophil counts had prominent power in predicting fatal 189 outcomes in patients with covid-19. neutrophil chemotaxis and transmigration are essential 190 components for host defense during infections, but excessive neutrophil infiltration 191 contributes to deleterious inflammatory processes, 18 which might deeply interact with 192 cytokine storm during virus invasion. 193 the substantially depressed total lymphocytes in the non-survivor group indicated that 194 sars-cov-2 might act on t lymphocytes, and high replication of the virus leads to the 195 depletion of t lymphocytes, which suppresses the body's immunity. 19 in addition, patients 196 with severe illness are more likely to be co-infected with bacteria because of depressed 197 immune function, which is another possible reason for the increased level of neutrophils and 198 hscrp. further studies are necessary to elucidate the cytokine storm and immunologic 199 abnormality in sars-cov-2 infection. 200 we found that coagulation indicators might play a role in identifying severe cases as 201 well. we observed that d-dimer was negatively associated with in-hospital mortality. high specificity and ppv were demonstrated in clinical models in the validation cohort, as 244 opposed to the training cohort. we hypothesized that the probable reason was that there were 245 more deaths in patients with a history of hypertensive or coronary artery disease in the 246 validation cohort. more external validation is needed to demonstrate the robustness of the 247 model, and we currently recommend that clinical models with limited information only be 248 used for preliminary screening of high-risk populations. 249 by comparing the training and validation populations in table 1 , we had observed 250 significant differences between the two groups in age, symptoms, and examination index 251 wk and lc conceived and designed the study. wk, zp, cx analyzed the data, and wrote the first draft of the manuscript. wk, ly, zm, zx, xs and zh recruited patients, gathered data and participated in manuscript revision. lc provided study oversight and participated in manuscript revision. all authors had access to study data and approved the decision to submit the manuscript. we thank the patients and families who agreed to participate in this important study. we there was no funding directly relevant to this work. chengyun liu received a grant from the national natural science foundation of china (81974222) within the last 36 months, which was not directly related to this study. and all of the authors declare that they have no conflict of interest. roc curves of in-hospital mortality from logistic regression models of patients with clinical data (red ) and laboratory data (black) using bootstrap resampling (times = 500). roc = receiver operator characteristic. auc = area under the curve. spo2=peripheral capillary oxygen saturation. ast=aspartate aminotransferase. gfr= glomerular filtration rate. alb=albumin. glo=globulin. ck=creatine kinase. -data not collected in the validation cohort. auc=area under the curve. aic= akaike information criterion. *bootstrap resampling (times = 500). outbreak of covid-19-an urgent need for good science to silence our fears? nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster machine learning for the prediction of volume responsiveness in patients with oliguric acute kidney injury in critical care boosted tree model reforms multimodal magnetic resonance imaging infarct prediction in acute stroke transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (tripod) comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach innate immune responses in the ageing lung clinical features predicting mortality risk in patients with viral pneumonia: the mulbsta score genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding from gene to proteinexperimental and clinical studies of ace2 in blood pressure control and arterial hypertension preparing for the next influenza pandemic an interferon-γ-related cytokine storm in sars patients corticosteroid treatment ameliorates acute lung injury induced by 2009 swine origin influenza a (h1n1) virus in mice the trafficking protein jfc1 regulates rac1-gtp localization at the uropod controlling neutrophil chemotaxis and in vivo migration understanding the t cell immune response in sars coronavirus infection biochemical aspects of coronavirus replication and virus-host interaction abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province 25 national health commission. treatment scheme of covid-19 key: cord-346502-x2b0ao3q authors: arabi, yaseen m; shalhoub, sarah; mandourah, yasser; al-hameed, fahad; al-omari, awad; al qasim, eman; jose, jesna; alraddadi, basem; almotairi, abdullah; al khatib, kasim; abdulmomen, ahmed; qushmaq, ismael; sindi, anees a; mady, ahmed; solaiman, othman; al-raddadi, rajaa; maghrabi, khalid; ragab, ahmed; al mekhlafi, ghaleb a; balkhy, hanan h; al harthy, abdulrahman; kharaba, ayman; gramish, jawaher a; al-aithan, abdulsalam m; al-dawood, abdulaziz; merson, laura; hayden, frederick g; fowler, robert title: ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study date: 2019-06-25 journal: clin infect dis doi: 10.1093/cid/ciz544 sha: doc_id: 346502 cord_uid: x2b0ao3q background: the objective of this study was to evaluate the effect of ribavirin and recombinant interferon (rbv/rifn) therapy on the outcomes of critically ill patients with middle east respiratory syndrome (mers), accounting for time-varying confounders. methods: this is a retrospective cohort study of critically ill patients with laboratory-confirmed mers from 14 hospitals in saudi arabia diagnosed between september 2012 and january 2018. we evaluated the association of rbv/rifn with 90-day mortality and mers coronavirus (mers-cov) rna clearance using marginal structural modeling to account for baseline and time-varying confounders. results: of 349 mers patients, 144 (41.3%) patients received rbv/rifn (rbv and/or rifn-α2a, rifn-α2b, or rifn-β1a; none received rifn-β1b). rbv/rifn was initiated at a median of 2 days (q1, q3: 1, 3 days) from intensive care unit admission. crude 90-day mortality was higher in patients with rbv/rifn compared to no rbv/rifn (106/144 [73.6%] vs 126/205 [61.5%]; p = .02]. after adjusting for baseline and time-varying confounders using a marginal structural model, rbv/rifn was not associated with changes in 90-day mortality (adjusted odds ratio, 1.03 [95% confidence interval {ci}, .73–1.44]; p = .87) or with more rapid mers-cov rna clearance (adjusted hazard ratio, 0.65 [95% ci, .30–1.44]; p = .29). conclusions: in this observational study, rbv/rifn (rbv and/or rifn-α2a, rifn-α2b, or rifn-β1a) therapy was commonly used in critically ill mers patients but was not associated with reduction in 90-day mortality or in faster mers-cov rna clearance. the middle east respiratory syndrome (mers) is a severe respiratory infection caused by a novel coronavirus (mers-cov) and is associated with high mortality [1] . to date, there is no specific therapy of proven effectiveness for mers. based on prior experience with severe acute respiratory syndrome and on preclinical data, ribavirin and recombinant interferon (rbv/rifn) therapy has been used in managing patients with mers [2] [3] [4] . rbv is a guanosine analog that has antiviral activity against multiple rna viruses [4] . different preparations of recombinant rifns (rifn-α2a, rifn-α2b, rifn-β1a, and rifn-β1b) are active against mers-cov in vitro [5] . rbv and rifn at relatively high concentrations inhibited mers-cov replication in vero and llc-mk2 cells, but when used in combination, lower concentrations achieved comparable endpoints [2] . high doses of rbv/rifn-α2b administered 8 hours after inoculation of rhesus macaques with mers-cov resulted in reduced viral loads and was partially effective in preventing progression to pneumonia compared to animals that were untreated [3] . however, clinical data on rbv/rifn in mers have been limited to small single-center studies [6] [7] [8] [9] . in one report, all 5 patients who received rbv/rifn-α2b at a median of 19 days from admission died [7] . in a retrospective study (n = 32), patients who received rbv/ifn-α2a had a mortality rate of 85% compared with 64% in those who received rbv/ifn-β1a (p = .24) [8] . another study (n = 44) showed that rbv/rifn-α2a therapy compared to control was associated with a significant reduction in 14-day mortality (30% vs 71%; p = .004) but not in 28-day mortality (70% vs 83%; p = .054). recipients of rbv/rifn-α2a had a significant reduction (>2 g) in hemoglobin level, raising concerns of a previously described complication of hemolysis [9] . another study (n = 51) found that mers therapy using different regimens of rifn-β, rifn-α, rbv, and mycophenolate mofetil was associated with lower mortality on univariable analysis but not on multivariable analysis [10] . these studies have not provided clear evidence upon which to base treatment recommendations because of their nonrandomized design, inconsistent results, small sample sizes with limited power, and the lack of adjustment for unbalanced covariates. in addition, because of therapy initiation was not standardized, such studies are prone to 2 sources of bias: immortal time bias and indication bias. immortal time bias may occur because patients in the therapy group have survived for a period of time before receiving the therapy. because the outcome (death) could not possibly have occurred during this period in this group, this type of bias systematically underestimates adverse outcomes (eg, death) [11, 12] . indication bias occurs when the association of the therapy and outcome is caused by the indication for which the therapy was used and not to the therapy itself. if physicians typically prescribe certain therapy, such as rbv/rifn, only when the clinical condition is not improving or is worsening, standard multivariable analyses may overestimate the association with poor outcome. addressing both immortal time and indication bias in observational studies requires accounting for baseline and time-varying confounding associated with the decision to initiate treatment. the objective of this study was to examine the effect of rbv/ rifn therapy in a large cohort of critically ill patients with mers on the 90-day mortality and mers-cov rna clearance by accounting for baseline and time-varying confounders. some of these findings have been previously presented in an abstract form [13] . this retrospective analysis was conducted on a multicenter database of all critically ill patients with mers admitted to the intensive care units (icus) of 14 hospitals in 5 cities in saudi arabia between september 2012 and january 2018 [14] . details of the cohort have been reported previously [14] . some of the patients in the current analysis may have been included in previous single-center studies [8] [9] [10] . patient-level informed consent was not required. the institutional review boards (irbs) of all participating centers approved the study. participating centers followed the saudi arabian ministry of health guidelines for mers diagnostic testing. patients were defined to have mers if they had a positive mers-cov real-time reverse transcription polymerase chain reaction result (rrt-pcr) targeting amplifications of the upstream e protein (upe gene) and open reading frame 1a. specimens were obtained from nasopharyngeal swabs or sputum in nonintubated patients and from tracheal aspirates or bronchoalveolar lavage in intubated patients [15] . for patients who tested positive for mers-cov, follow-up respiratory samples were collected at the discretion of the treating teams approximately 1-2 times per week for infection control purposes. in the current analysis, we excluded patients who were enrolled in a randomized controlled trial (rct) for mers antiviral therapy that started enrolling patients in november 2017 (mers-cov infection treated with a combination of lopinavir/ritonavir and interferon β-1b [miracle] trial) [16] . the main exposure was rbv/rifn therapy, defined as the use of rbv/rifn combination, rbv alone, or rifn alone. the comparator group was the use of neither rbv nor rifn. three different types of rifns were used in the current cohort: rifn-α2a (pegasys, hoffmann-la roche, c/o genentech, south san francisco, california); rifn-α2b (peg-intron, merck sharp & dohme, whitehouse station, new jersey); and rifn-β1a (rebif, serono, rockland, massachusetts). commonly used dosing protocols for rbv/rifn for patients with mers in the participating hospitals are shown in supplementary table 1 . using standardized case report forms, we documented demographics, clinical presentation, underlying comorbidities, and final outcomes [17] . we collected sequential organ failure assessment (sofa) score and physiologic and laboratory parameters on icu days 1, 3, 7, 14, and 28 [17, 18] . we documented therapeutic interventions, including corticosteroids, mechanical ventilation, oxygen rescue therapies (nitric oxide, prone ventilation, high frequency oscillatory ventilation or extracorporeal membrane oxygenation), packed red blood cell transfusion, vasopressor therapy, and renal replacement therapy. the primary outcome was 90-day mortality. we also assessed time to mers-cov rna clearance in respiratory samples in patients who had at least 1 follow-up rrt-pcr performed after the diagnostic test. clearance was defined as the time from icu admission until the test was negative on 2 occasions, without a positive test afterward. to assess rbv/rifn safety profile, we evaluated serial levels of hemoglobin, white blood cell (wbc) count, platelet count, aspartate aminotransferase (ast), alanine aminotransferase (alt), bilirubin, international normalized ratio (inr), lactic acid, and creatinine. other secondary outcomes were icu and hospital mortality, and icu and hospital length of stay. we compared baseline characteristics, interventions, and outcomes of patients who received rbv/rifn to those who did not, using χ 2 test or fisher exact test for categorical variables and student t test or mann-whitney u test for continuous variables. for serial measurements, we tested differences between the 2 groups over time using repeated-measures analysis of variance with bonferroni correction for multiple comparisons and with no imputation for missing values. we constructed 3 models to assess the association of rbv/ rifn therapy with 90-day mortality adjusting for baseline characteristics and time-varying confounders. these analytical models were detailed in a previous study [19] . first, we created a logistic regression model adjusting for the following a priori baseline variables of clinical interest and all significant variables at the univariable level (p ≤ .2). these variables included diabetes, liver disease, renal disease, malignancy, sofa score on the first day of icu admission, source of infection, and year (before july 2014 and after) by applying the proc genmod procedure (sas software). second, we created a cox proportional hazards model adjusting for the same covariates and accounting for the rbv/ rifn therapy as a time-varying covariate. third, we created a marginal structural model analysis with inverse probability of treatment weighting to account for timevarying confounders that are likely to influence the decision to initiate rbv/rifn therapy and at the same time may be associated with mortality [20] [21] [22] [23] . we calculated stabilized weights for the probability that each subject received the treatment, censored on the day of therapy, icu discharge, or 28-day mortality, whichever came first. we included in this model selected baseline characteristics as well as time-dependent variables (sofa on the index day of rbv/rifn initiation and the previous day, ventilation status on the index day and on the previous day [0: not ventilated, 1: noninvasive ventilation, 2: invasive mechanical ventilation, 3: oxygen rescue therapy], hemoglobin, wbc count, ast and creatinine on the index day, and corticosteroid therapy on the index day). we included corticosteroid therapy as a time-varying covariate, as we have shown previously that corticosteroid therapy in critically ill mers patients might prolong mers-cov rna clearance although it was not associated with difference in mortality [19] . because these values were recorded on days 1, 3, 7, 14, and 28, we imputed missing values for the remaining days (supplementary methods). we used a weight-trimming approach to deal with extreme weights; weights <5th percentile value were fixed at the 5th percentile value and weights >95th percentile value were fixed at the 95th percentile value. this process continued until the average weight reached approximately 1. then, we used a weighted regression model using these weights taking in consideration the repeated-measures nature of the data to estimate the association of rbv/rifn with 90-day mortality. we carried out sensitivity analyses examining the association of rbv therapy alone compared to no rbv and rifn therapy alone compared to no rifn. to account for the possible variation by site, we carried out a sensitivity analysis using a logistic regression model adjusting for clustering by centers in addition to the previously mentioned baseline variables. we further evaluated the association of different types of rifn (rifn-α2a, rifn-α2b, and rifn-β1a) on 90-day mortality using a similar logistic regression model and adjusting for clustering by center. we also created 2 models to assess the association of rbv/ rifn therapy with mers-cov rna clearance adjusting for baseline characteristics and time-varying confounders. first, we carried a cox proportional hazards model adjusting for the same baseline covariates mentioned earlier and accounting for the rbv/rifn therapy as a time-varying covariate. we censored patients if they never cleared mers-cov rna or at the time of last rrt-pcr test. second, we created a marginal structural cox proportional hazards model incorporating the stabilized weights to estimate the effect of rbv/rifn therapy on mers-cov rna clearance in a similar approach to the marginal structural model used for 90-day mortality. analyses were conducted using sas version 9.4 software (sas institute, cary, north carolina). the study was approved by the national guard health affairs irb and by the irbs of all participating sites. informed consent was waived by the irb because of the retrospective nature of the study. of the 355 patients with mers in the cohort, 6 patients were excluded because they were enrolled in the miracle trial. the remaining cohort included 349 patients, of whom 144 (41.3%) received rbv/rifn therapy (table 1) . patients in the 2 groups were similar in age, sex, body mass index, and source of admission (table 1) . comorbidities were common in both those who received rbv/rifn and those who did not (84.0% vs 78.0%; p = .17). patients who received rbv/rifn were more likely to have diabetes (58.3% vs 42.0%; p = .003) and chronic renal disease (36.8% vs 27.3%; p = .06), but less likely to have chronic liver disease (2.1% vs 7.8%; p = .02). of note, the rbv/rifn recipients had lower sofa scores than those not treated (median, 8 [q1, q3: 5, 11] vs 10 [q1, q3: 6, 13]; p = .01; table 1 ). rbv/rifn therapy was initiated at a median of 2 days (q1, q3: 1, 3) from icu admission, which corresponded to 5.0 days (q1, q3: 2.0, 9.0) from hospital admission and 9.0 (q1, q3: 6.0, 12.0) from onset of symptoms (table 2 and figure 1 ). of these patients, 117 (81.3%) patients received rbv/rifn combination, 18 (12.5%) rbv alone, and 9 (6.3%) rifn alone. a total of 73 (57.9%) received rifn-α2a, 22 (17.5%) received rifn α-2b, 31 (24.6%) received rifn-β1a, and none received rifn-β1b ( table 2 ). the use of rbv/rifn therapy and the type of rifn varied by site ( supplementary figures 1 and 2) . during icu stay, the provision of mechanical ventilation, oxygen rescue therapies, renal replacement therapy, and vasopressor therapy was similar in the 2 groups (table 3) . during the icu stay, patients who received rbv/rifn therapy were more likely to receive corticosteroid therapy compared with those who did not receive rbv/rifn (59.7% vs 44.9%; p = .006; table 3 ). crude 90-day mortality was higher in patients who received rbv/rifn therapy compared to those who did not analyses of rbv therapy vs no rbv and rifn vs no rifn were consistent with results to the primary analysis, with no significant association with 90-day mortality or mers-cov rna clearance using marginal structural modeling (table 4 ). when the logistic regression model was adjusted for clustering by centers in addition to the previously mentioned baseline variables, there remained no association of rbv/ rifn with 90-day mortality. examining different types of rifn on 90-day mortality using a similar logistic regression model and adjusting for clustering by center showed similar results (supplementary table 2 ). there were no differences between the 2 groups group over time in hemoglobin, wbc count, platelet count, ast, alt, bilirubin, inr, lactic acid, or creatinine (supplementary figure 3) . however, patients treated with rbv/rifn received more blood transfusions compared with those who were not treated with rbv/rifn (58/144 [40.3%] vs 58/205 [28.3%]; p = .02; table 3 ). while benefit of rbv/rifn was suggested by preclinical studies, our observational study that accounted for baseline and time-varying differences among 349 critically ill patients with mers treated with rbv/rifn, or not, demonstrates that rbv/ rifn was not associated with decreased mortality or with faster mers-cov rna clearance. what are the possible explanations for lack of clinical and virological benefit? first, it has been shown that the rbv concentrations required to inhibit mers-cov replication are much higher than clinically achievable concentrations with oral dosing [5] . second, the lack of rifn effectiveness may be related to the type used. one study examined the in vitro mers-cov susceptibility to different rifn preparations (rifn-α2b, rifn-γ, rifn-universal, rifn-α2a, rifn-β) and found that rifn-β had the strongest mers-cov inhibition, at 41 times lower than the previously reported 50% inhibitory concentration (56.08 u/ml) of rifn-α2b [5] . another in vitro study found that serum concentrations achievable at therapeutic doses of rifn-β-1b were 3-4 times higher than the in vitro inhibitory concentrations of mers-cov, whereas those of other rifn preparations and rbv were lower than inhibitory levels [24] . of note, none of the patients in the current cohort received rifn-β-1b. an rct (miracle) is currently recruiting patients examining the effect of a combination of lopinavir/ritonavir and rifn-β-1b on mortality of hospitalized patients with mers [16] . third, the positive, but modest, effect observed in previous rhesus macaque experiments occurred after very early treatment (8 hours after inoculation of with mers-cov) and with the administration of high doses of rbv/rifn-α2b. in contrast, it took a median of 5 days for patients in our cohort to present to the hospital and another 4 days to start therapy. to assess safety profile of rbv/rifn, we compared the levels of hemoglobin, wbc count, platelet count, ast, alt, bilirubin, inr, lactate, and creatinine and we found no difference between the 2 groups over the icu stay. however, these data should be interpreted in the context of the potential for time-varying confounding. of note, we found that patients who were treated with rbv/rifn received more blood transfusions than patients who were not treated with rbv/rifn, which is consistent with the findings of a previous study [9] . these changes may be related to hemolysis induced by rbv therapy. our study demonstrates that not accounting for time-varying confounding can substantially influence the results of observational studies. our study found an association of rbv/rifn with higher crude mortality on crude analysis, with adjustment for baseline characteristics alone (by logistic regression) and with adjustment for baseline characteristics including the time to initiation of rbv/rifn (by cox proportional hazards analysis). ultimately, we did not find evidence that rbv/rifn therapy was associated with reduced mers mortality when adjusting for baseline and time-dependent covariates using a marginal structural model. this finding suggests that much of the observed increased mortality may have been related to confounding due to indication bias, and calls for caution when interpreting observational studies that do not account for timevarying confounders. our study examined rbv/rifn therapy in a large multicenter cohort of critically ill patients with mers. limitations include its retrospective nature and lack of randomization (and therefore inevitable initial imbalance in potential confounders). although marginal structural models adjust for time-varying confounding, unmeasured confounders cannot be entirely excluded. quantitative data on viral loads were not available. because practices for repeating mers-cov rrt-pcr varied among centers and because of competing risk of mortality, sufficient data to assess mers-cov rna clearance were available for only about 50% of the patients. rcts remain the best approach to derive the most unbiased estimates of treatment effect. most patients included in this study were diagnosed with mers prior to the launch of the first therapeutic rct mentioned earlier (miracle). since then, efforts have focused on considering eligible patients in this trial. .71 logistic regression and cox proportional hazards regression models were adjusted for the following variables: diabetes with chronic complications, liver disease, renal disease, any malignancy including leukemia or lymphoma, sequential organ failure assessment (sofa) score on day 1, source of infection (community-acquired/healthcare worker, hospital-acquired/non-healthcare worker, hospital-acquired), and year (before 1 july 2014 and after). for logistic regression, hosmer-lemeshow goodness-of-fit test was used to assess the model fitness, and p values were not significant for all analyses. for marginal structural model, adjustment was made for the same afore mentioned baseline characteristics and for the following time-varying covariates: sofa on the index day of rbv/rifn initiation and the previous day, ventilation status on the index day and on the previous day (0: not ventilated, 1: noninvasive ventilation, 2: invasive mechanical ventilation, 3: oxygen rescue therapy), hemoglobin, white cell count, aspartate aminotransferase and creatinine on the index day, and corticosteroid therapy on the index day. for cox proportional hazards regression model and marginal structural cox proportional hazards model, we censored patients if they never cleared mers-cov rna or at the time of last real-time reverse-transcription polymerase chain reaction test. abbreviations: ahr, adjusted hazard ratio; aor, adjusted odds ratio; ci, confidence interval; mers-cov, middle east respiratory syndrome coronavirus; rbv, ribavirin; rifn, recombinant interferon. in conclusion, rbv/rifn therapy (rbv and/or rifn-α2a, rifn-α2b, or rifn-β1a) is not associated with reduction in 90-day mortality or with faster mers-cov rna clearance. future studies should test the antiviral and clinical effectiveness of newer antiviral interventions that show more promising results in relevant animal models [25, 26] . middle east respiratory syndrome inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia immortal time bias in pharmaco-epidemiology effectiveness of inhaled corticosteroids in chronic obstructive pulmonary disease: immortal time bias in observational studies effect of ribavirin and interferon on the outcome of critically ill patients with mers saudi critical care trial group. critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections miracle trial group. treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial international severe acute respiratory and emerging infection consortium (isaric) use of the sofa score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. working group on "sepsis-related problems" of the european society of intensive care medicine saudi critical care trial group. corticosteroid therapy for critically ill patients with middle east respiratory syndrome canadian critical care trials group h1n1 collaborative. the influence of corticosteroid treatment on the outcome of influenza a(h1n1pdm09)-related critical illness marginal structural models to estimate the causal effect of zidovudine on the survival of hiv-positive men analysis of longitudinal observational data using marginal structural models marginal structural models and causal inference in epidemiology broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-348350-pac9ha4q authors: martin-blondel, g; ruiz, s; murris, m; faguer, s; duhalde, v; eyvrard, f; izopet, j; mansuy, j m; rolland, y; delavigne, k; guimbaud, r; pugnet, g; conil, j m; georges, b; delobel, p; minville, v; silva sifontes, s; concordet, d; gandia, p title: hydroxychloroquine in covid-19 patients: what still needs to be known about the kinetics date: 2020-05-11 journal: clin infect dis doi: 10.1093/cid/ciaa558 sha: doc_id: 348350 cord_uid: pac9ha4q different dosage regimens of hydroxychloroquine are used to manage covid-19 patients, without information on the pharmacokinetics in this population.blood samples (n=101) were collected from 57 covid-19 patients for 7 days and concentrations were compared with simulated kinetic profiles.hydroxychloroquine exposure is low and cannot be predicted by other populations. based on the in vitro activity against sars-cov-2 and preliminary clinical data, hydroxychloroquine is currently used in the management of covid-19 patients [1] [2] [3] . results of ongoing clinical trials are eagerly awaited. in the meantime, the efficacy as well as the dosage of hydroxychloroquine is highly debated. in their pilot study, gautret et al. [2] administered the standard dosage used for treating systemic lupus erythematosus (sle), which is 200 mg x 3 / day, as hydroxychloroquine is mainly prescribed for this indication [4] . because of the severity of covid-19 and the pharmacokinetics of hydroxychloroquine in sle patients [5] , a loading dose was rapidly included in the new hospital regimens to optimize drug distribution in tissues and more precisely in the lungs. due to the lack of information on the plasma/blood concentrations required in covid-19 patients to induce a virological/clinical effect, in many european countries these concentrations are monitored in patients whether or not they are included in clinical research protocols. our aim was to determine whether or not the pharmacokinetics in sle patients as described by morita et al. [6] can be applied to covid-19 patients. this is essential information for further exploration of the pharmacokinetic-pharmacodynamic relationship. according to the guidelines established by the french national team ac43-anrs/stp-sfpt in march/april 2020, blood samples were collected at different time points (from 48h to 192h) during clinical management of covid-19 patients after treatment initiation and within 30 minutes before drug administration. plasma concentrations were determined using a validated chromatographic analytical method which presents a lower limit of quantification of 0.05 mg/l, and an intra-and interday variability < 4% and <10% respectively. to ensure that these concentrations were in keeping with the expected values at least in sle patients for the same dosage regimens, we used the population pharmacokinetic model published by morita et al. [6] hydroxychloroquine is mainly eliminated by hepatic metabolism (i.e. cyp3a4 and cyp2c8) with no reported difference between the japanese and caucasian populations concerning the prevalence of polymorphisms for these two cytochromes [7] . therefore, the ethnicity of the patients in morita's study [6] should not have a high impact on kinetics. we simulated the expected kinetic profiles in plasma (n=10,000) taking into account the significant covariate (i.e. body weight) which mainly explains the inter-individual variability of total clearance. this study was entered in the toulouse university hospital register of retrospective studies (registration number: rniph 2020-33) and is covered by mr-004 (cnil number: 2206723 v 0). it was approved by toulouse university hospital and ethical requirements were entirely respected. m a n u s c r i p t 4 the average trough concentrations (+/-standard deviation) collected from day 3 to day 9 for 57 patients (101 blood samples) are shown in table 1 , whether or not they were treated in an intensive care unit. currently, there are no clear guidelines for an optimal dosage regimen. consequently, different dosage regimens were applied over the past weeks and were updated based on data that emerged: regimen 1 (200 mg x 3/day), regimen 2 (400 mg x 2 on day 1 followed by 200 mg x 3/day), regimen 3 (400 mg x 2 on day 1 followed by 400 mg x 1/day) and regimen 4 (600 mg x 2 followed by 400 mg x 1 /day). for regimen 4, which was exclusively used for patients in intensive care units, tablets were crushed and administered by feeding tube. for each regimen applied at the toulouse university hospital, an example of the expected kinetic profiles for a body weight of 80 kg (the median weight in our population) are presented in figure 1 with the corresponding serum concentration for each patient. co-prescriptions were checked for each patient included, in order to assess a possible influence on hydroxychloroquine pharmacokinetics. none of the patients had medications that could increase or decrease the oral bioavailability and / or clearance of hydroxychloroquine. the concentrations measured in covid-19 patients show that hydroxychloroquine exposure tends to be low and in most instances lower than the values reported in sle patients, in particular for the standard regimen of "200 mg x 3/day". these preliminary results confirm that the pharmacokinetic behavior in covid-19 patients cannot be predicted by the sle population or by rheumatoid arthritis patients as recently reported by perinel et al [8] . furthermore, given the apparently large volume of distribution of hydroxychloroquine, morita's model [6] , which is based on plasma concentrations, presents a major limitation for accurately predicting hydroxychloroquine accumulation in deep tissue. it is a major additional argument to urgently assess hydroxychloroquine pharmacokinetics in covid-19 patients using both blood and plasma matrices. moreover, monitoring hydroxychloroquine concentrations in bronchoalveolar fluids of covid-19 patients could provide additional information on the degree of hydroxychloroquine lung impregnation [9, 10] for a short period of treatment (i.e. few days). in fact, data exist from lung tissue [11] and bal [12] for antibiotics such as macrolides that accumulate in deep tissues. therefore, it would be logical to consider bal as "quality control" that provides information on the degree of hydroxychloroquine exposure in the lung. there is no established efficacy threshold for the moment. therefore, the only relevant element to take into account when monitoring hydroxychloroquine concentrations seems to be toxicity and more precisely cardiotoxicity. a number of arguments suggest that a plasma concentration of 1 mg/l should not be exceeded, as initially suggested by the french national team, ac43-anrs/stp-sfpt. however, this concentration should be interpreted with caution because covid-19 patients may (i) receive co-prescriptions which can worsen the cardiotoxicity of hydroxychloroquine (for example, azithromycin), (ii) have preexisting heart problems along with covid-19 (ema/202483/2020) and/or (iii) have hypokalemia.if this is the case, monitoring for heart problems by placing patients on telemetry to track ekg over the course of treatment, as is the current practice for icu patients or at the university of washington medical center [13] , is not an option to be overlooked. m a n u s c r i p t 5 on april 2 nd 2020, for an unknown reason, the french ministry of health imposed a dosage regimen identical to that used in sle (no loading dose and 200 mg x 3/day) for patients treated outside the context of a clinical trial. given (i) the variability of the physiopathological status of covid-19 patients, (ii) the complex tissue distribution kinetics of hydroxychloroquine and (iii) the hydroxychloroquine concentration required to clear 100% of sars-cov-2 in vitro (≈ 7 mg/l) [13] , it does not seem reasonable to impose the same dosing regimen on the entire covid-19 population without any further pharmacokinetic-pharmacodynamic information. in conclusion, a full hydroxychloroquine kinetic exploration is currently needed for covid-19 patients. m a n u s c r i p t 6 m a n u s c r i p t 8 0.14 ± 0.13 [10] 0.10 ± 0.05 [5] 0.19 ± 0.06 [5] 0.16 ± 0.08 [2] 0.18 ± 0.06 [2] 62. 5 nd nd 85-125 [4] 400 mg x 2 at day 1 400 mg x 1 /day 0.09 ± 0.12 [9] 0.11 ± 0.06 [6] 0.13 ± 0.10 [9] 0.09 ± 0.03 [6] 0.13 ± 0.14 [6] 0.16 ± 0.07 [3] 0.07 ± 0.04 [3] nd: no data. when a concentration was lower than the limit of quantification (0.05 mg/l), it was set at 0.025 mg/l. a c c e p t e d m a n u s c r i p t 9 figure 1 : hydroxychloroquine plasma kinetic profiles (n=10000 per regimen) simulated with the population pharmacokinetic model published by morita et al. [6] for a body weight of 80 kg and for different dosage regimens (200 mg x 3 /day ; 400 mg x 2 at day 1 followed by 200 mg x 3 /day ; 400 mg x 2 at day 1 followed by 400 mg x 1 /day; 600 mg x 2 at day 1 followed by 400 mg x 1/day). the median kinetic profile is indicated by the white curve. the "extreme" profiles found in less than 5% and 95% of the population are indicated by the black curves. the red line corresponds to the toxic plasma concentration. the black squares represent observed trough concentrations (i.e. within 30 minutes before drug administration) for patients who weigh approximately 80 kg (the median weight in our population). to facilitate the interpretation of the figure, all the trough concentrations that are presented are for the same moment before administration. no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease-19 (covid-19) therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases steady-state pharmacokinetics of hydroxychloroquine in patients with cutaneous lupus erythematosus population pharmacokinetics of hydroxychloroquine in japanese patients with cutaneous or systemic lupus erythematosus characterization of adme genes variation in roma and 20 populations worldwide towards optimization of hydroxychloroquine dosing in intensive care unit covid-19 patients chloroquine: a review animal toxicity and pharmacokinetics of hydroxychloroquine sulfate pharmacokinetics and lung distribution of macrolide antibiotics in sepsis model rats steady-state intrapulmonary concentrations of moxifloxacin, levofloxacin, and azithromycin in older adults hydroxychloroquine for treatment of sars-cov-2 infection? improving our confidence in a model-based approach to dose selection we would like to thank prof. pierre-louis toutain for his expertise in the pharmacokinetic field, dr. michel lavit, dr. thomas lanot and patrick seraissol for their expertise in the analytical field, eni losha for his help in collecting data and the staff of technicians at the pharmacokinetics and toxicology laboratory of toulouse university hospital. a c c e p t e d m a n u s c r i p t this study was supported by internal funding. none to declare a c c e p t e d m a n u s c r i p t 7 key: cord-355618-7kfxc2w1 authors: mcateer, john; yildirim, inci; chahroudi, ann title: the vaccines act, deciphering vaccine hesitancy in the time of covid19 date: 2020-04-13 journal: clin infect dis doi: 10.1093/cid/ciaa433 sha: doc_id: 355618 cord_uid: 7kfxc2w1 nan a c c e p t e d m a n u s c r i p t since the covid19 pandemic first hit wuhan, china in december 2019, scientists have been racing to develop and test novel vaccines to protect against sars-cov-2. the speed of scientific discovery related to covid19 is unprecedented. with several vaccine candidates already being tested in clinical trials, we pose the question: what will the vaccine hesitant do in the face of this pandemic? vaccine hesitancy has vexed the practice of immunization ever since cowpox was used as a vaccine for smallpox by edward jenner in 1796 1 . skepticism about vaccines is a timeworn concept with shifting ideologies that reflect historical events and individual belief systems reflective of different societal periods. while the term "antivax" is ascribed to contemporary vaccine skeptics, "hesitant" is often a better descriptor for the majority of parents who contemplate or request delaying vaccines for their children or deviating from the recommended immunization schedule. the term hesitant avoids polarization into "pro" versus "anti" vaccination factions. hesitancy implies, however, that minds can be swayed towards acceptance, and as pediatricians armed with the latest data, we are frustrated that these facts and figures do not always convince parents in our exam rooms and those we encounter on social media that vaccination is right for their children. how and why does this hesitancy shift from physician-guided acceptance of vaccines to their refusal? vaccine hesitancy is a complex issue. one important consideration is that vaccine hesitancy has evolved in response to the complicated but ultimately successful history of vaccine science. vaccines are a victim of their own success, turning once devastating diseases into distant memories. hesitant parents, as a result, have shifted their focus to the perceived risks of vaccination as fewer people witness the consequences of forgoing vaccines in developed countries. however, some vaccine-preventable diseases are now making a comeback, which is putting our nation's children at risk. outbreaks of vaccine-preventable diseases are occurring at an alarming rate across the country, especially in geographic pockets with poor immunization rates 2 . in 2019 alone there were 1,282 confirmed cases of measles in the united states, the greatest number reported in this country since 1992 3 . these outbreaks led to the possibility that the united states would lose measles elimination status, initially awarded almost 20 years ago. despite remarkable advances in vaccine science, vaccine hesitancy has now become a recognized public health threat with potentially disastrous complications. anti-immunization sentiment is at an all-time high and the medical community has recognized the need to address it as a public health emergency, with research, action, and advocacy. the nature of hesitancy directly confronts our fundamental belief system as pediatric scientists, which is based on disease prevention and anticipatory guidance. vaccine hesitancy is one of the most challenging controversies we have faced in our history. this problem is personal to all of us. pediatricians and vaccinologists have been disbelieved and attacked. vaccine hesitancy has the potential to harm physician-patient relationships. it is difficult to comprehend that some of the greatest triumphs of medical science are being eroded by misinformation and distrust. to address vaccine hesitancy, we need to reframe our approach and focus on understanding the root causes of this complicated problem. on the surface, the idea of hesitancy is perplexing given the overwhelming scientific evidence for the safety and efficacy of vaccines. vaccine hesitancy is not a superficial issue with a universal solution, however. rather, it is a multifaceted, deeply complex construct that may be rooted in the moral composition that guides our daily decision making. research has shown that several distinct values are associated with vaccine hesitancyparticularly purity, liberty, and a c c e p t e d m a n u s c r i p t anti-authority 4 . combined with the historical amnesia of the consequences of vaccinepreventable diseases, vaccine hesitancy becomes easier to understand as a belief system grounded in certain moral values. by considering these values, rather than relying solely on vaccine safety and efficacy data, some researchers believe we may be able to more effectively address vaccine hesitancy. although each family is unique, there are likely common drivers for vaccine hesitancy and these, we submit, are being inadequately addressed. hesitant families frequently express concern about vaccine safety, but even this issue has multiple layers, including fears regarding potential links to autism (now thoroughly refuted 5 ), learning difficulties, and chronic illnesses, as well as a perceived lack of safety testing prior to approval for use 6 . a famous quote from bernard shaw applies here: "the greatest problem in communication is the illusion that it has been accomplished." properly identifying and completely responding to fears regarding vaccines within the confines of a 15-minute well visit is next to impossible. furthermore, how we discuss and communicate about vaccines, as much as the evidence-based facts provided, may be very important. we need a systematic assessment of factors affecting vaccine uptake on a continuing basis to develop the most effective and efficient communication strategies. as a medical community, we must investigate the determinants of vaccine hesitancy and the continuously evolving challenges it entails in order to tailor evidence-based strategies to overcome the problem with sustainable interventions. in a step forward in the fight against vaccine misinformation, facebook announced on september 4 th , 2019 that user searches for vaccine-related content will be directed to either the united states centers for disease control and prevention (cdc) or the world health organization (who) websites for accurate information regarding vaccines. in his statement of support 7 , the who director-general dr. tedros ghebreyesus asserted that "these online efforts must be matched by tangible steps by governments and the health sector to promote trust in vaccination and respond to the needs and concerns of parents." in order to adequately respond to these needs and concerns, which differ depending on the cultural, societal, and personal beliefs of a particular region, the who recommends that each country take steps to develop an understanding of vaccine hesitancy at a local level on an ongoing basis 8 . unfortunately, such a surveillance program in the united states (and in many other countries, for that matter) does not exist. as such, much of the information we have about vaccine hesitancy is retrospective and anecdotal. the absence of federal investment in understanding vaccine hesitancy was clear during the 2019 measles outbreaks. as cases increased nationally, cdc officials tracked cases and exposures across state lines. however, insight into the vulnerability of exposed communities was varied, and depended solely on the quality and attention state governments paid to evaluating mmr immunization rates in those communities. new york state, for example, quickly realized reluctance to immunize was centered in specific orthodox jewish communities in brooklyn and rockland counties and was rooted in concerns about potential pork products in vaccines. with local governments, the state created public awareness campaigns led by rabbis urging vaccination. other areas, absent this granular understanding, could simply react to burgeoning cases and missed opportunities that may have prevented the spread of cases to 31 states. in recognition of these missed opportunities and in response to declining immunization rates and increasing national skepticism on the safety of vaccines, congress has introduced bipartisan legislation to expand research into vaccine hesitancy. spearheaded by congresswoman and pediatrician dr. kim schrier, the vaccine awareness campaign to m a n u s c r i p t champion immunization nationally and enhance safety (vaccines) act (h.r. 2862) would provide federal funding for vaccine hesitancy surveillance and authorization of a public awareness campaign to increase public confidence in vaccines 9 . the bipartisan bill introduced in may 2019 will amend the public health service act of 1944 to support a rigorous scientific approach to learn more about the factors that drive vaccine hesitancy and will fund national surveillance of hesitant regions. while commendable, it should be noted that the requested resources in the current version of the vaccines act are inadequate to fund serious communications campaigns, given that some of the areas with the highest hesitancy also have the most expensive media markets. the bill does include a vital provision to support awareness campaigns tailored to specific communities that could prove to be an essential tool in the fight to protect children from vaccine-preventable diseases. signing this bill into law will provide a framework to better understand and respond to vaccine hesitancy in the united states. current research suggests that the drivers of vaccine hesitancy depend heavily on personal experiences and belief systems 4, 8 . having an adequately federally-funded surveillance program will permit analysis of contextual influences that promote and perpetuate reluctance at the local level. high-risk populations can be identified and specifically targeted based on their societal and cultural belief systems. awareness campaigns can be tailored to specific locales to address identified concerns regarding vaccines. studying all these factors will expand our armamentarium in preventing the rise of vaccine-preventable diseases. the vaccines act is currently in the first stage of the legislative process. it has been assigned to the house energy and commerce committee where the chairs will determine if the bill will pass to the house floor 9 . the odds of any bill becoming law at this stage are collectively low (around 3% since 2015) and even worse without strong public support 10 . it is thus of paramount importance for the scientific community to come together in support of this legislation. vaccine hesitancy remains a persistent global threat and is a concept we are only beginning to understand. we have been lagging in our response, in part because the epidemic of vaccine hesitancy has personally challenged our motivations and values as doctors, scientists, advocates, and often, family members and friends. while we wonder if the covid19 pandemic will begin to change minds regarding the overwhelming value of immunization, we fear that as time elapses and memories fade, a sars-cov-2 vaccine may join the ranks of mmr in eliciting hesitancy from some groups. the bipartisan vaccines act is an important step in supporting evidence-based research into vaccine hesitancy. it is a call for the scientific community to shift our focus towards understanding the underlying causes of this epidemic. as this bill goes through the legislative process, we strongly urge the scientific and medical communities to contact their representatives to voice support for a fully resourced vaccines act. we believe that all children and their families deserve it. none of the authors has any potential conflicts of interest to disclose. vaccine hesitancy, history, and human nature: the 2018 stanley a. plotkin lecture association between vaccine refusal and vaccine-preventable diseases in the united states: a review of measles and pertussis increase in measles cases: united states association of moral values with vaccine hesitancy measles, mumps, rubella vaccination and autism: a nationwide cohort study countering antivaccination attitudes world health organization. immunization, vaccines and biologicals-improving vaccine demand and addressing hesitancy statistics and historical comparison a c c e p t e d m a n u s c r i p t key: cord-341359-c34gyuv6 authors: larson, derek t; sherner, john h; gallagher, kia m; judy, cynthia l; paul, madison b; mahoney, alexandra m; weina, peter j title: clinical outcomes of coronavirus disease 2019 with evidence-based supportive care date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa678 sha: doc_id: 341359 cord_uid: c34gyuv6 calls for adherence to evidence-based medicine have emerged during the initial wave of the covid-19 pandemic but reports of outcomes are lacking. this retrospective study of an institutional cohort including 135 patients with confirmed covid-19 demonstrates positive outcomes when organizational standards of care consist of evidence-based supportive therapies. since the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1, 2] and the subsequent coronavirus disease 2019 (covid-19) pandemic, the publication of scientific data regarding clinical outcomes has been rapid, but with contrasting practice standards. for the bedside clinician, determination of optimal care has been complicated by the various standards presented that, to date, have been either unreported in publications or a combination of investigational antivirals, antibiotics, immune globulins, and immunomodulatory medications [3, 4] , each cohort complicated by varying local case fatality rates that range from 0.1% in denmark to 6.8% in italy [5, 6] . due to confounding by these variables, the benefits and harms of individual therapies remain largely unknown. following widespread use of therapeutic agents with limited data to support their use, calls were made to focus on the evidence-based care that has been the foundation of modern medical therapy for most acute respiratory viral infections [7, 8] . our study is the first report of covid-19 outcomes when institutional standards of care consist solely of known evidence-based practices of supportive care. this retrospective, observational case series included all symptomatic patients diagnosed with sars-cov-2 by reverse-transcriptase polymerase chain reaction (rt-pcr) at a single military community hospital in virginia. cases diagnosed between 6 march 2020 and 22 april 2020 were included. availability of tests allowed liberal testing strategies, and patients with minimal symptoms were included. no therapeutic trials were available, and institutional policies led to a nonuse of all investigational therapies, including expanded use of antivirals, off-label use of antiinflammatory medications, empirically therapeutic anticoagulation protocols, and investigational devices. antibiotics were prescribed according to the attending physicians' clinical judgment, and thromboprophylaxis with low-molecular-weight heparin was prescribed based on risk stratification using the padua prediction score. disease severity was classified according to historical methods [2] , and patients with only subjective dyspnea were managed as outpatients if living conditions were appropriate. the intensive care unit (icu) transfer policy included those who required ≥5 l of oxygen support. airway management was based on usual factors and clinical judgment; an explicit "early intubation" strategy was not pursued. all patients were screened for signs or symptoms compatible with covid-19 [2] . we tested 1099 patients using rt-pcr, including 75 inpatients, and 135 (12.3%) tested positive. of those, 88 (65.2%) were male, 54 (40%) were active-duty, and the median age was 46.5 years (interquartile range [iqr], 33-56; table 1 ). two pediatric patients were diagnosed with covid-19 and fully recovered as outpatients. data on comorbidities was available for all but 2 of our patients, and those reported were typical of chronic health conditions seen in a community hospital. incidence of coronary artery disease, chronic kidney, liver disease, and significantly immunocompromised patients each represented less than 5% of our population, whereas obesity, hypertension, and diabetes represented 37%, 25.2%, and 10.4%, respectively. of those diagnosed with covid-19, 21 (15.6%) had severe enough disease to necessitate hospitalization and 6 (4.4%) required care in the icu. among inpatients, 14 (66.7%) received antibiotics for community-acquired pneumonia, including 11 (52.4%) with azithromycin, all of which had radiologic findings consistent with acute pulmonary infiltrates. due to a preexisting health system policy, 2 of these patients who were intubated in the emergency department were transferred for mandated cohorting of all intubated covid patients at the local tertiary care hospital. both of these patients survived after prolonged icu stays and having received multiple investigational treatments in addition to supportive care. four patients were managed in our icu, 3 of whom required support via high-flow nasal cannula (30-40 l/min; 60%-100% fraction of inspired oxygen), with an average length of icu stay of 6.5 days. patients treated in our icu had median peak c-reactive protein of 18.7 mg/dl (iqr, 12.1-21.2), ferritin of 2981 ng/ml (iqr, 1040.5-3405), and a mean d-dimer of 2.66 µg/ml. at the time of manuscript preparation, all patients had been discharged from the hospital except 1 who, despite clinical stability, requires continued hospitalization only for infectioncontrol purposes. no investigational therapies were prescribed in our hospital. overall, no cases of septic shock or secondary infection were diagnosed, no admitted patients progressed to a level of respiratory failure that necessitated intubation during their hospital stay, and none died. our study demonstrates favorable outcomes for patients with mild to moderately severe covid-19 disease when evidence-based supportive care is considered the institutional standard. the 2 patients who were intubated and transferred both survived after extensive icu courses. both received off-label therapeutic agents at the receiving hospital; therefore, our data do not provide any insight regarding supportive-only care in patients on mechanical ventilation. notably, however, no patient's condition deteriorated sufficiently while an inpatient to necessitate endotracheal intubation, and none died. we believe this is attributable to multiple factors, including a focus on supportive care that is well established to benefit patients and a conservative intubation strategy. our population likely also benefited from having later occurrence of widespread community transmission as lessons learned from regions previously affected were incorporated into local care, and care was provided in a facility that was not taxed by an overwhelming surge. limitations of this study are notable for being retrospective in design, a younger population, and the lack of long-term follow-up data. future studies of other populations that are treated with only supportive care are needed to evaluate the benefits and harms of investigational therapeutics and their effects against the morbidity and mortality associated with covid-19. notes a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical features and short-term outcomes of 221 patients with covid-19 in wuhan effectiveness of convalescent plasma therapy in severe covid-19 patients an epidemiological study on covid-19: a rapidly spreading disease characteristics of covid-19 infection in beijing the risks of prescribing hydroxychloroquine for treatment of covid-19-first, do no harm covid-19-a reminder to reason acknowledgments. the authors acknowledge the frontline healthcare providers and support staff at fort belvoir community hospital for their enduring and tireless efforts that were required to adapt to the coronavirus disease 2019 epidemic while continuing the provision of quality care.disclaimer. the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of fort belvoir community hospital, the defense health agency, department of defense, or us government.potential conflicts of interest. the authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. key: cord-353862-7xe3fvd5 authors: li, na; han, lefei; peng, min; lv, yuxia; ouyang, yin; liu, kui; yue, linli; li, qiannan; sun, guoqiang; chen, lin; yang, lin title: maternal and neonatal outcomes of pregnant women with covid-19 pneumonia: a case-control study date: 2020-03-30 journal: clin infect dis doi: 10.1093/cid/ciaa352 sha: doc_id: 353862 cord_uid: 7xe3fvd5 background: the ongoing epidemics of coronavirus disease 2019 (covid-19) have caused serious concerns about its potential adverse effects on pregnancy. there are limited data on maternal and neonatal outcomes of pregnant women with covid-19 pneumonia. methods: we conducted a case-control study to compare clinical characteristics, maternal and neonatal outcomes of pregnant women with and without covid-19 pneumonia. results: during january 24 to february 29, 2020, there were sixteen pregnant women with confirmed covid-19 pneumonia and eighteen suspected cases who were admitted to labor in the third trimester. two had vaginal delivery and the rest took cesarean section. few patients presented respiratory symptoms (fever and cough) on admission, but most had typical chest ct images of covid-19 pneumonia. compared to the controls, covid-19 pneumonia patients had lower counts of white blood cells (wbc), neutrophils, c-reactive protein (crp), and alanine aminotransferase (alt) on admission. increased levels of wbc, neutrophils, eosinophils, and crp were found in postpartum blood tests of pneumonia patients. there were three (18.8%) and three (16.7%) of the mothers with confirmed or suspected covid-19 pneumonia had preterm delivery due to maternal complications, which were significantly higher than the control group. none experienced respiratory failure during hospital stay. covid-19 infection was not found in the newborns and none developed severe neonatal complications. conclusion: severe maternal and neonatal complications were not observed in pregnant women with covid-19 pneumonia who had vaginal delivery or caesarean section. mild respiratory symptoms of pregnant women with covid-19 pneumonia highlight the need of effective screening on admission. in december 2019, an outbreak of the 2019 coronavirus disease (covid-19) associated pneumonia was reported in wuhan, a mega city with an 11 million population in central china, and soon spread to other cities in china and overseas [1] . the causative pathogen was identified as a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] . as of 25 march 2020, covid-19 has caused more than 420,000 confirmed cases and 18,887 deaths globally, including 81,852 confirmed cases and 3,287 deaths in china [2] . in response to this fast-spreading epidemic, the chinese government has locked down the epicenter wuhan city since 23 january 2020, and implemented a series of social distancing measures such as strict traffic restrictions, forbidden social gatherings, closure of residential communities [3] . the epidemiological data in china showed that most cases had mild symptoms, with the overall case fatality rate of 2.3%. although sars-cov-2 appears less virulent than two previous zoonotic coronaviruses sars-cov and mers-cov, it was far more efficient to transmit between close contacts [4] . particularly, this novel coronavirus has caused special concerns in pregnant women, because both sars-cov and mers-cov have been found to cause severe complications in pregnant women [5, 6] . several reports on suspicious vertical transmission of this virus have further increased such concerns [7] . although recent laboratory studies and clinical reports did not find strong evidence to support a vertical transmission route, the possibility still cannot be completely ruled out [8] [9] [10] . clinical and epidemiological features of covid-19 infection have been widely reported [11] [12] [13] [14] [15] . however, clinical reports on maternal and neonatal outcomes of pregnant women with sars-cov-2 infection remain sparse. an earlier study by chen et al reported nine pregnant women with covid-19 pneumonia, who took cesarean section in a tertiary hospital of wuhan [8] . these patients showed clinical symptoms similar to non-pregnant patients with covid-19 pneumonia. they also claimed that there was no evidence of vertical transmission. to date, none of previous studies have investigated the adverse effects of covid-19 infection on pregnancy, by comparing maternal and neonatal outcomes of pregnant women with covid-19 pneumonia to those without pneumonia. we retrospectively reviewed medical records of pregnant women who were admitted into the hubei provincial maternal and child health center, a tertiary hospital in wuhan with 1,900 hospital beds, during january 24 -february 29, 2020. we followed the clinical diagnosis criteria for covid-19 pneumonia in the new coronavirus pneumonia prevention and control program (5th edition) by the national health commission of china [16] . throat swabs were collected from all these patients and sent to the laboratory of the wuhan center for disease control and prevention for tests of sars-cov-2 using the standard kit (biogerm, shanghai, china). diagnosis criteria of covid-19 infection include 1) typical chest ct imaging of patchy shadowing and ground-glass opacity, and 2) positive in reverse transcription polymerase chain reaction (rt-pcr) tests for sars-cov-2. however, previous studies argued that false negative cases might be common for covid-19 infection cases due to low virus titers, sampling at late stage of illness, and inappropriate swabbing sites [8] . given overloaded healthcare systems and limited test capacities during our study period, we might have missed some covid-19 cases if solely relying on laboratory tests. therefore, in this study we also included the suspected patients with typical chest ct imaging but negative in rt-pcr tests. eleven pregnant women who were tested positive for sars-cov-2 were classified as laboratory confirmed case group, and eighteen with typical chest ct imaging but tested negative in rt-pcr tests as suspected case group. the control group of pregnant women without pneumonia during hospital stay were randomly selected from the medical records by an investigator (mp), who was not involved in statistical analysis. only those aged 25-35 years were selected to match the age range of cases. we selected 121 women who were admitted during the same period (control 2020 group). considering the potential adverse effects of mental stress caused by city lockdown and severe epidemics, we also included the second control group of 121 women admitted during january 24 -february 11, 2019 (control 2019 group). blood test results were also retrieved from medical records. two case groups underwent blood tests every three days but two control groups only took once on admission. clinical characteristics, laboratory test results, maternal and neonatal outcomes were collected from medical records and reviewed independently by two investigators (yxl and yo). fisher's exact tests and mann-whitney u tests were used to compare the group difference for categorical and continuous variables, respectively. friedman tests were used to test for the difference of blood test results across time within the same subjects. all data analysis was conducted using r version 3.6.2. demographic characteristics of two case groups and two control groups are shown in table 1 . the age of confirmed cases ranged 26-37 years and all were in the third trimester. two confirmed cases (12.5%) and one suspected case had chronic conditions of hypertension, polycystic ovary syndrome and hepatitis b. their gestational weeks on admission ranged from 33 weeks plus 6 days to 40 weeks plus 4 days. around 70% of two case groups had other maternal complications, significantly higher than the controls (31-33%). all these complications were developed before diagnosis of pneumonia. fourteen patients had caesarean section, because confirmed or suspected covid pneumonia has become one indication for caesarean section in our hospital since 24 january 2020. two patients had vaginal delivery because neither presented any respiratory symptoms when admitted for full-term labor. one of them had fever two days after childbirth and another had ct images of patchy shadowing in the right lung on the same day of labor. there were 22 patients who took emergency cesarean section (12 confirmed and 10 suspected cases) because of active labor at the time of admission, and eight had scheduled cesarean section (3 confirmed and 5 suspected cases). in addition to pneumonia, eleven out of 16 confirmed cases had gestational complications on admission, including gestational diabetes mellitus (n=3), premature rupture of membranes (1), gestational hypertension (3), hypothyroidism (2), preeclampsia (1) and sinus tachycardia (1) . only one of them had more than one complications (gestational diabetes mellitus and hypertension). among three confirmed cases with preterm delivery, two were caused by premature rupture of membranes, and one by placental bleeding. two suspected cases had preterm delivery due to gestational hypertension/preeclampsia, and one suspected case due to placenta previa. none of confirmed covid-19 patients reported an exposure history. four were admitted with fever for investigation and eight developed fever after childbirth ( table 2) . none presented other respiratory symptoms on admission nor during hospital stay. two of the patients with suspected covid-19 pneumonia reported cough, sore throat, dyspnea, diarrhea and vomiting. all patients took chest ct scans. seven of confirmed cases had typical image of pneumonia in both lungs and eight in single lung. seventeen out of eighteen suspected patients had either both lungs or single lung affected. compared to the controls, two case groups had slightly lower counts of white blood cells (wbc), neutrophils, c-reactive protein (crp) and alanine aminotransferase (alt) on admission, although none reached statistical significance and most were marginally beyond the normal range (table 3) . lymphocytes, eosinophils and aspartate aminotransferase (ast) were comparable between the cases and controls. an increase of wbc, neutrophils, and crp were observed in the first postpartum blood test of confirmed cases, followed by a decrease in the second postpartum test (figure 1 ). but this transient change was not found in suspected cases. lymphocytes remained at the lower end of normal range in two case groups. all covid-19 pneumonia patients received antibiotics and four patients received antivirals during hospital stay. all of them have been discharged or transferred to the designated hospitals for covid-19 patients, and the length of stay in our hospital ranged from 3 to 26 days, with a median of 6.5 days. none were admitted into the intensive care unit (icu) because of covid-19 pneumonia or severe maternal complications. sixteen patients with confirmed covid-19 pneumonia gave birth to seventeen babies (fifteen singletons and two twins). two singletons were born prematurely due to premature rupture of membranes and placental abruption. there were 23.5% and 21.1% premature birth among the newborns born to mothers with confirmed or suspected covid-19 pneumonia, significantly higher than those from the controls (5.8% and 5.0% in the 2020 and 2019 controls) ( table 4 ). low birth weight also occurred more often in infants of two case groups (17.6% and 10.5%) than in those of two control groups (2.5%). newborns from the cases and controls showed no significant differences in key neonatal indicators including gestational age at birth, apgar score at 5 minutes, and intrauterine fetal distress. of three newborns with intrauterine fetal distress, two were from the covid-19 confirmed mothers, one of whom also had sinus tachycardia. one case of fetal distress was from the mother who had suspected covid-19 pneumonia but no other comorbidity. no events of severe neonatal asphyxia and deaths occurred in these newborns. to reduce contact transmission, all covid-19 patients were immediately moved to isolation wards after delivery or cesarean section, and their newborns were taken care by other family members. three newborns (including two twins), who were delivered by caesarean section, took throat swabs at 4 and 14 days after birth. all of them tested negative for sars-cov-2. to our best knowledge, this is the first case-control study to comprehensively compare maternal and neonatal outcomes of pregnant women with covid-19 pneumonia, to those with non-covid-19 pneumonia and without pneumonia. we found that sars-cov-2 infection caused generally mild respiratory symptoms in pregnant women. clinical signs and symptoms mainly included fever and pneumonia, but other respiratory symptoms were less common. our results echo the findings of a previous study in pregnant women with sars in hong kong, reporting that fever was the dominant presenting symptom [17] . however, it is of note that most patients did not have any symptoms on admission. for the purpose of screening for suspected cases, we asked all pre-laboring pregnant women to take low-dose chest ct scans with their abdominal region covered, and found that 2.1% fulfilled the criteria of covid-19 pneumonia (patchy shadowing and ground glass opacity in single or both lungs). this highlights the need of enhancing screening for covid-19 pneumonia on admission, as well as strengthening infection control measures in obstetric wards during the epidemics. compared to other covid-19 pneumonia patients, pregnant women generally had no or mild respiratory symptoms. none of our patients developed severe respiratory complications to require critical care. laboratory investigations on admission found lower counts of wbc, neutrophils, crp, and alt in pregnant women, compared to the non-pneumonia controls. these findings are consistent with those reported in other hospitalized covid-19 patients who often had lymphopenia and decreased wbc [11] . slightly increased wbc, neutrophils, eosinophils and crp were found in postpartum blood tests. we also notice that confirmed and suspected cases shared similar dynamic profiles, suggesting that laboratory test results might not be very useful in making differential diagnosis. cesarean section in one tertiary hospital in wuhan [8] . in our study, in addition to fourteen cesarean section patients, we also reported two pregnant woman who had a full-term vaginal delivery and were confirmed with covid-19 pneumonia on the day of delivery. we observed a higher incidence rate of premature delivery in confirmed cases (18.8%), but none was due to severe maternal respiratory failure. this rate was higher than in suspected patients (16.7%) and in two control groups (~5%) of our study, but lower than the rate of 44% in confirmed covid-19 pneumonia patients reported by chen et al. [8] all these events of preterm delivery were triggered by gestational complications such as premature rupture of membranes and placental bleeding, which might not be directly related to covid-19 pneumonia. we did not observe any deaths or events of severe complications associated with covid-19 pneumonia that required critical care in the pregnant women and newborns. hence, the adverse effects of covid-19 pneumonia on pregnancy appear less severe than those of sars-cov and mers-cov. there were three pregnant women died during the 2003 sars outbreak in hong kong, and preterm delivery was as high as 80% [18] . although no maternal deaths were recorded in the mers-cov outbreak, more than half of their newborns required critical care and nearly 30% eventually died [19] . zhu et al reported ten newborns born to mothers with covid-19 pneumonia in wuhan, and there was one newborn who died from multiple organ failure and disseminated intravascular coagulation (dic), and another had dic but recovered. however, none of these ten newborns tested positive for sars-cov-2 [20] . a previous study also reported that sars-cov infection could increase the risk of preterm delivery in the second trimester and spontaneous abortion in the first trimester [17] . since all patients in our study and others were in the third trimester, the potential adverse effect of sars-cov-2 infection in the first and second trimesters remains to be investigated. in response to this unprecedented covid-19 outbreak in wuhan, confirmed and suspected covid-19 infection has been included as one indication for cesarean section in our hospital, because there was only one negative pressure operation room suitable for airborne precautions. two patients had vaginal delivery in positive pressure labor rooms before they were diagnosed with covid-19 pneumonia. no transmission events occurred in the doctors and midwives, who were wearing a full set of personal protective equipment (n95 respirators, protective gown, coveralls, gloves and goggles) during the delivery procedure. healthcare workers need to stay vigilant against covid-19 infection when there is an epidemic in neighborhood or pregnant women have a travel history to an epidemic area within 14 days. as suggested by favre et al, vaginal delivery could be considered for the benefit of patients, when there is a labor room properly equipped for airborne precautions [21] . all healthcare workers in close contacts should strictly adhere to contact and airborne precautions in addition to standard precautions. similar to two previous reports of nine and one pregnant women with confirmed covid-19 infection [8, 22] , we did not find any evidence to support the vertical transmission of sars-cov-2 from mother to fetus via placenta or during cesarean section. however, there was one newborn in wuhan who was born to the mother with covid-19 pneumonia under emergent cesarean section and tested positive for sars-cov-2 at 36 hours after birth [23] . none of these studies have detected the virus in breast milk, cord blood or placenta. therefore, there is limited evidence of vertical transmission via placenta or during cesarean section. our study also added some evidence to suggest that the risk of vertical transmission during vaginal delivery might also be trivial. there were two patients with vaginal delivery, one of them had symptom onset two days after delivery and another had delivery during the course of illness. neither of their newborns had respiratory systems after birth. unfortunately, none of them gave us the consent to collect the respiratory specimens of their neonates. given the small sample size of our study, the possibility of vertical transmission during vaginal delivery still cannot be ruled out. there are a few caveats in our study. first, this is a retrospective case control study from one single center, which could be subject to recall bias and selection bias. second, we collected the data of sixteen pregnant women with laboratory confirmed covid-19 pneumonia and eighteen suspected cases with typical ct imaging. although this is the largest number of pregnant women with covid-19 pneumonia in literature so far, the sample size is still relatively small. nevertheless, given the ongoing global pandemic caused by sars-cov-2, we believe our study could be one of important clinical studies to guide clinical diagnosis and treatment to this vulnerable group. in this study, we did not find any evidence to suggest that covid-19 pneumonia causes lyang and mp originated and designed the study. ylv, kl lyue, ql and yo contributed to data collection and clean. lh conducted data analysis. nl, lh, gs, lc and lyang interpreted the findings and drafted the manuscript. all the authors proved the final version of this manuscript. all authors have completed the icmje uniform disclosure form at www.icmje.org/coi_disclosure.pdf . ly is supported by the alibaba (china) -hong kong polytechnic university collaborative research fund. other authors declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. the ethical approval has been obtained from the ethics committee of the hubei provincial maternal and child health center. apgar score at 5 minutes after birth, mean (sd) intrauterine fetal distress (n, %) 2 (11.7%) 1 (5.3%) 0.593 6 (5.0%) 0.256 6 (5.0%) 0.256 a p value of fisher's exact tests and mann-whitney u tests, the laboratory confirmed cases as reference group. b babies who were born weighing less than 2,500 grams. c babies who were born before the start of the 37th week of pregnancy of mothers. a novel coronavirus from patients with pneumonia in china coronaviruses: genome structure, replication, and pathogenesis the novel coronavirus originating in wuhan, china: 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with 2019 novel coronavirus-infected pneumonia in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of coronavirus disease 2019 in china new coronavirus pneumonia prevention and control program a case-controlled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome severe acute respiratory syndrome and pregnancy middle east respiratory syndrome coronavirus during pregnancy: report of two cases & review of the literature clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia guidelines for pregnant women with suspected sars-cov-2 infection. the lancet infectious diseases a case of 2019 novel coronavirus in a pregnant woman with preterm delivery a case report of neonatal covid-19 infection in china all data and materials used in this work are available based on reasonable request to the corresponding author. key: cord-330742-m5xx8861 authors: qian, jie; zhao, lin; ye, run-ze; li, xiu-jun; liu, yuan-li title: age-dependent gender differences of covid-19 in mainland china: comparative study date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa683 sha: doc_id: 330742 cord_uid: m5xx8861 background: the ongoing pandemic of novel coronavirus disease 2019 (covid-19) is challenging global public health system. sex-differences in infectious diseases are a common but neglected problem. methods: we used the national surveillance database of covid-19 in mainland china to compared gender differences in attack rate (ar), proportion of severe and critical cases (pscc) and case fatality rate (cfr) in relation to age, affected province, and onset-to-diagnosis interval. results: the overall ar was significantly higher in female population than in males (63.9 versus 60.5 per million persons; p ˂ .001). by contrast, pscc and cfr were significantly lower among female patients (16.9% and 4.0%) than among males (19.5% and 7.2%), with ors of 0.87 and 0.57, respectively (both p ˂ .001). the female-to-male differences were age-dependent, which were significant among people aged 50–69 years for ar, and in the patients of 30-years or older for both pscc and cfr (all p ≤ .001). the ar, pscc and cfr varied greatly from province to province. however, female-to-male differences in ar, pscc and cfr were significant in the epicenter, hubei province, where 82.2% confirmed cases and 97.4% deaths occurred. after adjusting for age, affected province and onset-to-diagnosis interval, the female-to-male difference in ar, pscc and cfr remained significant in multivariate logistic regression analyses. conclusions: we elucidate an age-dependent gender dimorphism for covid-19, in which the females have higher susceptibility but lower severity and fatality. further epidemiological and biological investigations are required to better understand the sex-specific differences for effective interventions. the coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first reported in wuhan, china in december 2019 [1] , and is leading to a global health crisis. considering the gender differences in infectious diseases of humans are a common but neglected global health problem [2] , there is a greatly need to investigate the specific question as regard to covid-19. global health 50/50, an independent health equity research organization based at university college london, compiled sex-disaggregated infection and mortality data available from tens of affected countries, and implied that the male patients were more likely to die than the female patients. however, data have so far provided no clear pattern in terms of who is more likely to become infected with sars-cov-2 [3] . furthermore, no data on severity of the disease are available for them to do the comparative analyses. in mainland china, some reports have mentioned differences in fatality between male and female patients using data only from early reported cases or hospital settings [4] [5] [6] . in this study, we used the surveillance data containing all confirmed cases in mainland china as of april 28, 2020 to evaluated gender-specific differences in attack rate, proportion of severe and critical cases, and case fatality in relation to age, affected province and onset-to-diagnosis interval, in order to provide evidence-based guidance for more effective and equitable interventions and treatments. a c c e p t e d m a n u s c r i p t 5 according to the diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7), which was updated by national health commission & state administration of traditional chinese medicine on march 3, 2020 (supplemental material) [7] , confirmed cases were patients who had related epidemiological history and clinical manifestations, with one of the following etiological or serological evidences: sars-cov-2 nucleic acid detected by specific real-time rt-pcr assay, viral gene sequence homologous to sars-cov-2, specific igm and/or igg are detectable in serum, or a 4-fold increase in igg titer in convalescent serum compared with the acute phase. among the confirmed cases, if patients had mild symptoms but no sign of pneumonia on imaging, they were defined as mild cases. if patients presented fever and respiratory symptoms with radiological findings of pneumonia, they were defined as moderate cases. if adult patients met any of the following criteria, i.e. respiratory distress (≧30 breaths per minute, bpm ), oxygen saturation ≤ 93% at rest, or arterial partial pressure of oxygen (pao 2 ) / fraction of inspired oxygen (fio 2 ) ≦300 mm hg (l mm hg = 0.133 kpa), they were defined as severe cases. the criteria for severe child cases were as following: respiratory distress (≥ 60 bpm for infants aged below 2 months, ≥ 50 bpm for infants aged 2-12 months, ≥ 40 bpm for children aged 1-5 years, and rr ≥ 30 bpm for children above 5 years old), oxygen saturation ≤ 92% at rest, having labored breathing, cyanosis or intermittent apnea, showing lethargy and convulsion, or having difficulty for feeding and signs of dehydration. critical cases were defined if they had respiratory failure requiring mechanical ventilation, shock, or other organ failure that requires cares in the intensive care unit. we collected data of all confirmed covid-19 cases reported to the china information system for diseases control and prevention (cisdcp), official reports by the national, a c c e p t e d m a n u s c r i p t 6 provincial, and municipal health commissions as of april 28, 2020. the surveillance data included the information on age, sex, occupation, residence location, date of illness onset, date of diagnosis, and disease classification. according to the regulations issued by the central government of mainland china, all the confirmed patients should be admitted to either general hospitals or temporary cabin hospitals until recovery from covid-19 or death. the disease classification was duly updated according to the change in clinical manifestations of each case. as this study constituted public health surveillance rather than research in human beings, ethical approval from institutional review boards was not required. all the information regarding individual persons had been anonymized. we to estimate the differences between groups, the student's t test for a continuous variable, and the chi-square test or a fisher's exact test for a categorical variable were used where appropriate. the administrative divisions including provinces, autonomous regions, and municipalities of china were all referred to as provinces in the paper for simplicity. we evaluated the association between gender and ar in each age group and affected province, and then estimated risk ratio (rr) and its 95% ci by woolf method. we compared the a c c e p t e d m a n u s c r i p t 7 psccs and cfrs between female and male patients in each age group and affected province, and then estimated odds ratio (or) and its 95% ci by maximum likelihood method. the gender difference in either pscc or cfr was validated by the multivariate logistic regression analysis using spss software (version 18.0) by including gender as an independent variable and age group, affected province, and onset-to-diagnosis interval as co-variables. a two-sided p value less than 0.05 was considered to be significant. as of april 28,2020, a total of 82,858 confirmed case were reported, of which 41,580 (50.2%) were female ( table 1 ). the median age of the patients was 51 years (iqr 39-63), with a mean (±sd) of 51.0 ± 16.7 years. there was no significant difference in age distribution between female and male patients. the number of health care workers (hcws) was 3402, accounting for 4.1% (95% ci, 4.0-4.2%) of total cases. the female cases (1956) among hcws outnumbered males (1446) (female, 57.5%; male, 42.5%; p < .001). the mean (±sd) time from illness onset to diagnosis was 9.5 ± 7.4 days. the onset-to-diagnosis interval was significantly longer among female cases than among male cases. the overall ar was 62.2 per million persons (95% ci, 61.7-62.6), which was significantly higher in female population than in males (63.9 versus 60.5 per million persons; p ˂ .001). among the confirmed cases, 12,366 (14.9%) were severe and 2696 (3.3%) were critical, with an overall pscc of 18.2%. the pscc was significantly lower in females (7017 / 41580, 16.9%) than in males (8045 / 41278, 19.5%) with a female-to-male or was 0.87 (95% ci, 0.84-0.89; p < .001). the overall cfr was 5.6% (4633 / 82858), which was significantly lower among female patients (1681/41580, 4.0%) than among male patients (2952 / 41278, 7.2%), with an or of 0.57 (95%ci 0.53-0.60; p < .001). the pscc (15.4%) and cfr (2.1%) among a c c e p t e d m a n u s c r i p t 8 verhcws were significantly lower than other cases (18.3% and 5.7%; p < .001). similarly, pscc and cfr were significantly lower in female (13.8% and 1.1%) than males hcws (19.3% and 4.4%) ( both p < .001). the overall ar of covid-19 was significantly increased with age (χ² for trend test, p < .001), with people over 60 years having a 9.9 times higher than those under 30 years of age table 2 ). the older were the patients, the more severe were their illness. the pscc was continuously increased with age (χ² for trend test, p < .001). the psccs were lower in female than male cases in all age groups except 20-29 years. the female-to-male ors were significantly in the age groups older than 30 years (all p < .001) (figure 1b, supplemental table 3 ). the cfr sharply grew with age (χ² for trend test, p < .001). similar with psccs, cfrs were lower among female patients in all age groups, and the gender differences in cfr were significant in the patients 30-years or older (all p < .001) ( figure 1c , supplemental table 4 ). table 5 ). the pscc was lower among female than among male cases in all provinces except shandong province. the female-to-male differences in pscc were statistically significant in hubei, zhejiang, guangdong, hunan and jiangxi provinces ( figure 2b , supplementary table 6 ). 97.4% of all dead cases occurred in hubei province, where significant female-to-male difference in cfr was observed, with an or of 0.55 (95% ci 0.52-0.58; p < .001) ( figure 2c , supplementary table 7 ). the cfr of female patients was comparable to that of male patients in rest of china (or = 1.2; 95% ci 0.8-1.6; p = .37). we conducted a multivariate logistic regression to validate the gender-differences in pscc and cfr by adjusting for age, affected province, and onset-to-diagnosis interval, which were significant in the univariate analyses. considering both pscc and cfr were increased with age group and onset-to-diagnosis interval, we included them as continuous co-variables, while affected province as categorical co-variables in the models. we revealed that female-tomale or for pscc remained significant after adjusting for those possible confounding variables, with an adjusted or of 0.80 (95% ci 0.77-0.83). the older age and longer onsetto-diagnosis interval was also identified as risk predictor for severity of illness (both p < .001) ( table 2) . similarly, the adjusted female-to-male or of cfr was 0.53 with a 95% ci of 0.49-0.57 (p < .001). in the finale model for cfr, the or for age was 1.10 (95% ci 1.10-a c c e p t e d m a n u s c r i p t 10 1.11) with each 10-year increase (p < .001), and or for onset-to-diagnosis interval was 1.01 (95% ci 1.01-1.02) for each day longer (p < .001) ( table 2) . as the world responds to the unprecedented pandemic of covid-19, it is critical to recognize the populations at high-risk for sars-cov-2 infection and disease severity for creating effective surveillance and target interventions. because gender is a determinant of health [8] , understanding the extent to which outbreaks affect women and men differently is a fundamental step to evaluating the primary and secondary effects of a health emergency on individuals and communities [9] . we did comparative analyses using the national surveillance data containing all confirmed covid-19 cases of mainland china. the agedependent gender differences in incidence, severity and fatality of covid-19 imply that more intensive public health surveillance and preventions should focus on women older than 50 years especially in the epicenter to control the transmission more efficiently. on the other hand, more attentions should be provided to male patients especially those over 30 years of age for enhanced clinical managements. furthermore, our findings on gender differences have also provided evidences for addressing the health needs of men and women equally, so as to help policy maker and societies prevent future human tragedies [10, 11] . at first glance, covid-19 seems to occur equally among women and men. because there are more men than women in the general population of mainland china, we look at the ar by taking sex constitution into consideration. as a result, the female tendency is significant especially in hubei province, where 82.2% cases occurred. the gender difference in ar in mainland china is age-dependent, with the peak in individuals aged 50-69 years. this is disparate from that in the republic of korea, where the highest rate is among people aged 20-39 years, with a much greater female-to-male ration of nearly 2:1 [12] . these m a n u s c r i p t 11 findings in the two early affected countries imply that women are in general more likely infected by sars-cov-2, especially in some specific age groups. the infection of sars-cov-2 is primarily through angiotensin converting enzyme 2 (ace2) receptor, which serves as a gateway for the virus's entry into tissues [13] . the ace2 gene is located on the x chromosome, therefore female individuals should have higher ace2 levels [14] , which might be the reason for more susceptible to sars-cov-2 infection in comparison to males. further investigations into ace2 enzyme activity in correlation with sex is required to verify the hypothesis. in addition to the biological factors, age-related social and behavioral factors might have contributed to the age-dependent gender difference in covid-19 morbidity. lack of adherence to social distancing and self-quarantine recommendations initiated by korean health authorities is supposed to be the risk factor for the higher infection rates among the young adults and teenagers as well as the shincheonji religious community [12] . lockdown of city and closure of schools to control covid-19 transmission in china might have increased risk of sars-cov-2 infection in women, who have provided cares in families and communities. as seen in outbreak of ebola virus disease in west african during 2014 to 2016, women were more likely to be infected, given their predominant roles as caregivers within families and as front-line health-care workers [15] . the higher covid-19 morbidity in female than male population might also come from more likely seeing a doctor after symptom onset. an example is that the incidence rate of zika virus disease for persons seeking care was higher among women than among men during the 2007 outbreak in micronesia [16] . systematically investigations are required to understand whether observed age-dependent gender difference in ar is due to differences in infection rates, development of disease, seeking medical care, or reporting bias. a c c e p t e d m a n u s c r i p t 12 our analyses revealed that both pscc and cfr were lower among female patients than among male patients in nearly all provinces and all age groups. these findings are consist with the results of previous reports based on hospital data in china and in other affected countries [5, 12, 17, 18] . the reasons for gender difference in the severity and fatality of covid-19 might be attributed to underlying comorbidity and higher risk behaviors such as smoking [3, 4, 19] . the higher female proportion of hcws might have some contribution to the gender difference in pscc and cfr, because hcws tend to have less severe illness as observed in our study and in the united states [20] . female individuals generally have stronger innate and adaptive immune responses than males, because the x-chromosome contains more copy numbers of immune-related genes [21] , which might lead to more prompt clearance of sars-cov-2 in women, and subsequently decrease the severity and fatality of the disease. in addition, sex-dependent production of steroid hormones may contribute to gender specific disease outcomes after virus infections [22, 23] . a recent observation that the female patients have higher level of igg antibody against saes-cov-2 compared with male patients [24] , provides direct evidence for sex differences in immune responses. further investigations on the association between stronger immune response and less severity in female are warranted. sex-differences in ace2 might also play a role in pathogenesis, because ace2 can protect against lung damage through its anti-inflammatory function [13] . therefore, the higher ace2 levels among women are supposed to protect them from more severe disease [14] . the study had some limitations. first, we used the database of cisdcp, in which the individual characteristics relevant to gender, such as socioeconomic status, comorbidity, and immunological condition, were not recorded. lack of such information has prohibited us from further investigating their possible impacts on gender differences. second, we did comparative analyses using the surveillance data, which did not include the information on a c c e p t e d m a n u s c r i p t 13 the clinical managements. unfortunately, we could not compare any treatments given that may confound the results regarding pscc and cfr between female and male patients. in fact, treatments in different hospitals and areas varied, and even in the same hospital the treatments might be different among female and male patients. third, missed diagnosis is avoidable due to lack of health facilities and /or laboratory capacity in the early stage of the outbreak. this situation certainly have led to under-estimates of covid-19 burdens, and might cause bias in some specific groups. in conclusion, this report raises awareness about the age-based gender differences in incidence, severity and fatality of covid-19. interestingly, the females might be more prone to get the disease, but less likely for a poor or fatal outcome. the age-dependent gender dimorphism in covid-19 might contribute to various factors, and deserves further investigations on immune responses and other biological mechanisms for sex differences. policies and public health efforts have rarely addressed the gendered impacts of disease outbreaks [25] . our gender analyses using the data from the first outbreak country have not only got insight into the gender differences, but also provided evidences for target treatment, jq, xjl and yll designed the study, performed the main data analysis, and wrote the paper. jq, lz, rzy and xjl managed the data and did the statistical analysis. this work is supported by the natural science foundation of china (81673238) and peking union medical college education fund. we acknowledge the china cdc for their valuable assistance in coordinating data collection. union medical college education fund. we declare that we have no conflicts of interest. clinical features of patients infected with 2019 novel coronavirus in wuhan, china sex differences in infectious diseases-common but neglected epidemic update and risk assessment of 2019 novel coronavirus -china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series diagnosis and treatment protocol for novel coronavirus pneumonia addressing sex and gender in epidemic-prone infectious diseases on behalf of the gender and covid-19 working group. covid-19: the gendered impacts of the outbreak better science with sex and gender: facilitating the use of a sex and gender-based analysis in health research the gendered dimensions of covid-19 disparities in age-specific morbidity and mortality from sars-cov-2 in china and the republic of korea soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy? sex differences in angiotensin-converting enzyme modulation of ang (1-7) levels in normotensive wky rats a gendered human rights analysis of ebola and zika: locating gender in global health emergencies zika virus outbreak on yap island, federated states of micronesia gender differences in patients with covid-19: focus on severity and mortality clinical characteristics of coronavirus disease 2019 in china sex difference and smoking predisposition in patients with covid-19 characteristics of health care personnel with covid-19 -united states the x chromosome and sex-specific effects in infectious disease susceptibility sex differences in susceptibility to viral infection igg antibody between male and female covid-19 patients: a possible reason underlying different outcome between sex overcoming the "tyranny of the urgent": integrating gender into disease outbreak preparedness and response pscc (%) (95% ci) cfr (%) (95% ci) abbreviations: iqr, interquartile range; sd, standard deviation ar, attack rate; pscc, proportion of severe and critical cases; cfr, case fatality 3 rate a c c e p t e d m a n u s c r i p t key: cord-354943-wxhbwcfr authors: guo, li; ren, lili; yang, siyuan; xiao, meng; chang, de; yang, fan; dela cruz, charles s; wang, yingying; wu, chao; xiao, yan; zhang, lulu; han, lianlian; dang, shengyuan; xu, yan; yang, qi-wen; xu, sheng-yong; zhu, hua-dong; xu, ying-chun; jin, qi; sharma, lokesh; wang, linghang; wang, jianwei title: profiling early humoral response to diagnose novel coronavirus disease (covid-19) date: 2020-03-21 journal: clin infect dis doi: 10.1093/cid/ciaa310 sha: doc_id: 354943 cord_uid: wxhbwcfr background: the emergence of coronavirus disease 2019 (covid-19) is a major healthcare threat. the current method of detection involves a quantitative polymerase chain reaction (qpcr)–based technique, which identifies the viral nucleic acids when present in sufficient quantity. false-negative results can be achieved and failure to quarantine the infected patient would be a major setback in containing the viral transmission. we aim to describe the time kinetics of various antibodies produced against the 2019 novel coronavirus (sars-cov-2) and evaluate the potential of antibody testing to diagnose covid-19. methods: the host humoral response against sars-cov-2, including iga, igm, and igg response, was examined by using an elisa-based assay on the recombinant viral nucleocapsid protein. 208 plasma samples were collected from 82 confirmed and 58 probable cases (qpcr negative but with typical manifestation). the diagnostic value of igm was evaluated in this cohort. results: the median duration of igm and iga antibody detection was 5 (iqr, 3–6) days, while igg was detected 14 (iqr, 10–18) days after symptom onset, with a positive rate of 85.4%, 92.7%, and 77.9%, respectively. in confirmed and probable cases, the positive rates of igm antibodies were 75.6% and 93.1%, respectively. the detection efficiency by igm elisa is higher than that of qpcr after 5.5 days of symptom onset. the positive detection rate is significantly increased (98.6%) when combining igm elisa assay with pcr for each patient compared with a single qpcr test (51.9%). conclusions: the humoral response to sars-cov-2 can aid in the diagnosis of covid-19, including subclinical cases. a novel coronavirus severe acute respiratory syndrome coronavirus 2 (sars-cov-2) causing coronavirus disease in 2019 (covid-19) emerged as a major pandemic [1, 2] . with more than 110 000 confirmed cases and over 4000 deaths as of 11 march 2020, this pandemic surpassed the severe acute respiratory syndrome coronavirus (sars-cov) of 2003 [3] . coronaviruses are frequent causes of respiratory infections in which 6 major species are known to cause human infections besides the sars-cov-2. these species include the highly pathogenic sars-cov and middle east respiratory syndrome coronavirus (mers-cov), along with less virulent species that include nl63, 229e, oc43, and hku1 [4] . the epidemiology, etiology, and clinical characteristics of covid-19 have recently been described in detail [5] [6] [7] [8] [9] [10] [11] . the current methods of diagnosis of covid-19 includes detection of the virus by genomic techniques using either polymerase chain reaction (pcr)-based methods or deep sequencing [7, 12, 13] . however, these detection methods heavily rely on the presence of the viral genome in sufficient amounts at the site of sample collection that can be amplified. missing the time window of viral replication can provide false-negative results. similarly, an incorrect sample collection can limit the usefulness of the quantitative pcr (qpcr)-based assay. a false-negative diagnosis can have grave consequences, especially at this stage of the pandemic, by allowing infected patients to spread the infection and hampering the efforts to contain the spread of the virus [14] . in such conditions, additional screening methods that can detect the presence of infection despite lower viral titers can be highly beneficial to ensure timely diagnosis of all infected patients. detection of the production of antibodies, especially immunoglobulin (ig) m, which are produced rapidly after the infection, can be a tool that can be combined with pcr to enhance detection sensitivity and accuracy. however, currently, the extent and the time kinetics of the humoral response against sars-cov-2 are not known. in this study, we demonstrate the time kinetics of the antibody response to sars-cov-2 in infected patients. we further demonstrate that combining the antibody testing with qpcr can significantly improve the diagnosis of covid-19. the full-length nucleocapsid (n) genes of sars-cov-2 were amplified from a bronchoalveolar lavage fluid (balf) specimen of a patient infected with sars-cov-2 and cloned into prokaryotic expression vector pet30a (+) (novagen, san diego, ca, usa). the resultant plasmids were transformed into escherichia coli bl21 (de3) to express the recombinant n proteins (rnps) according to the manufacturer's protocol. the 6x histidine-tagged proteins were then purified by using hitrap sp ff and histrap hp columns (ge healthcare, waukesha, wi, usa) to greater than 90% purity. the identity of the purified protein was confirmed by western blot analysis using an anti-6x histidine monoclonal antibody (sigma, st louis, mo, usa). the rnps of human cov-229e, -nl63, -oc43, -hku1, sars-cov, and mers-cov were expressed and purified as described previously [15] . in this study, a total of 208 blood samples were collected from 2 cohorts. in the first cohort, we recruited a total of 101 inpatients from wuhan hospitals during the early phase of the pandemic in january 2020. paired throat swab and blood samples were taken from each patient. among them, 43 (20 severe and 23 mild to moderate) were confirmed virus positive (referred as "confirmed cases" [ccs]) by deep sequencing or a qpcr assay with a detection limit of 1 copy/μl, as previously reported [1, 2] , while 58 cases (5 severe and 53 mild to moderate) suspected to be infected with sars-cov-2 based on clinical manifestation, chest radiography imaging, and epidemiology but no virus were detected by deep sequencing or a qpcr assay (referred as "probable cases" [pcs]). a total of 69 blood samples were taken from the 43 ccs (2 serial samples from 26 patients with a 4-day interval and 1 sample from the remaining 17 patients), while 100 plasma samples were collected from 58 pcs (2 blood samples from 42 pcs and 1 single blood sample from 16 pcs). the second cohort included a total of 39 hospitalized ccs recruited from beijing hospitals (8 severe and 31 mild to moderate cases), with 1 blood sample provided from each patient. all of the blood samples were taken between 1 and 39 days of the disease onset. in addition, a family cluster including 6 individuals over 3 generations was enrolled to validate our detection method. another 135 plasma samples collected in 2018 from adult patients with acute lower respiratory tract infections (alrtis) and 150 plasma samples obtained from healthy adults in 2018-2019 during regular health check-ups in wuhan city were used as controls. the plasma samples positive for human cov-229e, -nl63, -oc43, -hku1, and sars-cov were obtained as previously reported [15] . purified rnps of human cov-229e, -nl63, -oc43, -hku1, and sars-cov were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) gels and transferred to a nitrocellulose membrane (pall, port washington, ny, usa). human plasma samples positive for these coronaviruses were applied to detect cross-reactivity between these n proteins and antibodies. goat antihuman irdye fluor 800-labeled igg secondary antibody was used at a dilution of 10 000 (li-cor, lincoln, ne, usa). the membranes were scanned by using the odyssey infrared imaging system (li-cor). we developed an indirect enzyme-linked immunosorbent assay (elisa) protocol for detecting igm, iga, and igg antibodies against sars-cov-2 using purified rnps as coating antigens. the elisa protocol was developed as previously reported [16] . the concentration of the coated rnps and plasma dilutions for this elisa were optimized using chessboard titration tests. to determine the cutoff values for the elisas, we determined the mean values and sds of plasma from healthy individuals. the optimal coating concentration of antigen and optimal plasma dilutions were 0.1 μg/ml and 1:200, respectively. the cutoff values were determined by calculating the mean absorbance at 450 nm (a450) of the negative sera plus 3-fold the sd values, which were 0.13, 0.1, and 0.30 for igm, iga, and igg, respectively. the viral nucleocapsid gene sequences were aligned using the clustalw program using mega software (version 7.0.14). phylogenetic trees were constructed by means of neighborjoining methods with the use of mega software (version 7). the viral full-genome sequences were deposited in gisaisd (no. epi_isl_402123) and the genome warehouse in the national genomics data center, beijing institute of genomics (big), chinese academy of sciences (accessible at https://bigd. big.ac.cn/gwh). this study was approved by the ethical review board of institute of pathogen biology, chinese academy of medical sciences and peking union medical college. written informed consent was waived in the light of this emerging infectious disease of high clinical relevance. for healthy volunteers and patients suffering from common respiratory infections, written informed consent was obtained from each person before enrollment. plasma antibody titers are expressed as geometric mean titers (gmts) and compared using the student's t test by using the spss software program version 19.0. we used a nonlinear regression (third-order polynomial) model to fit the positive rate of andibodies. p < .05 was considered to be statistically significant. to evaluate the potential cross-reactivities of n proteins between sars-cov-2 and other human coronaviruses, we examined the reactivities of the sars-cov-2 rnp and human plasma with positive antibodies against nl63, 229e, oc43, hku1, and sars-cov, respectively, by using western blot and elisa assays. western blot analysis showed that there was no cross-reactivity of sars-cov-2 rnp with human plasma positive for igg antibodies against nl63, 229e, oc43, and hku1. however, a strong cross-reactivity was observed between sars-cov-positive human plasma and sars-cov-2 rnp, and specific bands of 45 kd in size were observed ( figure 1a) . the cross-reactivities were also confirmed by using elisas where sars-cov-positive human plasma presented high a450 with sars-cov-2 rnp ( figure 1b) . these results indicate cross-reactivity of antibodies to n proteins between the sars-cov and sars-cov-2. pairwise analysis showed the sars-cov-2 nucleocapsid gene had 46.1%, 27.6%, 26.5%, 20.0%, and 19.1%, amino acid (aa) sequence homology with mers-cov, hku1, oc43, nl63, and 229e, respectively. interestingly, 90.5% aa sequence homology was observed with sars-cov, which may explain the cross-reactivity between the 2 viruses ( figure 2 ). first, we examined the specificity of the elisa by using 135 plasma samples from patients with alrtis and 150 healthy individuals whose samples were collected in 2018-2019 in wuhan city. no anti-sars-cov-2 igm, iga, and igg antibodies were detected in these samples (data not shown). the antibody levels were then evaluated in the plasma samples of ccs and pcs. the appearance of igm, iga, and igg antibodies against sars-cov-2 was positive as early as day 1 after the symptom onset ( figure 3a) . the endpoint titration for antibodies against sars-cov-2 rnp was determined by calculating the a450 of a 2-fold serial dilution of each plasma sample for igm, iga, and igg ( figure 3b ). compared with the titers at days 0-7 (gmt, 400), the igm antibody levels increased between days 8 and 14 (gmt, 535.8; p = .000; student's t test) but did not increase further between days 15 and 21 (gmt, 536.31; p = .992) or after day 21 (gmt, 565.69; p = .719). iga antibody levels increased from days 0-7 (gmt, 400) to days 8-14 (gmt, 597.24; p = .000); however, there was no increase after this time point (gmt at day 15-21 = 723.28, p = .156; or gmt at day >21 = 831.41, p = .538). the igg antibodies were detected on days 0-7 (gmt, 490.45), increased on days 8-14 (gmt, 1325.6; p = .000), continued to increase until days 15-21 (gmt, 2690.87; p = .000), and reached a plateau by day 21 (gmt, 2974.83; p = .72). the igm-positive rate to sars-cov-2 was 75.6% (62/82) in ccs and 93.1% (54/58) in pcs ( figure 4a ). further, we fitted curves of the pcr-positive rate and igg and igm elisa positive rates on different days after symptom onset ( figure 4b ). the detection rates between days 26 and 39 pso are not shown due to the limited number of patients on each day. based on the fitted curve, the pcr-positive rate was more than 90% on 1-3 days pso, then declined to less than 80% (95% confidence interval [ci], 57.1-95.7%) at day 6 and less than 50% (95% ci, 23.7-59.5%) after 14 days pso. overall, the detection rate by qpcr was higher than the igm elisa before 5.5 days pso, while the detection efficiency by igm elisa was higher than that of the pcr method after 5.5 days of symptom onset. overall, the positive detection rate was only 51.9% in a single pcr test, but significantly increased (98.6%) when an igm elisa assay was applied to pcr-negative patients ( figure 4c ). to further evaluate the performance of anti-sars-cov-2 igm in covid-19 diagnosis, we analyzed the data from the 26 ccs who had 2 serial plasma samples together with paired throat swabs within a 4-day interval. among them, 7 patients were pcr negative when testing the first swab, but 6 patients were igm positive. all 7 patients were pcr positive for the second swab. these results suggest that igm elisa can increase the positive detection rate when combined with the pcr method and can be used for the early diagnosis of covid-19 infections. to prospectively evaluate the validity of igm elisa in the diagnosis of covid-19, we used the igm elisa to diagnose a family cluster involving 2 patients and 4 close contacts ( table 1 ). the grandparents returned to beijing from wuhan on 22 january 2020. the grandmother started having symptoms including fever (38.0℃) and dry cough, and chest computed tomography (ct) showed a bilateral patchy shadow on 2 february. the grandfather experienced a stuffy and runny nose on 3 february. the other 4 members in the family had no significant clinical symptoms. the pcr assay and chest ct of their close contacts, including their daughter, son-in-law, grandson, and granddaughter, were performed on 5 february. the grandmother was found to be positive on viral rna detection by qpcr. the grandfather and daughter only presented abnormal findings on chest ct but were negative for viral rna when tested by pcr. the grandson presented with pcr positivity with a normal chest ct. the son-in-law and granddaughter were pcr negative and showed normal lungs by ct imaging. the grandfather, daughter, son-in-low, and granddaughter were also negative for sars-cov-2 after the repeat pcr on the next day. however, the igm antibodies were detected in the grandmother, grandfather, daughter, grandson, and granddaughter. these findings indicate the significant and improved efficacy of covid-19 detection when igm measurements are used to assess subclinical subjects. covid-19 has emerged as a major healthcare challenge globally [13, 14, 16, 17] . a key aspect of limiting this virus spread is to ensure early and accurate diagnosis of the viral infection and appropriate quarantine for those infected. the current methods of diagnosis by qpcr or deep sequencing-based technologies rely on the presence of replicating virus in sufficient amounts to ensure that sufficient quantities of virus are collected [7] . this method often fails to detect the viral infection if the collection procedure is not optimal, or if the patient has low viral load due to an early stage of the disease or suppression by host immunity, or if the samples were obtained at a late stage in the course of infection. in this study, we first demonstrated that current sars-cov-2-positive plasma did not show any cross-reactivity with other coronaviruses, with the exception of sars-cov. it is highly unlikely that these patients were pre-infected with sars-cov during the last epidemic in 2002, as we measured igm reactivity, which is unlikely to last that long. second, the number of infections with sars-cov was limited to 8096 worldwide [18] , which only represents a tiny fraction of the chinese population. however, given the sequence homology between these 2 viruses (>90%), the cross-reactivity is not surprising. we propose conducting antibody testing when a qpcr test is negative despite other indications of covid-19, including symptoms and epidemiology. our data show that the supplementary igm test can provide better sensitivity than a qpcrbased method alone. this is especially important at this stage of the pandemic, where proper diagnosis is essential to limit the virus spread. although a more sensitive qpcr may improve detection efficacy, the efficacy may still be limited due to various biological reasons as discussed above. however, the more highly sensitive qpcr assays can further improve the detection method when combined with igm elisas. we primarily used a cross-sectional sample of specimens to determine the kinetics of sars-cov-2 antibodies. this is one of the limitations of our study because each patient has different kinetics for the development of antibodies. the median time of appearance of antibodies is therefore affected by factors such as when the specimen was collected and when the symptom onset took place in each individual patient. further studies are warranted that use longitudinal sample collection in an unbiased manner to test the kinetics of the antibody response. it is worth noting that approximately 22.0% (18/82) of the patients who were confirmed to be positive by qpcr were found to be negative by the igm antibody tests. among the 18 patients, 13 cases were enrolled within less than 7 days after symptom onset, which is probably when the antibodies were not produced in sufficient amounts. two patients were severe cases and their samples were collected at 19 and 22 days after symptom onset, respectively, suggesting a possible failure of these patients to generate the antibody response, which may have contributed to the disease severity. the remaining 3 patients provided samples between days 9 and 17 after symptom onset. these data suggest that the time course as well as host factors can contribute toward the antibody response to the virus. to confirm the applicability of our method, we prospectively tested a family cluster, with 2 members who were confirmed to be infected by sars-cov-2 while the remaining members tested negative using pcr. in contrast, the antibody testing confirmed the viral infections in the 2 patients and in 3 of the 4 close contacts, which were clinically corroborated in 3 of the family members. moreover, after a variable period of time, one expects the pcr result to become negative as people stop shedding viruses. presumably, igg antibodies will remain positive. this would permit the use of serological tools to better understand the overall rate of infection in the community-including the rate of asymptomatic infections. this could potentially enable us to identify healthcare workers who have been infected in the past and might be less prone to becoming infected on the job. our data also show the ability to detect subclinical infections effectively, a much-needed tool to stop the spread of infection, which is currently spreading at an alarming rate in europe and the united states. in addition, the higher speed of antibody testing can be useful and applied in remote areas where qpcr assays cannot be performed. we demonstrated the time kinetics of the humoral response during covid-19 and provided evidence that this can aid in the diagnosis of covid-19, including in subclinical cases. clinical features of patients infected with 2019 novel coronavirus in wuhan, china identification of a novel coronavirus causing severe pneumonia in human: a descriptive study coronavirus disease 2019 (covid-19) situation report-23 2020. world health organization epidemiology, genetic recombination, and pathogenesis of coronaviruses china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china novel coronavirus-important information for clinicians a new coronavirus associated with human respiratory disease in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia inwuhan, china: a descriptive study genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr transmission of 2019-ncov infection from an asymptomatic contact in germany antibody against nucleocapsid protein predicts susceptibility to human coronavirus infection pandemic potential of 2019-ncov novel coronavirus (2019-ncov) early-stage importation risk to europe summary of probable sars cases with onset of illness from 1 acknowledgments. the authors thank the clinicians who contributed to sample collection and transportation.disclaimer. the funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; key: cord-354011-v9t2b2ca authors: benkouiten, samir; charrel, rémi; belhouchat, khadidja; drali, tassadit; salez, nicolas; nougairede, antoine; zandotti, christine; memish, ziad a.; al masri, malak; gaillard, catherine; parola, philippe; brouqui, philippe; gautret, philippe title: circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage date: 2013-10-01 journal: clin infect dis doi: 10.1093/cid/cit446 sha: doc_id: 354011 cord_uid: v9t2b2ca background. the hajj is the oldest and largest annual mass gathering in the world and may increase the risk of spread of respiratory viruses. methods. we performed a prospective survey among a cohort of pilgrims departing from marseille, france, to mecca in the kingdom of saudi arabia (ksa) for the 2012 hajj season. nasal swabs were collected from participants and tested for 11 respiratory viruses by real-time reverse transcription polymerase chain reaction. results. of 165 participants sampled before departing to the ksa, 8 (4.8%) were positive for at least 1 virus (5 rhinovirus, 1 influenza c, 1 adenovirus, and 1 enterovirus). seventy symptomatic pilgrims underwent additional nasal swabs during their pilgrimage in the ksa, of which 27 (38.6%) were positive for at least 1 virus (19 rhinovirus, 6 influenza a, 1 influenza c, 1 respiratory syncytial virus b, 1 metapneumovirus, 1 adenovirus, and 1 enterovirus). this was significantly higher than the 4.8% who were positive before departing for the ksa (p < .001). of 154 pilgrims sampled before leaving the ksa, 17 (11%) were positive for at least 1 virus (13 rhinovirus, 3 adenovirus, 2 influenza b, and 1 enterovirus), which was also significantly higher than the percentage of positive pilgrims (4.8%), before departing for the ksa (p = .040). conclusions. this study suggests a rapid acquisition of respiratory viruses among pilgrims during their stay in the ksa, most notably rhinovirus, and highlights the potential of spreading these infections in the pilgrims' home countries upon their return. the hajj and its rituals are physically demanding and usually last for 1 week, but the period of pilgrimage is up to 1 month. on arrival in mecca, the holiest city in islam, hajj pilgrims start their pilgrimage by visiting the grand mosque for the circumambulation (tawaf ) of the kaaba. the pilgrims then move, during subsequent days, to different sacred places located around the city of mecca, including mina, arafat, and the muzdalifah valleys, where they reside for several nights in tent camps [4] (figure 1 ). later, most pilgrims leave mecca for the city of medina to visit islam's secondholiest site, the mosque of the prophet. the hajj presents major public health and infection control challenges. in addition to fatigue and extreme weather conditions [4] (in mecca, during october the average temperature is >38°c during the day and >25°c at night, with a monthly rainfall averaging 3 mm), which increase the susceptibility of pilgrims to airborne infections, inevitable overcrowding within a confined area of individuals from different parts of the world and close contact with others greatly increase the risk of acquiring or spreading infectious diseases during the pilgrims' stay [5] ; in particular, acute respiratory infections, which are among the leading cause of acute illnesses worldwide, can spread among the pilgrims. respiratory tract infections are very common during the hajj [1, 6] and account for most of the hospital admissions during this period [7, 8] . it has been estimated that more than one-third of pilgrims will experience respiratory symptoms during their stay [9] . a range of pathogens can cause acute respiratory infections, but respiratory viruses were found to be the most common etiology of upper respiratory tract infections among pilgrims in several surveys [1] . this study, including sample collection and laboratory methods, was conducted among a cohort of pilgrims departing from marseille, france, to mecca in the ksa for the 2012 hajj season. they were tested for the most common respiratory viruses, with the aim of elucidating the dynamics of viral circulation among pilgrims. participants were recruited between august and october 2012 from a private specialized travel agency in the city of marseille, france, which organizes travel to mecca. pilgrims who planned to take part in the 2012 hajj season were asked to participate in the study on a voluntary basis if they were 18 years of age or older and were able to provide consent. we conducted a cohort survey of participants who were followed up and sampled before departing to the ksa, during their pilgrimage in the ksa, and just before leaving the ksa. upon inclusion, the participants were questioned by arabicspeaking investigators using a standardized pretravel questionnaire, which included demographic data and medical history. a posttravel questionnaire, which collected travel-associated diseases, vaccination status, and compliance with protective behaviors, was completed by a french muslim arabic-speaking medical doctor who traveled with the pilgrims. this survey was administered during a face-to-face interview just prior to returning to france or via telephone after returning to france. if we were unable to contact the pilgrim after 3 attempts, we considered the pilgrim lost to follow-up. health problems that occurred during the stay in the ksa were monitored and recorded by the medical doctor. a cough was defined as the occurrence of a cough with or without sputum. subjective fever was defined as a pilgrim's report of feeling feverish. for the purpose of this study, influenza-like illness (ili) was defined according to the presence of the triad of a cough, sore throat, and subjective fever [10] . this study was approved by our local ethics committee under number 13-017 and by the saudi ethics committee. it was performed in accordance with the good clinical practices recommended by the declaration of helsinki and its amendments. all participants gave written informed consent. anterior nare swabs were systematically collected from each participant using a commercial rigid cotton-tipped swab applicator (sigma virocult, mw950s, wiltshire, england), in the month before departing from france and in the 3 days before leaving the ksa. in a number of cases, the medical doctor also collected additional nasal samples at the onset of symptoms during the pilgrimage in the ksa (within 2 weeks after their arrival). all the samples collected during the study were placed in viral transport media (virocult virus transport medium) at the point of collection and kept at room temperature (stabilized by air conditioning to 20°c, in france and in the ksa) before being transported to the marseille laboratory for storage in a −80°c freezer. each sample was tested for the following viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr): influenza a (flua) [11] , influenza b (flub) [11] , influenza c (fluc), and a/2009/h1n1 [12] viruses; human respiratory syncytial virus a and b (rsvb) [13] ; human metapneumovirus (hmpv) [14] ; human rhinovirus (hrv) [15] ; ms2 bacteriophage; human adenovirus (hadv) [16] ; and human enterovirus (hev) [17] . total nucleic acids were purified from a 400-μl sample volume and were spiked with ms2 + t4 bacteriophage as an internal control [18] , using the biorobot ez1 xl with the virus mini kit v2.0 (both from qiagen, courtaboeuf, france) according to the manufacturer's instructions. each sample was tested independently in a 25-μl reaction containing 5 μl of rna, 12.5 μl of 2× buffer (iscripttm one-step rt-pcr kit for probes, bio-rad), 1 μl of reverse transcriptase/taq, 400 nm concentration of each primer, and 160 nm of probe. the reactions were performed using a c1000tm thermal cycler (cfx96tm real-time system, bio-rad, marnes-la-coquette, france). the following cycling conditions were applied: 50°c for 10 minutes, followed by 95°c for 5 minutes; and then 45 cycles of 95°c for 15 seconds and 60°c for 30 seconds. the presence of inhibitors was determined using ms2 and t4 bacteriophage-specific detection systems, as previously reported [18] . we hypothesized that several factors may influence the outcome of respiratory symptoms or virus portage during the stay, including age, preventive measures, and underlying chronic diseases. the pearson χ 2 test and fisher exact test, as appropriate, were applied to analyze the categorical variables. p values of ≤.05 were considered significant. statistical analyses were performed using spss software, version 17.2. a total of 169 participants were recruited to take part in the study, of whom 167 responded to the pretravel questionnaire. table 1 summarizes their baseline demographics and characteristics. participants' mean age was 59.3 years (sd, 12.4; range, 21-83 years), with a male-to-female sex ratio of 0.6 to 1. they were predominately born in north africa (90.4%), and most of the foreign-born individuals reported living in france for >20 years. most of the participants reported living in marseille and the surrounding cities. more than half of the participants (57.5%) reported suffering from at least 1 chronic disease, including diabetes (27.5%), hypertension (26.3%), chronic respiratory disease (7.8%), and chronic cardiac disease (7.2%). a total of 137 posttravel questionnaires were completed, representing a total response rate of 81.5%, and 46.0% of these were telephone-administered. the mean time between return from the ksa and administration of the questionnaire by telephone was 24 days (range, 15-33 days). before departing to the ksa, none of the pilgrims presented acute respiratory symptoms at the time they were sampled. pilgrims stayed in the ksa for 4 weeks, and the vast majority (90.4%) of them suffered from at least 1 respiratory symptom during their stay. a cough was the most frequently reported complaint (83.4% of respondents), followed by sore throat (79.7%), rhinorrhea (68.5%), myalgia (46.5%), feverishness (45.4%), dyspnea (19.6%), conjunctivitis (15.2%), and diarrhea (15.7%). of the pilgrims who reported respiratory symptoms during their stay in the ksa, 41.3% met the criteria for selfreported ili. the onset of respiratory symptoms peaked at 5-7 days after the arrival of the pilgrims in mecca and declined thereafter. a second peak of smaller amplitude occurred on days 5 and 6 after arrival in mina, shortly after moving from arafat and muzdalifah. both peaks immediately occurred after performing the tawaf in mecca. finally, only 2% of the pilgrims who were systematically sampled before leaving the ksa reported respiratory symptoms during the 3 days prior to leaving the ksa. regarding preventive measures, 45.6% of participants reported receiving a seasonal influenza vaccination in the past year and 35.9% reported receiving a pneumococcal vaccination in the past 5 years. during their stay in the ksa, 55.1% of pilgrims reported using facemasks, 87.6% using disposable handkerchiefs, 40.3% frequent hand-washing, and 46.3% using hand sanitizer. pilgrims who reported frequent hand-washing during their stay, compared with those who reported using typical handwashing habits, more frequently reported feverishness (55.6% vs 37.5%; odds ratio [or], 2.08; 95% confidence interval [ci], 1.03-4.20; p = .039) and ili symptoms (53.7% vs 32.5%; or, 2.41; 95% ci, 1.18-4.90; p = .014). rhinorrhea was more frequently reported by pilgrims who declared that they were not vaccinated against influenza in 2012 compared to vaccinated pilgrims (75.8% vs 56.8%; or, 2.39; 95% ci, 1.14-5.01; p = .020). dyspnea was more frequently reported by those with chronic respiratory disease compared to other pilgrims (55.6% vs 17.3%; or, 5.97; 95% ci, 1.48-24.02; p = .015). the majority of pilgrims (79.5%) sought healthcare from a doctor during their stay in the ksa, and 19.2% consulted a doctor after their return to france. four pilgrims (3%) were hospitalized (1 in the ksa and 3 upon returning to france), 1 for respiratory tract infection, 1 for ili symptoms, 1 for nephritic colic, and 1 for vomiting. no deaths occurred. of the 169 participants enrolled in the study, 165 (97.6%) underwent a systematic pre-hajj nasal swab before traveling to the ksa, and 154 (91.1%) underwent a systematic post-hajj nasal swab in the 3 days before leaving the ksa (89.3% underwent both pre-and post-hajj nasal swabs). a total of 70 pilgrims (41.4%) also underwent an additional nasal swab at the onset of acute respiratory symptoms during their pilgrimage in the ksa. the collection dates of the samples and results are shown in figures 2, 3, and 4 . before departing to the ksa, 8 participants (4.8%) were positive for at least 1 virus (5 hrv, 1 fluc, 1 hadv, and 1 hev), without coinfection. during the pilgrimage in the ksa, among the 70 symptomatic pilgrims who were sampled, 27 (38.6%) were positive for at least 1 virus (19 hrv, 6 flua, 1 fluc, 1 rsvb, 1 hmpv, 1 hadv, and 1 hev), and there were 3 double infections (flua/fluc, rsvb/hrv, and hrv/hev). this was significantly higher than the 4.8% of pilgrims who were positive before departing to the ksa (p < .001). the prevalence of hrv was significantly higher during the hajj than before (27.1% vs 3%; p < .001). of the 19 pilgrims positive for hrv during their pilgrimage, 2 were positive before traveling to the ksa. among the 57 pilgrims (41.0%) who met criteria for self-reported ili during the hajj, 40 (70.2%) were sampled, of whom 15 (37.5%) were virus-positive. the overall prevalence of respiratory viruses during the pilgrimage was significantly higher in pilgrims who reported more frequent hand-washing than usual during their stay compared to those who reported usual hand-washing (53.6% vs 23.3%; or, 3.79; 95% ci, 1.23-11.69; p = .018). no respiratory symptoms reported during the stay in the ksa were significantly associated with specific viral detection. before leaving the ksa, 17 pilgrims (11%) were positive for at least 1 virus (13 hrv, 3 hadv, 2 flub, and 1 hev), with 2 double infections (hrv/hadv and hrv/hev), and this was significantly higher than the 4.8% of pilgrims who were positive before departing to the ksa (p = .040). the prevalence of hrv was significantly higher before leaving the ksa than before departing to the ksa (8.4% vs 3%; p = .036). of the 13 pilgrims positive for hrv before leaving the ksa, 3 were positive during their pilgrimage. the overall prevalence of respiratory viruses before leaving the ksa was significantly higher in individuals who reported using hand sanitizer during their stay compared to the remaining pilgrims (16.4% vs 5.6%; or, 3.28; 95% ci, .97-11.07; p = .045). all the pilgrims who were viruspositive before leaving the ksa complained of at least 1 respiratory symptom during their stay. this is the first prospective longitudinal study investigating respiratory viruses from nasal specimens taken before departing for the ksa, during the pilgrimage in the ksa, and just before leaving the ksa in a single cohort of pilgrims, whether symptomatic or not. other studies have been conducted either among symptomatic pilgrims recruited in the ksa [19, 20] , among separate populations of arriving and departing pilgrims [21] , and in returned pilgrims only [22] , or were limited to the influenza virus [23] [24] [25] . nine pilgrims out of 10 experienced respiratory symptoms during their stay in the ksa, with a cough and sore throat as the most common symptoms. ili symptoms were reported by 41.0% of total pilgrims. an increase of respiratory symptoms was observed twice, following the tawaf, which is performed in the grand mosque in mecca in highly overcrowded conditions. an 8-fold increase in the overall prevalence of respiratory viruses was observed between samples obtained before departing from france (4.8% of pilgrims) and samples obtained from ill pilgrims after performing their first tawaf in mecca (38.6% of pilgrims). in our study, hrv was the most frequent virus detected from symptomatic pilgrims (27%). this result is in accordance with studies conducted worldwide where hrv has been recognized as the most frequent virus responsible for the common cold in adults [26, 27] . this is not unexpected, as rhinoviruses are nonenveloped viruses that are more resistant in the environment than enveloped viruses. symptoms of hrv infection are generally mild and limited to the upper respiratory tract. in contrast, lower respiratory symptoms associated with hrv infection are prominent in patients who have underlying asthma or other chronic lung disease [28] . in the present study, 7.8% of pilgrims reported chronic respiratory diseases; therefore, prevention must be reinforced in this high-risk population. there have been arguments [26] regarding whether rhinoviruses are spread chiefly from infected person to healthy individuals by direct contact to the fingers of healthy individuals by a handshake or indirect contact from the hands of an infected person to an intermediary surface [29] , or through the aerosol route [30] . our findings showed that more frequent hand-washing was significantly associated with feverishness and symptoms of ili and with a higher prevalence of respiratory viruses. however, these results may indicate a reverse causation, as pilgrims with symptoms or who believe themselves to be at greater risk may have washed their hands more frequently. a recent study found that hand disinfection did not reduce hrv infection or hrv-related common cold illnesses [31] . the prevalence of hrv paralleled the increase of respiratory clinical symptoms, with a higher prevalence in pilgrims sampled in mecca city during the first week of the pilgrimage. influenza viruses ranked second after hrv among symptomatic pilgrims (10.0%), and 3 patients with influenza virus infection reported receiving a seasonal influenza vaccination prior to the hajj pilgrimage. other viruses were rarely found in our study. we previously reported that the novel coronavirus middle east respiratory syndrome (mers-cov; formerly known as hcov-emc) was not detected during the 2012 hajj season among the cohort pilgrims described here [32] . other coronaviruses and parainfluenza viruses were not considered in this study and should be investigated in future studies. more than 1 out of 10 pilgrims tested positive for overall respiratory viruses just before leaving the ksa, which indicates a high potential for spreading viral pathogens into pilgrims' home countries. given that a significant proportion of french pilgrims also travel to their country of birth in north africa immediately after returning to france from the hajj [33] , there is also a potential for spreading these viruses beyond france. the main limitation of this study is that, although the vast majority of pilgrims reported respiratory symptoms during their stay in ksa, the sampling by the medical doctor was limited to those who sought medical consultation from her. however, some symptomatic pilgrims were found to be negative for respiratory viruses. for those sampled at mina and medina, this was likely due to delayed medical consultation, as the specimen was collected several weeks after the onset of symptoms. second, symptom occurrence was collected retrospectively and was based on self-reporting; thus, the date of the medical consultation did not necessarily correspond to the selfreported date of the onset of respiratory symptoms. third, samples that were obtained at the beginning of the pilgrimage in the ksa were stored at room temperature (20°c) for periods up to 30 days before being processed, which may have been resulted in the degradation of genetic material. this may have likely contributed to underestimating the frequencies of infection. alternative strategies to better preserve the samples must be considered, among which ethanol or nucleic acid lysis buffer [34] could be used. fourth, a technical point is that hrv sequencing was not performed to determine how often new hrv infections were acquired during the stay in the ksa. finally, we cannot demonstrate that the viruses detected from nasal swabs were responsible for the symptoms, as nasal carriage in asymptomatic pilgrims was observed in some cases, and symptoms may have resulted from infection by other viruses [35] , or possibly bacteria [36] , that were not investigated in our study. although our results cannot be extrapolated to all pilgrims, our study illustrates the rapid acquisition of respiratory viruses among pilgrims during their stay in the ksa, particularly rhinovirus, and demonstrates the potential for spreading these infections to pilgrims' home countries upon their return. financial support. this work was supported by the marseille public hospitals authority (aorc). potential conflicts of interest. all authors: no reported conflicts. respiratory tract infections during the annual hajj: potential risks and mitigation strategies global public health implications 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(h1n1) infection among 2009 hajj pilgrims from southern iran: a real-time rt-pcr-based study human rhinoviruses clinical effects of rhinovirus infections pathogenesis of rhinovirus infection transmission of rhinovirus colds by self-inoculation aerosol transmission of rhinovirus colds a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms travel reported by pilgrims from marseille evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification the viral etiology of an influenza-like illness during the 2009 pandemic bacteria and viruses that cause respiratory tract infections during the pilgrimage (haj) season in makkah, saudi arabia key: cord-340317-gwqy6u9x authors: dora, amy v; winnett, alexander; fulcher, jennifer a; sohn, linda; calub, feliza; lee-chang, ian; ghadishah, elham; schwartzman, william a; beenhouwer, david o; vallone, john; graber, christopher j; goetz, matthew bidwell; bhattacharya, debika title: using serologic testing to assess the effectiveness of outbreak control efforts, serial pcr testing, and cohorting of positive sars-cov-2 patients in a skilled nursing facility date: 2020-08-28 journal: clin infect dis doi: 10.1093/cid/ciaa1286 sha: doc_id: 340317 cord_uid: gwqy6u9x we characterized serology following a nursing home outbreak where residents were serially tested by rt-pcr and positive residents were cohorted. when tested 46-76 days later, 24/26 rt-pcr-positive residents were seropositive; none of the 124 rt-pcr-negative residents had confirmed seropositivity, supporting serial sars-cov-2 rt-pcr testing and cohorting in nursing homes. m a n u s c r i p t 4 since the emergence of 2019 coronavirus disease in the united states in january 2020, skilled nursing facilities (snf) have been a repeated site of sars-cov-2 (the virus that causes covid-19) outbreaks [1, 2] . as older populations are the most severely affected by covid-19 [3] , the ability to identify and contain these outbreaks is critical. despite numerous outbreaks, the performance of serologic testing for sars-cov-2 in residents of skilled nursing facilities (snf) is not well described; its utility in epidemiologic sero-surveillance studies and outbreak reporting is under evaluation. to assess unrecognized sars-cov-2 exposure, we reviewed serologic testing in a cohort of snf residents who had been serially tested for sars-cov-2 infection by nasopharyngeal swab rt-pcr following a covid-19 outbreak at this snf [1] . study design and setting. from march 28 -30, symptom-based rt-pcr testing identified 3 covid-19 cases at a snf at the veterans affairs greater los angeles healthcare system west los angeles (wla) campus [1] . in response, all remaining snf patients (n=96) underwent universal testing with nasopharyngeal rt-pcr (roche cobas 6800) for sars-cov-2, repeated approximately weekly on each ward and discontinued when all ward residents tested negative. in addition, staff and residents underwent temperature and symptoms screening, visitors were restricted, and universal masking of staff was initiated. between march 29-april 6, 16 additional cases were identified [1] . these 19 patients were transferred to the acute care hospital for treatment or a designated covid recovery unit (cru) with cohorted staff, located within the snf [4] . no further cases were identified upon additional serial testing the weeks of april 13 and april 20, and serial testing was discontinued. surveillance testing on may 11 and may 18 did not yield any cases. by june, a c c e p t e d m a n u s c r i p t 5 all cases had been transferred back to the snf or cru. an additional 9 patients from the community who were diagnosed with covid-19 by rt-pcr and treated in the acute care hospital were transferred to the cru by june 5 and were included in this analysis. snf staff were tested once between march 29 and april 10; 8/136 were positive and self-isolated at home [1] . including two of the known rt-pcr positive cases, were excluded as they were on hospice, refused blood draw, received convalescent plasma, or had died. serologic testing for igg antibody to sars-cov-2 spike (s1/s2) protein was performed using the diasorin liaison  assay due to availability at our facility. as our objective was to identify potentially missed cases of covid-19 during serial surveillance testing, samples from residents with historically negative sars-cov-2 rt-pcr tests with diasorin igg-positive results were re-tested on a second serologic testing platform (abbott sars-cov-2 igg immunoassay) for igg antibody specific to sars-cov-2 nucleocapsid. there were an additional 124 residents in the snf who underwent serial sars-cov-2 rt-pcr testing and were persistently negative. of this cohort, 69 were residing in the wla snf and 44 were residing at sacc at the time of the initial covid-19 outbreak in march (figure) . among these 124 snf residents with negative rt-pcr, all but two were seronegative for igg antibody to sars-cov-2 spike protein by the diasorin assay. the two seropositive residents had no detectable igg antibody to sars-cov-2 nucleocapsid when reflex tested by the abbott assay performed the same day. one of these residents had 7 previous negative rt-pcr results; the other had 3. the latter was transferred to the wla snf from a non-va acute care hospital, where he had been admitted for gastrointestinal and other symptoms attributed to urinary tract infection and was thus not present during the initial wla snf outbreak. all 47 sacc residents were igg negative. in a cohort of 150 nursing home residents tested for sars-cov-2 infection by rt-pcr, including 26 previously diagnosed with covid-19, the sensitivity of the diasorin assay was 92% (24/26) and the specificity was 98% (122/124). the two individuals seropositive by diasorin assay despite multiple negative rt-pcr tests likely represented false positive results, as reflex serologic testing using the abbott assay indicated seronegativity. the absence of confirmed seropositivity in the rt-pcr negative cohort supports that universal and serial rt-pcr testing and early cohorting strategies in the snf were effective at reducing further sars-cov-2 transmission. multiple factors may affect sensitivity and specificity of serologic testing to identify sars-cov-2 infection, including temporal dynamics in antibody response and possibly immunosenescence. sars-cov-2 antibody development (igg and igm) has been a c c e p t e d m a n u s c r i p t 8 demonstrated in the first 2 weeks of illness [5, 6] , with igg more reliably detectable two weeks after symptom onset and persisting at least 3-4 weeks into the illness [5, 7, 8] . there is a paucity of data using igg to evaluate history of infection two months later, as was performed in our study. the role of immunosenescence and covid-19 igg response also remains unclear but may be especially pertinent in older snf residents. despite these factors, diasorin assay performance was within the confidence intervals reported by the manufacturer [9]. although rt-pcr is the most common method of diagnosing covid-19, serologic testing has been shown to aid in retrospective diagnosis [5, 10] . rt-pcr may be falsely negative due to inconsistent sampling, time and temperature of specimen transport, inhibitors to nucleic acid amplification, and kinetics of viral shedding [11] . our objective was to use serology to identify covid-19 cases missed by rt-pcr, as was observed in an epidemiologic investigation in singapore where serological testing provided laboratory evidence linking two clusters that rt-pcr results had not [10] . though our cohort had two residents who were rt-pcr negative but seropositive by diasorin, they likely do not represent cases missed by rt-pcr, as reflex testing was negative by the abbott assay, which is unlikely to yield an overlapping false positive result [12] . the utility of serological testing has also been highlighted by large scale epidemiological studies [8] and to retrospectively evaluate outbreak control strategies [10] . our results support that the infection control strategies employed during the wla snf covid-19 outbreak were effective, including universal rt-pcr testing of residents and staff, and serial rt-pcr testing of residents with subsequent rapid isolation and cohorting of positive individuals, which limited transmission of infection. additionally, the establishment of a cru allowed cohorting of clinically stable residents a c c e p t e d m a n u s c r i p t 9 with covid-19 [4] , which provided an ideal cohort to evaluate known positive patients using serology. limitations of this report include small cohort size and a predominantly male, older population with a specific comorbidity profile that may be difficult to extrapolate to other populations. relatedly, the size of the cohort may also limit the ability to assess the true accuracy of pcr and serology tests on a population-wide scale. as the outbreak control methods included a multipronged infection prevention/control approach, we are unable to exclude the contributions of these methods in preventing further spread of cases and note that testing resources can limit the utility of specific diagnostic tools. serial rt-pcr testing and rapid cohorting and isolation in a dedicated covid-19 unit are effective measures to suppress further covid-19 infection in a skilled nursing facility. serologic evidence further supports no new cases of covid-19 after initial case-finding activities. m a n u s c r i p t universal and serial laboratory testing for sars-cov-2 at a long-term care skilled nursing facility for veterans -los characteristics of u.s. nursing homes with covid-19 cases covid-19) -united states establishment of a covid-19 recovery unit in a veteran affairs (va) post-acute facility antibody detection and dynamic characteristics in patients with covid-19 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study the role of serology for covid-19 control: population, kinetics and test performance do matter serological assays for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) connecting clusters of covid-19: an epidemiological and serological investigation role of serology in the covid-19 pandemic side by side comparison of three fully automated sars-cov-2 antibody assays with a focus on specificity a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-350972-0n4dumgg authors: sing, chor-wing; tan, kathryn c b; wong, ian c k; cheung, bernard m y; cheung, ching-lung title: long-term outcome of short-course high-dose glucocorticoids for sars: a 17-year follow-up in sars survivors date: 2020-07-16 journal: clin infect dis doi: 10.1093/cid/ciaa992 sha: doc_id: 350972 cord_uid: 0n4dumgg use of high-dose glucocorticoids for coronavirus disease 2019 (covid-19; caused by sars-cov-2) is controversial because of safety concerns. we examined long-term consequences in severe acute respiratory syndrome (sars; caused by sars-cov-1) survivors. results showed that high-dose glucocorticoids greatly increased long-term risk of avascular necrosis, but not other major diseases. high-dose glucocorticoids have been widely used in critically-ill patients to suppress inflammation response. 1 while short-term outcomes of such treatment have been reported 2 , long-term clinical outcomes are unknown. hong kong had an outbreak of severe acute respiratory syndrome (sars; caused by sars-cov-1) in 2003, resulting in 1,755 cases and 299 deaths 3 short course of very-high-dose (vhd) glucocorticoids was used for the treatment of sars, especially to prevent cytokine storm. in the recent pandemic of coronavirus disease 2019 (covid-19; caused by sars-cov-2), glucocorticoids has been suggested but there is controversy because of safety concerns. to understand the long-term consequences of vhd glucocorticoids, we studied the clinical outcomes of sars survivors after 17 years. we identified sars survivors using an electronic medical record database, clinical data analysis and reporting system (cdars) of the hong kong hospital authority, the details of which have been described elsewhere. 4 patients who i) were screened positive for sars during the outbreak from 22 feb to 2 jun, 2003 3 ; ii) received glucocorticoid therapy during hospitalization; and iii) recovered and discharged, were included. in the analysis of each outcome-of-interest, we excluded patients with history of that outcome. clinical data including demographics, diagnosis and prescription records were retrieved. patients who did not receive glucocorticoid therapy were not included in the analysis due to the concern on treatment allocation bias. these patients were generally frail or in poor comorbid, leading to potential bias to the long-term outcomes. a c c e p t e d m a n u s c r i p t 4 exposure and outcome exposure was defined by the daily dose of glucocorticoid (or its equivalent prednisone dose) prescribed for each patient. glucocorticoid doses were classified according to the recommendation by eular. 5 patients receiving vhd or pulse regimen (>100 mg prednisone equivalent a day) were compared with patients receiving low-to-high-dose regimen (lthd; ≤100 mg prednisone equivalent a day). outcomes of interest were common diseases and diseases associated with long-term use of glucocorticoid (table 1 ). these diseases were defined using icd-9 codes (supplementary table 1 ). patients were followed from the date of discharge until the occurrence of an outcome-ofinterest, death or study end (31 dec 2019), whichever was earlier. cox-proportional hazard regression was used to examine the association between use of glucocorticoid and clinical outcomes. hazard ratio (hr) and 95% confidence interval (ci), adjusted for age, sex, and charlson comorbidity index were estimated. sensitivity analysis was conducted using propensity score (ps) matching. details of the ps matching were provided in supplementary methods. additional analysis was performed for outcome showing significant association with exposure. common risk factors for the outcome at baseline (i.e. on/before patient discharge) were further adjusted in the cox model. the follow-up was censored at the occurrence of risk factors, in addition to death and study end. statistical analysis was performed using r (version 3.5.2). a p-value<0.05 was considered statistically significant. a c c e p t e d m a n u s c r i p t 5 we identified 1,326 sars survivors in cdars. of these, 184 survivors who did not received glucocorticoid were excluded, resulting in 1,142 sars survivors (vhd: 850; lthd: 292) in the analysis. overall, the mean±sd age of the cohort was 38±15.2 and 40% was male. there is no significant difference in age and gender between the groups but the mean charlson comorbidity index in vhd was significantly lower than that in lthd (supplementary table 2 table 2 ). over a median follow-up of 16.7 years, we observed a much higher incidence of avascular necrosis (avn) in vhd than lthd (4.83 vs 0.67 per 1000 person-year). a significant 7.5fold increased risk of avn (hr 7.50; 95% ci 2.34-24.04; p<0.001) in the vhd, compared with the lthd, was estimated. there was no significant association in other clinical outcomes. similar findings were observed in the ps-matching cohort. (table 1 in this retrospective study of sars survivors with 17 years of follow-up, a short-course use of vhd glucocorticoids was not associated with major diseases except avn. the association between high-dose glucocorticoids and avn in sars patients has been reported. 6, 7 however, these studies had a short follow-up time ranging from 6 months to 3 years. study by hui et al. reported that among 41 patients developed avn, 11 (27%) of them were diagnosed with avn after 3-years of follow-up. in our study, 48% (30 out of 62) patients developed avn 3 years after their recovery from sars, suggesting that the risk persisted for a long time. thus, long-term monitoring for avn is necessary even the use of high-dose glucocorticoids is short. osteoporotic fracture is a well-known adverse effect of chronic use of glucocorticoids. a study using national claim data showed that short course of low dose glucocorticoids (median dose 20mg/day) was associated with increased risk of fractures by 83%, compared with nonusers, within first 30 days of drug initiation. 8 our study did not show such association. the discrepancy could be due to the heterogeneity of study design. in addition, bone remodeling is a slow process and thus, occurrence of fracture in a short period of time should be unlikely. more studies are warranted to examine the risk of fractures in short-term use of glucocorticoids. similarly, it is well-reported that chronic use of glucocorticoids is associated with diabetes mellitus (dm) but the effect of short course use is unknown. in this study, the incidence of dm in vhd was higher than that in lthd (4.54 vs 3.77 per 1000 person-years) but the association was not significant. a prospective study in patients with perioperative use of high-dose glucocorticoids showed that hyperglycemia was observed but the condition was amid the covid-19 pandemic, glucocorticoids are once again being considered for selected patients. 10, 11 given that cytokine storm is the most life-threatening condition in sars-cov-2, vhd glucocorticoids may be used in the treatment. our study showed that short-course of vhd glucocorticoids was not associated with major diseases. however, given the high risk of avn, long-term surveillance and clinical care in patients is recommended. a multi-center study in us showed that early short-course of glucocorticoids had a significant reduction of death, mechanical ventilator use, and length of stay in hospital. 10 a recent report from the recovery trial showed that dexamethasone improved survival in patients requiring oxygen or mechanical ventilation, compared to usual care alone. 12 the trial used 6mg dexamethasone once daily for 10 days. the daily dose was equivalent to 40mg prednisone which is classified as lthd in this study. our study provides useful safety data on the potential long-term comorbidity if glucocorticoids therapy is needed. strengths of this report include a long-term follow-up in a large sars cohort. in addition, hong kong hospital authority implemented a follow-up programme for sars survivors. thus, under-diagnosis of comorbidity should be minimal. conversely, there were limitations. first, this study may have limited power in detecting small effect size. thus, studies with larger sample sizes are warranted to verify our findings. second, data on excess alcohol use, a common risk factor for avn, was not available. nevertheless, the per capita alcohol consumption in hong kong according to government statistics in 2010 was 2.6 litre/year (https://www.change4health.gov.hk/), compared to 9.2 litre/year in us and 11.6 litre/year in uk (who statistics https://www.who.int/substance_abuse/activities/gsrah/en/). therefore, alcohol as a risk factor for avn is relatively less important in hong kong. there is no study in hong kong reporting significant difference of alcohol consumption between vhd and lthd glucocorticoids users. thus, confounding by alcohol use should be minimal. third, further exposure of glucocorticoid during the follow-up might bias the results of short-course use of glucocorticoids. to address this concern, we conducted a post-hoc analysis by excluding patients with further exposure of glucocorticoids (n=337) and the results remained consistent (hr 8.33; 95% ci 2.01-34.58; p = 0.004). m a n u s c r i p t 11 corticosteroids in severe pneumonia one-year outcomes in survivors of the acute respiratory distress syndrome sars in hong kong: from experience to action. hong kong sar october association between dabigatran vs warfarin and risk of osteoporotic fractures among patients with nonvalvular atrial fibrillation standardised nomenclature for glucocorticoid dosages and glucocorticoid treatment regimens: current questions and tentative answers in rheumatology avascular necrosis of bone in severe acute respiratory syndrome avascular osteonecrosis after treatment of sars: a 3-year longitudinal study short term use of oral corticosteroids and related harms among adults in the united states: population based cohort study effect of high-dose dexamethasone on patients without diabetes during elective neurosurgery: a prospective study early short course corticosteroids in hospitalized patients with covid-19 expert consensus on the use of corticosteroid in patients with 2019-ncov pneumonia zhonghua jie he he hu xi za zhi [chinese journal of tuberculosis and respiratory diseases effect of dexamethasone in hospitalized patients with covid-19: preliminary report. medrxiv m a n u s c r i p t 8 in conclusion, our study shows clearly that short-term use of vhd glucocorticoid greatly increases the risk of avn, but fortunately not the risk of other major diseases like cardiovascular disease or cancer. covid-19 affects patients of all ages. doctors must therefore weigh up the potential benefits against the long-term risks whenever high-dose glucocorticoids are contemplated.a c c e p t e d m a n u s c r i p t 9 acknowledgement:the study protocol was approved by the institutional review boards of the university of hong kong and hong kong hospital authority (hkha); reference: uw20-172 there is no funding source for the study. no author has conflict of interest in the study.a c c e p t e d m a n u s c r i p t 10 key: cord-292094-vmsdhccp authors: mandell, lionel a.; wunderink, richard g.; anzueto, antonio; bartlett, john g.; campbell, g. douglas; dean, nathan c.; dowell, scott f.; file, thomas m.; musher, daniel m.; niederman, michael s.; torres, antonio; whitney, cynthia g. title: infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults date: 2007-03-01 journal: clin infect dis doi: 10.1086/511159 sha: doc_id: 292094 cord_uid: vmsdhccp nan improving the care of adult patients with communityacquired pneumonia (cap) has been the focus of many different organizations, and several have developed guidelines for management of cap. two of the most widely referenced are those of the infectious diseases society of america (idsa) and the american thoracic society (ats). in response to confusion regarding differences between their respective guidelines, the idsa and the ats convened a joint committee to develop a unified cap guideline document. the guidelines are intended primarily for use by emergency medicine physicians, hospitalists, and primary care practitioners; however, the extensive literature evaluation suggests that they are also an appro-reprints or correspondence: dr. lionel a. mandell priate starting point for consultation by specialists. substantial overlap exists among the patients whom these guidelines address and those discussed in the recently published guidelines for health care-associated pneumonia (hcap). pneumonia in nonambulatory residents of nursing homes and other long-term care facilities epidemiologically mirrors hospital-acquired pneumonia and should be treated according to the hcap guidelines. however, certain other patients whose conditions are included in the designation of hcap are better served by management in accordance with cap guidelines with concern for specific pathogens. 1. locally adapted guidelines should be implemented to improve process of care variables and relevant clinical outcomes. (strong recommendation; level i evidence.) it is important to realize that guidelines cannot always account for individual variation among patients. they are not intended to supplant physician judgment with respect to particular patients or special clinical situations. the idsa considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances. enthusiasm for developing these guidelines derives, in large part, from evidence that previous cap guidelines have led to improvement in clinically relevant outcomes. consistently beneficial effects in clinically relevant parameters (listed in table 3) followed the introduction of a comprehensive protocol (including a combination of components from table 2) that increased compliance with published guidelines. the first recommendation, therefore, is that cap management guidelines be locally adapted and implemented. documented benefits. 2 . cap guidelines should address a comprehensive set of elements in the process of care rather than a single element in isolation. (strong recommendation; level iii evidence.) 3. development of local cap guidelines should be directed toward improvement in specific and clinically relevant outcomes. (moderate recommendation; level iii evidence.) almost all of the major decisions regarding management of cap, including diagnostic and treatment issues, revolve around the initial assessment of severity. site-of-care decisions (e.g., hospital vs. outpatient, intensive care unit [icu] vs. general ward) are important areas for improvement in cap management. hospital admission decision. 4 . severity-of-illness scores, such as the curb-65 criteria (confusion, uremia, respiratory rate, low blood pressure, age 65 years or greater), or prognostic models, such as the pneumonia severity index (psi), can be used to identify patients with cap who may be candidates for outpatient treatment. (strong recommendation; level i evidence.) 5. objective criteria or scores should always be supplemented with physician determination of subjective factors, including the ability to safely and reliably take oral medication and the availability of outpatient support resources. (strong recommendation; level ii evidence.) 6. for patients with curb-65 scores у2, more-intensive treatment-that is, hospitalization or, where appropriate and available, intensive in-home health care services-is usually warranted. (moderate recommendation; level iii evidence.) physicians often admit patients to the hospital who could be well managed as outpatients and who would generally prefer to be treated as outpatients. objective scores, such as the curb-65 score or the psi, can assist in identifying patients who may be appropriate for outpatient care, but the use of such scores must be tempered by the physician's determination of additional critical factors, including the ability to safely and reliably take oral medication and the availability of outpatient support resources. icu admission decision. 7 . direct admission to an icu is required for patients with septic shock requiring vasopressors or with acute respiratory failure requiring intubation and mechanical ventilation. (strong recommendation; level ii evidence.) 8. direct admission to an icu or high-level monitoring unit is recommended for patients with 3 of the minor criteria for severe cap listed in table 4. (moderate recommendation; level ii evidence.) in some studies, a significant percentage of patients with cap are transferred to the icu in the first 24-48 h after hospitalization. mortality and morbidity among these patients appears to be greater than those among patients admitted directly to the icu. conversely, icu resources are often overstretched in many institutions, and the admission of patients with cap who would not directly benefit from icu care is also problematic. unfortunately, none of the published criteria for severe cap adequately distinguishes these patients from those for whom icu admission is necessary. in the present set of guidelines, a new set of criteria has been developed on the basis of data on individual risks, although the previous ats criteria format is retained. in addition to the 2 major criteria (need for mechanical ventilation and septic shock), an expanded set of minor criteria (respiratory rate, 130 breaths/min; arterial oxygen pressure/fraction of inspired oxygen (pao 2 /fio 2 ) ratio, !250; multilobar infiltrates; confusion; blood urea nitrogen level, 120 mg/dl; leukopenia resulting from infection; thrombocytopenia; hypothermia; or hypotension requiring aggressive fluid resuscitation) is proposed (table 4). the presence of at least 3 of these criteria suggests the need for icu care but will require prospective validation. 9. in addition to a constellation of suggestive clinical features, a demonstrable infiltrate by chest radiograph or other imaging technique, with or without supporting microbiological data, is required for the diagnosis of pneumonia. (moderate recommendation; level iii evidence.) recommended diagnostic tests for etiology. 10 . patients with cap should be investigated for specific pathogens that would significantly alter standard (empirical) management decisions, when the presence of such pathogens is suspected on the basis of clinical and epidemiologic clues. (strong recommendation; level ii evidence.) recommendations for diagnostic testing remain controversial. the overall low yield and infrequent positive impact on clinical care argue against the routine use of common tests, such as blood and sputum cultures. conversely, these cultures may have a major impact on the care of an individual patient and are important for epidemiologic reasons, including the antibiotic susceptibility patterns used to develop treatment guidelines. a list of clinical indications for more extensive diagnostic testing (table 5) was, therefore, developed, primarily on the basis of 2 criteria: (1) when the result is likely to change individual antibiotic management and (2) when the test is likely to have the highest yield. 11 . routine diagnostic tests to identify an etiologic diagnosis are optional for outpatients with cap. (moderate recommendation; level iii evidence.) 12. pretreatment blood samples for culture and an expectorated sputum sample for stain and culture (in patients with a productive cough) should be obtained from hospitalized patients with the clinical indications listed in table 5 but are optional for patients without these conditions. (moderate recommendation; level i evidence.) 13. pretreatment gram stain and culture of expectorated sputum should be performed only if a good-quality specimen can be obtained and quality performance measures for collection, transport, and processing of samples can be met. (moderate recommendation; level ii evidence.) 14. patients with severe cap, as defined above, should at least have blood samples drawn for culture, urinary antigen tests for legionella pneumophila and streptococcus pneumoniae performed, and expectorated sputum samples collected for culture. for intubated patients, an endotracheal aspirate sample should be obtained. (moderate recommendation; level ii evidence.) the most clear-cut indication for extensive diagnostic testing is in the critically ill cap patient. such patients should at least have blood drawn for culture and an endotracheal aspirate obtained if they are intubated; consideration should be given to more extensive testing, including urinary antigen tests for l. pneumophila and s. pneumoniae and gram stain and culture of expectorated sputum in nonintubated patients. for inpatients without the clinical indications listed in table 5, diagnostic testing is optional (but should not be considered wrong). empirical antimicrobial therapy. empirical antibiotic recommendations (table 7) have not changed significantly from those in previous guidelines. increasing evidence has strengthened the recommendation for combination empirical therapy for severe cap. only 1 recently released antibiotic has been added to the recommendations: ertapenem, as an acceptable b-lactam alternative for hospitalized patients with risk factors for infection with gram-negative pathogens other than pseudomonas aeruginosa. at present, the committee is awaiting further evaluation of the safety of telithromycin by the us food and drug administration before making its final recommendation regarding this drug. recommendations are generally for a class of antibiotics rather than for a specific drug, unless outcome data clearly favor one drug. because overall efficacy remains good for many classes of agents, the more potent drugs are given preference because of their benefit in decreasing the risk of selection for antibiotic resistance. 19 . a b-lactam plus a macrolide (strong recommendation; level i evidence) (preferred b-lactam agents include cefotaxime, ceftriaxone, and ampicillin; ertapenem for selected patients; with doxycycline [level iii evidence] as an alternative to the macrolide. a respiratory fluoroquinolone should be used for penicillin-allergic patients.) increasing resistance rates have suggested that empirical therapy with a macrolide alone can be used only for the treat-ment of carefully selected hospitalized patients with nonsevere disease and without risk factors for infection with drug-resistant pathogens. however, such monotherapy cannot be routinely recommended. inpatient, icu treatment 20. a b-lactam (cefotaxime, ceftriaxone, or ampicillin-sulbactam) plus either azithromycin (level ii evidence) or a fluoroquinolone (level i evidence) (strong recommendation) (for penicillin-allergic patients, a respiratory fluoroquinolone and aztreonam are recommended.) 21 . for pseudomonas infection, use an antipneumococcal, antipseudomonal b-lactam (piperacillin-tazobactam, cefepime, imipenem, or meropenem) plus either ciprofloxacin or levofloxacin (750-mg dose) or the above b-lactam plus an aminoglycoside and azithromycin or the above b-lactam plus an aminoglycoside and an antipneumococcal fluoroquinolone (for penicillin-allergic patients, substitute aztreonam for the above b-lactam). (moderate recommendation; level iii evidence.) 22 . for community-acquired methicillin-resistant staphylococcus aureus infection, add vancomycin or linezolid. (moderate recommendation; level iii evidence.) infections with the overwhelming majority of cap pathogens will be adequately treated by use of the recommended empirical regimens. the emergence of methicillin-resistant s. aureus as a cap pathogen and the small but significant incidence of cap due to p. aeruginosa are the exceptions. these pathogens occur in specific epidemiologic patterns and/or with certain clinical presentations, for which empirical antibiotic coverage may be warranted. however, diagnostic tests are likely to be of high yield for these pathogens, allowing early discontinuation of empirical treatment if results are negative. the risk factors are included in the table 5 recommendations for indications for increased diagnostic testing. risk factors for other uncommon etiologies of cap are listed in table 8, and recommendations for treatment are included in table 9 . pathogen-directed therapy. 23 definitions and classification. 38 . the use of a systematic classification of possible causes of failure to respond, based on time of onset and type of failure (table 11) , is recommended. (moderate recommendation; level ii evidence.) as many as 15% of patients with cap may not respond appropriately to initial antibiotic therapy. a systematic approach to these patients (table 11) will help to determine the cause. because determination of the cause of failure is more accurate if the original microbiological etiology is known, risk factors for nonresponse or deterioration (table 12) figure prominently in the list of situations in which more aggressive and/ or extensive initial diagnostic testing is warranted ( [2] . despite advances in antimicrobial therapy, rates of mortality due to pneumonia have not decreased significantly since penicillin became routinely available [3] . groups interested in approaches to the management of cap include professional societies, such as the american thoracic society (ats) and the infectious diseases society of america (idsa); government agencies or their contract agents, such as the center for medicare and medicaid services and the department of veterans affairs; and voluntary accrediting agencies, such as the joint commission on accreditation of healthcare organizations. in addition, external review groups and consumer groups have chosen cap outcomes as major quality indicators. such interest has resulted in numerous guidelines for the management of cap [4] . some of these guidelines represent truly different perspectives, including differences in health care systems, in the availability of diagnostic tools or therapeutic agents, or in either the etiology or the antibiotic susceptibility of common causative microorganisms. the most widely referenced guidelines in the united states have been those published by the ats [5, 6] and the idsa [7] [8] [9] . differences, both real and imagined, between the ats and idsa guidelines have led to confusion for individual physicians, as well as for other groups who use these published guidelines rather than promulgating their own. in response to this concern, the idsa and the ats convened a joint committee to develop a unified cap guideline document. this document represents a consensus of members of both societies, and both governing councils have approved the statement. purpose and scope. the purpose of this document is to update clinicians with regard to important advances and controversies in the management of patients with cap. the committee chose not to address cap occurring in immunocompromised patients, including solid organ, bone marrow, or stem cell transplant recipients; patients receiving cancer chemotherapy or long-term (130 days) high-dose corticosteroid treatment; and patients with congenital or acquired immunodeficiency or those infected with hiv who have cd4 cell counts !350 cells/mm 3 , although many of these patients may be infected with the same microorganisms. pneumonia in children (р18 years of age) is also not addressed. substantial overlap exists among the patients these guidelines address and those discussed in the recently published guidelines for health care-associated pneumonia (hcap) [10] . two issues are pertinent: (1) an increased risk of infection with drugresistant isolates of usual cap pathogens, such as streptococcus pneumoniae, and (2) an increased risk of infection with less common, usually hospital-associated pathogens, such as pseudomonas and acinetobacter species and methicillin-resistant staphylococcus aureus (mrsa). pneumonia in nonambulatory residents of nursing homes and other long-term care facilities epidemiologically mirrors hospital-acquired pneumonia and should be treated according to the hcap guidelines. however, certain other patients whose conditions are included under the designation of hcap are better served by management in ac-cordance with cap guidelines with concern for specific pathogens. for example, long-term dialysis alone is a risk for mrsa infection but does not necessarily predispose patients to infection with other hcap pathogens, such as pseudomonas aeruginosa or acinetobacter species. on the other hand, certain patients with chronic obstructive pulmonary disease (copd) are at greater risk for infection with pseudomonas species but not mrsa. these issues will be discussed in specific sections below. the committee started with the premise that mortality due to cap can be decreased. we, therefore, have placed the greatest emphasis on aspects of the guidelines that have been associated with decreases in mortality. for this reason, the document focuses mainly on management and minimizes discussions of such factors as pathophysiology, pathogenesis, mechanisms of antibiotic resistance, and virulence factors. the committee recognizes that the majority of patients with cap are cared for by primary care, hospitalist, and emergency medicine physicians [11] , and these guidelines are, therefore, directed primarily at them. the committee consisted of infectious diseases, pulmonary, and critical care physicians with interest and expertise in pulmonary infections. the expertise of the committee and the extensive literature evaluation suggest that these guidelines are also an appropriate starting point for consultation by these types of physicians. although much of the literature cited originates in europe, these guidelines are oriented toward the united states and canada. although the guidelines are generally applicable to other parts of the world, local antibiotic resistance patterns, drug availability, and variations in health care systems suggest that modification of these guidelines is prudent for local use. methodology. the process of guideline development started with the selection of committee cochairs by the presidents of the idsa [12] and ats [13] , in consultation with other leaders in the respective societies. the committee cochairs were charged with selection of the rest of the committee. the idsa members were those involved in the development of previous idsa cap guidelines [9] , whereas ats members were chosen in consultation with the leadership of the mycobacteria tuberculosis and pulmonary infection assembly, with input from the chairs of the clinical pulmonary and critical care assemblies. committee members were chosen to represent differing expertise and viewpoints on the various topics. one acknowledged weakness of this document is the lack of representation by primary care, hospitalist, and emergency medicine physicians. the cochairs generated a general outline of the topics to be covered that was then circulated to committee members for input. a conference phone call was used to review topics and to discuss evidence grading and the general aims and expectations of the document. the topics were divided, and committee members were assigned by the cochairs and charged with presentation of their topic at an initial face-to-face meeting, as well as with development of a preliminary document dealing with their topic. controversial topics were assigned to 2 committee members, 1 from each society. an initial face-to-face meeting of a majority of committee members involved presentations of the most controversial topics, including admission decisions, diagnostic strategies, and antibiotic therapy. prolonged discussions followed each presentation, with consensus regarding the major issues achieved before moving to the next topic. with input from the rest of the committee, each presenter and committee member assigned to the less controversial topics prepared an initial draft of their section, including grading of the evidence. iterative drafts of the statement were developed and distributed by e-mail for critique, followed by multiple revisions by the primary authors. a second face-to-face meeting was also held for discussion of the less controversial areas and further critique of the initial drafts. once general agreement on the separate topics was obtained, the cochairs incorporated the separate documents into a single statement, with substantial editing for style and consistency. the document was then redistributed to committee members to review and update with new information from the literature up to june 2006. recommended changes were reviewed by all committee members by e-mail and/or conference phone call and were incorporated into the final document by the cochairs. this document was then submitted to the societies for approval. each society independently selected reviewers, and changes recommended by the reviewers were discussed by the committee and incorporated into the final document. the guideline was then submitted to the idsa governing council and the ats board of directors for final approval. grading of guideline recommendations. initially, the committee decided to grade only the strength of the evidence, using a 3-tier scale (table 1) used in a recent guideline from both societies [10] . in response to reviewers' comments and the maturation of the field of guideline development [14] , a separate grading of the strength of the recommendations was added to the final draft. more extensive and validated criteria, such as grade [14] , were impractical for use at this stage. the 3-tier scale similar to that used in other idsa guideline documents [12] and familiar to many of the committee members was therefore chosen. the strength of each recommendation was graded as "strong," "moderate," or "weak." each committee member independently graded each recommendation on the basis of not only the evidence but also expert interpretation and clinical applicability. the final grading of each recommendation was a composite of the individual committee members' grades. for the final document, a strong recommendation required у6 (of 12) of the members to consider it to be strong and the majority of the others to grade it as moderate. the implication of a strong recommendation is that most patients should receive that intervention. significant variability in the management of patients with cap is well documented. some who use guidelines suggest that this variability itself is undesirable. industrial models suggesting that variability per se is undesirable may not always be relevant to medicine [15] . such models do not account for substantial variability among patients, nor do they account for variable end points, such as limitation of care in patients with end-stage underlying diseases who present with cap. for this reason, the committee members feel strongly that 100% compliance with guidelines is not the desired goal. however, the rationale for variation from a strongly recommended guideline should be apparent from the medical record. conversely, moderate or weak recommendations suggest that, even if a majority would follow the recommended management, many practitioners may not. deviation from guidelines may occur for a variety of reasons [16, 17] . one document cannot cover all of the variable settings, unique hosts, or epidemiologic patterns that may dictate alternative management strategies, and physician judgment should always supersede guidelines. this is borne out by the finding that deviation from guidelines is greatest in the treatment of patients with cap admitted to the icu [18] . in addition, few of the recommendations have level i evidence to support them, and most are, therefore, legitimate topics for future research. subsequent publication of studies documenting that care that deviates from guidelines results in better outcomes will stimulate revision of the guidelines. the committee anticipates that this will occur, and, for this reason, both the ats and idsa leaderships have committed to the revision of these guidelines on a regular basis. we recognize that these guidelines may be used as a measure of quality of care for hospitals and individual practitioners. although these guidelines are evidence based, the committee strongly urges that deviations from them not necessarily be considered substandard care, unless they are accompanied by evidence for worse outcomes in a studied population. 1. locally adapted guidelines should be implemented to improve process of care variables and relevant clinical outcomes. (strong recommendation; level i evidence.) enthusiasm for developing this set of cap guidelines derives, in large part, from evidence that previous cap guidelines have led to improvement in clinically relevant outcomes [17, [19] [20] [21] . protocol design varies among studies, and the preferable randomized, parallel group design has been used in only a small minority. confirmatory studies that use randomized, parallel groups with precisely defined treatments are still needed, but a consistent pattern of benefit is found in the other types of level i studies. documented benefits. published protocols have varied in primary focus and comprehensiveness, and the corresponding benefits vary from one study to another. however, the most impressive aspect of this literature is the consistently beneficial effect seen in some clinically relevant parameter after the introduction of a protocol that increases compliance with published guidelines. a decrease in mortality with the introduction of guidelinebased protocols was found in several studies [19, 21] . a 5-year study of 28,700 patients with pneumonia who were admitted during implementation of a pneumonia guideline demonstrated that the crude 30-day mortality rate was 3.2% lower with the guideline (adjusted or, 0.69; 95% ci, 0.49-0.97) [19] , compared with that among patients treated concurrently by nonaffiliated physicians. after implemention of a practice guideline at one spanish hospital [21] , the survival rate at 30 days was higher (or, 2.14; 95% ci, 1.23-3.72) than at baseline and in comparison with 4 other hospitals without overt protocols. lower mortality was seen in other studies, although the differences were not statistically significant [22, 23] . studies that documented lower mortality emphasized increasing the number of patients receiving guideline-recommended antibiotics, confirming results of the multivariate analysis of a retrospective review [24] . when the focus of a guideline was hospitalization, the number of less ill patients admitted to the hospital was consistently found to be lower. using admission decision support, a prospective study of 11700 emergency department (ed) visits in 19 hospitals randomized between pathway and "conventional" management found that admission rates among low-risk patients at pathway hospitals decreased (from 49% to 31% of patients in pneumonia severity index [psi] classes i-iii; p ! ) without differences in patient satisfaction scores or rate of .01 readmission [20] . calculating the psi score and assigning the risk class, providing oral clarithromycin, and home nursing follow-up significantly ( ) decreased the number of low-p p .01 mortality-risk admissions [25] . however, patient satisfaction among outpatients was lower after implementation of this guideline, despite survey data that suggested most patients would prefer outpatient treatment [26] . of patients discharged from the ed, 9% required hospitalization within 30 days, although another study showed lower readmission rates with the use of a protocol [23] . admission decision support derived from the 1993 ats guideline [5] recommendations, combined with outpatient antibiotic recommendations, reduced the cap hospitalization rate from 13.6% to 6.4% [23] , and admission rates for other diagnoses were unchanged. not surprisingly, the resultant overall cost of care decreased by half ( ). p p .01 protocols using guidelines to decrease the duration of hospitalization have also been successful. guideline implementation in 31 connecticut hospitals decreased the mean length of hospital stay (los) from 7 to 5 days ( ) [27] . an ed-p ! .001 based protocol decreased the mean los from 9.7 to 6.4 days ( ), with the benefits of guideline implementation p ! .0001 maintained 3 years after the initial study [22] . a 7-site trial, randomized by physician group, of guideline alone versus the same guideline with a multifaceted implementation strategy found that addition of an implementation strategy was associated with decreased duration of intravenous antibiotic therapy and los, although neither decrease was statistically significant [28] . several other studies used guidelines to significantly shorten the los, by an average of 11.5 days [20, 21] . markers of process of care can also change with the use of a protocol. the time to first antibiotic dose has been effectively decreased with cap protocols [22, 27, 29] . a randomized, parallel group study introduced a pneumonia guideline in 20 of 36 small oklahoma hospitals [29] , with the identical protocol implemented in the remaining hospitals in a second phase. serial measurement of key process measures showed significant improvement in time to first antibiotic dose and other variables, first in the initial 20 hospitals and later in the remaining 16 hospitals. implementing a guideline in the ed halved the time to initial antibiotic dose [22] . 2. cap guidelines should address a comprehensive set of elements in the process of care rather than a single element in isolation. (strong recommendation; level iii evidence.) common to all of the studies documented above, a comprehensive protocol was developed and implemented, rather than one addressing a single aspect of cap care. no study has documented that simply changing 1 metric, such as time to first antibiotic dose, is associated with a decrease in mortality. elements important in cap guidelines are listed in table 2. of these, rapid and appropriate empirical antibiotic therapy is consistently associated with improved outcome. we have also included elements of good care for general medical inpatients, such as early mobilization [30] and prophylaxis against thromboembolic disease [31] . although local guidelines need not include all elements, a logical constellation of elements should be addressed. in instituting cap protocol guidelines, the outcomes most relevant to the individual center or medical system should be addressed first. unless a desire to change clinically relevant outcomes exists, adherence to guidelines will be low, and institutional resources committed to implement the guideline are likely to be insufficient. guidelines for the treatment of pneumonia must use approaches that differ from current practice and must be successfully implemented before process of care and outcomes can change. for example, rhew et al. [32] designed a guideline to decrease los that was unlikely to change care, because the recommended median los was longer than the existing los for cap at the study hospitals. the difficulty in implementing guidelines and changing physician behavior has also been documented [28, 33] . clinically relevant outcome parameters should be evaluated to measure the effect of the local guideline. outcome parameters that can be used to measure the effect of implementation of a cap guideline within an organization are listed in table 3. just as it is important not to focus on one aspect of care, studying more than one outcome is also important. improvements in one area may be offset by worsening in a related area; for example, decreasing admission of low-acuity patients might increase the number of return visits to the ed or hospital readmissions [25] . almost all of the major decisions regarding management of cap, including diagnostic and treatment issues, revolve around the initial assessment of severity. we have, therefore, organized the guidelines to address this issue first. hospital admission decision. the initial management decision after diagnosis is to determine the site of care-outpatient, hospitalization in a medical ward, or admission to an icu. the decision to admit the patient is the most costly issue in the management of cap, because the cost of inpatient care for pneumonia is up to 25 times greater than that of outpatient care [34] and consumes the majority of the estimated $8.4-$10 billion spent yearly on treatment. other reasons for avoiding unnecessary admissions are that patients at low risk for death who are treated in the outpatient setting are able to resume normal activity sooner than those who are hospitalized, and 80% are reported to prefer outpatient therapy [26, 35] . hospitalization also increases the risk of thromboembolic events and superinfection by more-virulent or resistant hospital bacteria [36] . 4. severity-of-illness scores, such as the curb-65 criteria (confusion, uremia, respiratory rate, low blood pressure, age 65 years or greater), or prognostic models, such as the psi, can be used to identify patients with cap who may be candidates for outpatient treatment. (strong recommendation; level i evidence.) significant variation in admission rates among hospitals and among individual physicians is well documented. physicians often overestimate severity and hospitalize a significant number of patients at low risk for death [20, 37, 38] . because of these issues, interest in objective site-of-care criteria has led to attempts by a number of groups to develop such criteria [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] . the relative merits and limitations of various proposed criteria have been carefully evaluated [49] . the 2 most interesting are the psi [42] and the british thoracic society (bts) criteria [39, 45] . the psi is based on derivation and validation cohorts of 14,199 and 38,039 hospitalized patients with cap, respectively, plus an additional 2287 combined inpatients and outpatients [42] . the psi stratifies patients into 5 mortality risk classes, and its ability to predict mortality has been confirmed in multiple subsequent studies. on the basis of associated mortality rates, it has been suggested that risk class i and ii patients should be treated as outpatients, risk class iii patients should be treated in an observation unit or with a short hospitalization, and risk class iv and v patients should be treated as inpatients [42] . yealy et al. [50] conducted a cluster-randomized trial of low-, moderate-, and high-intensity processes of guideline implementation in 32 eds in the united states. their guideline used the psi for admission decision support and included recommendations for antibiotic therapy, timing of first antibiotic dose, measurement of oxygen saturation, and blood cultures for admitted patients. eds with moderate-to high-intensity guideline implementation demonstrated more outpatient treatment of low-risk patients and higher compliance with antibiotic recommendations. no differences were found in mortality rate, rate of hospitalization, median time to return to work or usual activities, or patient satisfaction. this study differs from those reporting a mortality rate difference [19, 21] in that many hospitalized patients with pneumonia were not included. in addition, eds with low-intensity guideline implementation formed the comparison group, rather than eds practicing nonguideline, usual pneumonia care. the bts original criteria of 1987 have subsequently been modified [39, 51] . in the initial study, risk of death was increased 21-fold if a patient, at the time of admission, had at least 2 of the following 3 conditions: tachypnea, diastolic hypotension, and an elevated blood urea nitrogen (bun) level. these criteria appear to function well except among patients with underlying renal insufficiency and among elderly patients [52, 53] . the most recent modification of the bts criteria includes 5 easily measurable factors [45] . multivariate analysis of 1068 patients identified the following factors as indicators of increased mortality: confusion (based on a specific mental test or disorientation to person, place, or time), bun level 17 mmol/l (20 mg/dl), respiratory rate у30 breaths/min, low blood pressure (systolic, !90 mm hg; or diastolic, р60 mm hg), and age у65 years; this gave rise to the acronym curb-65. in the derivation and validation cohorts, the 30-day mortality among patients with 0, 1, or 2 factors was 0.7%, 2.1%, and 9.2%, respectively. mortality was higher when 3, 4, or 5 factors were present and was reported as 14.5%, 40%, and 57%, respectively. the authors suggested that patients with a curb-65 score of 0-1 be treated as outpatients, that those with a score of 2 be admitted to the wards, and that patients with a score of у3 often required icu care. a simplified version (crb-65), which does not require testing for bun level, may be appropriate for decision making in a primary care practitioner's office [54] . the use of objective admission criteria clearly can decrease the number of patients hospitalized with cap [20, 23, 25, 55] . whether the psi or the curb-65 score is superior is unclear, because no randomized trials of alternative admission criteria exist. when compared in the same population, the psi classified a slightly larger percentage of patients with cap in the lowrisk categories, compared with the curb or curb-65 criteria, while remaining associated with a similar low mortality rate among patients categorized as low risk [56] . several factors are important in this comparison. the psi includes 20 different variables and, therefore, relies on the availability of scoring sheets, limiting its practicality in a busy ed [55] . in contrast, the curb-65 criteria are easily remembered. however, curb-65 has not been as extensively studied as the psi, especially with prospective validation in other patient populations (e.g., the indigent inner-city population), and has not been specifically studied as a means of reducing hospital admission rates. in eds with sufficient decision support resources (either human or computerized), the benefit of greater experience with the psi score may favor its use for screening patients who may be candidates for outpatient management [50, [57] [58] [59] . 5 . objective criteria or scores should always be supplemented with physician determination of subjective factors, including the ability to safely and reliably take oral medication and the availability of outpatient support resources. (strong recommendation; level ii evidence.) studies show that certain patients with low psi or curb-65 scores [20, 60, 61] require hospital admission, even to the icu [49, 62, 63] . both scores depend on certain assumptions. one is that the main rationale for admission of a patient with cap is risk of death. this assumption is clearly not valid in all cases. another is that the laboratory and vital signs used for scoring are stable over time rather than indicative of transient abnormalities. this is also not true in all cases. therefore, dynamic assessment over several hours of observation may be more accurate than a score derived at a single point in time. although advantageous to making decisions regarding hospital admission, sole reliance on a score for the hospital admission decision is unsafe. reasons for the admission of low-mortality-risk patients fall into 4 categories: (1) complications of the pneumonia itself, (2) exacerbation of underlying diseases(s), (3) inability to reliably take oral medications or receive outpatient care, and/or (4) multiple risk factors falling just above or below thresholds for the score [62] . use of the psi score in clinical trials has demonstrated some of its limitations, which may be equally applicable to other scoring techniques. a modification of the original psi score was needed when it was applied to the admission decision. an arterial saturation of !90% or an arterial oxygen pressure (pao 2 ) of !60 mm hg as a complication of the pneumonia, was added as a sole indicator for admission for patients in risk classes i-iii as an added "margin of safety" in one trial [42] . in addition to patients who required hospital admission because of hypoxemia, a subsequent study identified patients in low psi risk classes (i-iii) who needed hospital admission because of shock, decompensated coexisting illnesses, pleural effusion, inability to maintain oral intake, social problems (the patient was dependent or no caregiver was available), and lack of response to previous adequate empirical antibiotic therapy [64] . of 178 patients in low psi risk classes who were treated as inpatients, 106 (60%) presented with at least 1 of these factors. other medical or psychosocial needs requiring hospital care include intractable vomiting, injection drug abuse, severe psychiatric illness, homelessness, poor overall functional status [65] , and cognitive dysfunction [61, 66] . the psi score is based on a history of diseases that increase risk of death, whereas the curb-65 score does not directly address underlying disease. however, pneumonia may exacerbate an underlying disease, such as obstructive lung disease, congestive heart failure, or diabetes mellitus, which, by themselves, may require hospital admission [60, 67] . atlas et al. [25] were able to reduce hospital admissions among patients in psi risk classes i-iii from 58% in a retrospective control group to 43% in a psi-based intervention group. ten of 94 patients in the latter group (compared with 0 patients in the control population) were subsequently admitted, several for reasons unrelated to their pneumonia. also, the presence of rare illnesses, such as neuromuscular or sickle cell disease, may require hospitalization but not affect the psi score. the necessary reliance on dichotomous predictor variables (abnormal vs. normal) in most criteria and the heavy reliance on age as a surrogate in the psi score may oversimplify their use for admission decisions. for example, a previously healthy 25-year-old patient with severe hypotension and tachycardia and no additional pertinent prognostic factors would be placed in risk class ii, whereas a 70-year-old man with a history of localized prostate cancer diagnosed 10 months earlier and no other problems would be placed in risk class iv [42] . finally, patient satisfaction was lower among patients treated outside the hospital in one study with a psi-based intervention group [25] , suggesting that the savings resulting from use of the psi may be overestimated and that physicians should consider additional factors not measured by the psi. 6. for patients with curb-65 scores у2, more-intensive treatment-that is, hospitalization or, where appropriate and available, intensive in-home health care services-is usually warranted. (moderate recommendation; level iii evidence.) although the psi and curb-65 criteria are valuable aids in avoiding inappropriate admissions of low-mortality-risk patients, another important role of these criteria may be to help identify patients at high risk who would benefit from hospitalization. the committee preferred the curb-65 criteria because of ease of use and because they were designed to measure illness severity more than the likelihood of mortality. patients with a curb-65 score у2 are not only at increased risk of death but also are likely to have clinically important physiologic derangements requiring active intervention. these patients should usually be considered for hospitalization or for aggressive in-home care, where available. in a cohort of ∼3000 patients, the mortality with a curb-65 score of 0 was only 1.2%, whereas 3-4 points were associated with 31% mortality [45] . because the psi score is not based as directly on severity of illness as are the curb-65 criteria, a threshold for patients who would require hospital admission or intensive outpatient treatment is harder to define. the higher the score, the greater the need for hospitalization. however, even a patient who meets criteria for risk class v on the basis of very old age and multiple stable chronic illnesses may be successfully managed as an outpatient [23] . 7. direct admission to an icu is required for patients with septic shock requiring vasopressors or with acute respiratory failure requiring intubation and mechanical ventilation. (strong recommendation; level ii evidence.) the second-level admission decision is whether to place the patient in the icu or a high-level monitoring unit rather than on a general medical floor. approximately 10% of hospitalized patients with cap require icu admission [68] [69] [70] , but the indications vary strikingly among patients, physicians, hospitals, and different health care systems. some of the variability among institutions results from the availability of high-level monitoring or intermediate care units appropriate for patients at increased risk of complications. because respiratory failure is the major reason for delayed transfer to the icu, simple cardiac monitoring units would not meet the criteria for a highlevel monitoring unit for patients with severe cap. one of the most important determinants of the need for icu care is the presence of chronic comorbid conditions [68] [69] [70] [71] [72] . however, approximately one-third of patients with severe cap were previously healthy [73] . the rationale for specifically defining severe cap is 4-fold: • appropriate placement of patients optimizes use of limited icu resources. • transfer to the icu for delayed respiratory failure or delayed onset of septic shock is associated with increased mortality [74] . although low-acuity icu admissions do occur, the major concern is initial admission to the general medical unit, with subsequent transfer to the icu. as many as 45% of patients with cap who ultimately require icu admission were initially admitted to a non-icu setting [75] . many delayed transfers to the icu represent rapidly progressive pneumonia that is not obvious on admission. however, some have subtle findings, including those included in the minor criteria in table 4, which might warrant direct admission to the icu. • the distribution of microbial etiologies differs from that of cap in general [76] [77] [78] [79] , with significant implications for diagnostic testing and empirical antibiotic choices. avoidance of inappropriate antibiotic therapy has also been associated with lower mortality [80, 81] . • patients with cap appropriate for immunomodulatory treatment must be identified. the systemic inflammatory response/severe sepsis criteria typically used for generic sepsis trials may not be adequate when applied specifically to severe cap [82] . for example, patients with unilateral lobar pneumonia may have hypoxemia severe enough to meet criteria for acute lung injury but not have a systemic response. several criteria have been proposed to define severe cap. most case series have defined it simply as cap that necessitates icu admission. objective criteria to identify patients for icu admission include the initial ats definition of severe cap [5] and its subsequent modification [6, 82] , the curb criteria [39, 45] , and psi severity class v (or iv and v) [42] . however, none of these criteria has been prospectively validated for the icu admission decision. recently, these criteria were retrospectively evaluated in a cohort of patients with cap admitted to the icu [63] . all were found to be both overly sensitive and nonspecific in comparison with the original clinical decision to admit to the icu. revisions of the criteria or alternative criteria were, therefore, recommended. for the revised criteria, the structure of the modified ats criteria for severe cap was retained [6] . the 2 major criteriamechanical ventilation with endotracheal intubation and septic shock requiring vasopressors-are absolute indications for admission to an icu. in contrast, the need for icu admission is less straightforward for patients who do not meet the major criteria. on the basis of the published operating characteristics of the criteria, no single set of minor criteria is adequate to define severe cap. both the ats minor criteria [75] and the curb criteria [45] have validity when predicting which patients will be at increased risk of death. therefore, the ats minor criteria and the curb variables were included in the new proposed minor criteria (table 4) . age, by itself, was not felt to be an appropriate factor for the icu admission decision, but the remainder of the curb-65 criteria [45] were retained as minor criteria (with the exception of hypotension requiring vasopressors as a major criterion). rather than the complex criteria for confusion in the original curb studies, the definition of confusion should be new-onset disorientation to person, place, or time. three additional minor criteria were added. leukopenia (white blood cell count, !4000 cells/mm 3 ) resulting from cap has consistently been associated with excess mortality, as well as with an increased risk of complications such as acute respiratory distress syndrome (ards) [77, 79, [83] [84] [85] [86] [87] . in addition, leukopenia is seen not only in bacteremic pneumococcal disease but also in gram-negative cap [88, 89] . when leukopenia occurs in patients with a history of alcohol abuse, the adverse manifestations of septic shock and ards may be delayed or masked. therefore, these patients were thought to benefit from icu monitoring. the coagulation system is often activated in cap, and development of thrombocytopenia (platelet count, !100,000 cells/mm 3 ) is also associated with a worse prognosis [86, [90] [91] [92] . nonexposure hypothermia (core temperature, !36њc) also carries an ominous prognosis in cap [83, 93] . the committee felt that there was sufficient justification for including these additional factors as minor criteria. other factors associated with increased mortality due to cap were also considered, including acute alcohol ingestion and delirium tremens [79, 85, 94] , hypoglycemia and hyperglycemia, occult metabolic acidosis or elevated lactate levels [91] , and hyponatremia [95] . however, many of these criteria overlap with those selected. future studies validating the proposed criteria should record these factors as well, to determine whether addition or substitution improves the predictive value of our proposed criteria. with the addition of more minor criteria, the threshold for icu admission was felt to be the presence of at least 3 minor criteria, based on the mortality association with the curb criteria. selecting 2 criteria appears to be too nonspecific, as is demonstrated by the initial ats criteria [5] . whether each of the criteria is of equal weight is also not clear. therefore, prospective validation of this set of criteria is clearly needed. 9. in addition to a constellation of suggestive clinical features, a demonstrable infiltrate by chest radiograph or other imaging technique, with or without supporting microbiological data, is required for the diagnosis of pneumonia. (moderate recommendation; level iii evidence.) the diagnosis of cap is based on the presence of select clinical features (e.g., cough, fever, sputum production, and pleuritic chest pain) and is supported by imaging of the lung, usually by chest radiography. physical examination to detect rales or bronchial breath sounds is an important component of the evaluation but is less sensitive and specific than chest radiographs [96] . both clinical features and physical exam findings may be lacking or altered in elderly patients. all patients should be screened by pulse oximetry, which may suggest both the presence of pneumonia in patients without obvious signs of pneumonia and unsuspected hypoxemia in patients with diagnosed pneumonia [42, 97, 98] . a chest radiograph is required for the routine evaluation of patients who are likely to have pneumonia, to establish the diagnosis and to aid in differentiating cap from other common causes of cough and fever, such as acute bronchitis. chest radiographs are sometimes useful for suggesting the etiologic agent, prognosis, alternative diagnoses, and associated conditions. rarely, the admission chest radiograph is clear, but the patient's toxic appearance suggests more than bronchitis. ct scans may be more sensitive, but the clinical significance of these findings when findings of radiography are negative is unclear [99] . for patients who are hospitalized for suspected pneumonia but who have negative chest radiography findings, it may be reasonable to treat their condition presumptively with antibiotics and repeat the imaging in 24-48 h. microbiological studies may support the diagnosis of pneumonia due to an infectious agent, but routine tests are frequently falsely negative and are often nonspecific. a history of recent travel or endemic exposure, if routinely sought, may identify specific potential etiologies that would otherwise be unexpected as a cause of cap (see table 8 ) [100] . 10. patients with cap should be investigated for specific pathogens that would significantly alter standard (empirical) management decisions, when the presence of such pathogens is suspected on the basis of clinical and epidemiologic clues. (strong recommendation; level ii evidence.) the need for diagnostic testing to determine the etiology of cap can be justified from several perspectives. the primary reason for such testing is if results will change the antibiotic management for an individual patient. the spectrum of antibiotic therapy can be broadened, narrowed, or completely altered on the basis of diagnostic testing. the alteration in therapy that is potentially most beneficial to the individual is an escalation or switch of the usual empirical regimen because of unusual pathogens (e.g., endemic fungi or mycobacterium tuberculosis) or antibiotic resistance issues. broad empirical coverage, such as that recommended in these guidelines, would not provide the optimal treatment for certain infections, such as psittacosis or tularemia. increased mortality [80] and increased risk of clinical failure [81, 101] are more common with inappropriate antibiotic therapy. management of initial antibiotic failure is greatly facilitated by an etiologic diagnosis at admission. de-escalation or narrowing of antibiotic therapy on the basis of diagnostic testing is less likely to decrease an individual's risk of death but may decrease cost, drug adverse effects, and antibiotic resistance pressure. some etiologic diagnoses have important epidemiologic implications, such as documentation of severe acute respiratory syndrome (sars), influenza, legionnaires disease, or agents of bioterrorism. diagnostic testing for these infections may affect not only the individual but also many other people. although pneumonia etiologies that should be reported to public health officials vary by state, in general, most states' health regulations require reporting of legionnaires disease, sars, psittacosis, avian influenza (h5n1), and possible agents of bioterrorism (plague, tularemia, and anthrax). in addition, specific diagnostic testing and reporting are important for pneumonia cases of any etiology thought to be part of a cluster or caused by pathogens not endemic to the area. there are also societal reasons for encouraging diagnostic testing. the antibiotic recommendations in the present guidelines are based on culture results and sensitivity patterns from patients with positive etiologic diagnoses [102] . without the accumulated information available from these culture results, trends in antibiotic resistance are more difficult to track, and empirical antibiotic recommendations are less likely to be accurate. the main downside of extensive diagnostic testing of all patients with cap is cost, which is driven by the poor quality of most sputum microbiological samples and the low yield of positive culture results in many groups of patients with cap. a clear need for improved diagnostic testing in cap, most likely using molecular methodology rather than culture, has been recognized by the national institutes of health [103] . the cost-benefit ratio is even worse when antibiotic therapy is not streamlined when possible [104, 105] or when inappropriate escalation occurs [95] . in clinical practice, narrowing of antibiotic therapy is, unfortunately, unusual, but the committee strongly recommends this as best medical practice. the possibility of polymicrobial cap and the potential benefit of combination therapy for bacteremic pneumococcal pneumonia have complicated the decision to narrow antibiotic therapy. delays in starting antibiotic therapy that result from the need to obtain specimens, complications of invasive diagnostic procedures, and unneeded antibiotic changes and additional testing for false-positive tests are also important considerations. the general recommendation of the committee is to strongly encourage diagnostic testing whenever the result is likely to change individual antibiotic management. for other patients with cap, the recommendations for diagnostic testing focus on patients in whom the diagnostic yield is thought to be greatest. these 2 priorities often overlap. recommendations for patients in whom routine diagnostic testing is indicated for the above reasons are listed in retrospective studies of outpatient cap management usually show that diagnostic tests to define an etiologic pathogen are infrequently performed, yet most patients do well with empir-ical antibiotic treatment [42, 106] . exceptions to this general rule may apply to some pathogens important for epidemiologic reasons or management decisions. the availability of rapid point-of-care diagnostic tests, specific treatment and chemoprevention, and epidemiologic importance make influenza testing the most logical. influenza is often suspected on the basis of typical symptoms during the proper season in the presence of an epidemic. however, respiratory syncytial virus (rsv) can cause a similar syndrome and often occurs in the same clinical scenario [107] . rapid diagnostic tests may be indicated when the diagnosis is uncertain and when distinguishing influenza a from influenza b is important for therapeutic decisions. other infections that are important to verify with diagnostic studies because of epidemiologic implications or because they require unique therapeutic intervention are sars and avian (h5n1) influenza, disease caused by agents of bioterrorism, legionella infection, community-acquired mrsa (ca-mrsa) infection, m. tuberculosis infection, or endemic fungal infection. attempts to establish an etiologic diagnosis are also appropriate in selected cases associated with outbreaks, specific risk factors, or atypical presentations. 12. pretreatment blood samples for culture and an expectorated sputum sample for stain and culture (in patients with a productive cough) should be obtained from hospitalized patients with the clinical indications listed in the only randomized controlled trial of diagnostic strategy in cap has demonstrated no statistically significant differences in mortality rate or los between patients receiving pathogendirected therapy and patients receiving empirical therapy [108] . however, pathogen-directed therapy was associated with lower mortality among the small number of patients admitted to the icu. the study was performed in a country with a low incidence of antibiotic resistance, which may limit its applicability to areas with higher levels of resistance. adverse effects were significantly more common in the empirical therapy group but may have been unique to the specific antibiotic choice (erythromycin). the lack of benefit overall in this trial should not be interpreted as a lack of benefit for an individual patient. therefore, performing diagnostic tests is never incorrect or a breach of the standard of care. however, information from cohort and observational studies may be used to define patient groups in which the diagnostic yield is increased. patient groups in which routine diagnostic testing is indicated and the recommended tests are listed in table 5. blood cultures. pretreatment blood cultures yielded positive results for a probable pathogen in 5%-14% in large series of nonselected patients hospitalized with cap [104, 105, [109] [110] [111] . the yield of blood cultures is, therefore, relatively low (although it is similar to yields in other serious infections), and, when management decisions are analyzed, the impact of positive blood cultures is minor [104, 105] . the most common blood culture isolate in all cap studies is s. pneumoniae. because this bacterial organism is always considered to be the most likely pathogen, positive blood culture results have not clearly led to better outcomes or improvements in antibiotic selection [105, 112] . false-positive blood culture results are associated with prolonged hospital stay, possibly related to changes in management based on preliminary results showing gram-positive cocci, which eventually prove to be coagulasenegative staphylococci [95, 109] . in addition, false-positive blood culture results have led to significantly more vancomycin use [95] . for these reasons, blood cultures are optional for all hospitalized patients with cap but should be performed selectively (table 5). the yield for positive blood culture results is halved by prior antibiotic therapy [95] . therefore, when performed, samples for blood culture should be obtained before antibiotic administration. however, when multiple risk factors for bacteremia are present, blood culture results after initiation of antibiotic therapy are still positive in up to 15% of cases [95] and are, therefore, still warranted in these cases, despite the lower yield. the strongest indication for blood cultures is severe cap. patients with severe cap are more likely to be infected with pathogens other than s. pneumoniae, including s. aureus, p. aeruginosa, and other gram-negative bacilli [77-80, 95, 113, 114] . many of the factors predictive of positive blood culture results [95] overlap with risk factors for severe cap (table 4) . therefore, blood cultures are recommended for all patients with severe cap because of the higher yield, the greater possibility of the presence of pathogens not covered by the usual empirical antibiotic therapy, and the increased potential to affect antibiotic management. blood cultures are also indicated when patients have a host defect in the ability to clear bacteremia-for example, as a result of asplenia or complement deficiencies. patients with chronic liver disease also are more likely to have bacteremia with cap [95] . leukopenia is also associated with a high incidence of bacteremia [79, 95] . respiratory tract specimen gram stain and culture. the yield of sputum bacterial cultures is variable and strongly influenced by the quality of the entire process, including specimen collection, transport, rapid processing, satisfactory use of cytologic criteria, absence of prior antibiotic therapy, and skill in interpretation. the yield of s. pneumoniae, for example, is only 40%-50% from sputum cultures from patients with bacteremic pneumococcal pneumonia in studies performed a few decades ago [115, 116] . a more recent study of 100 cases of bacteremic pneumococcal pneumonia found that sputum specimens were not submitted in 31% of cases and were judged as inadequate in another 16% of cases [117] . when patients receiving antibiotics for 124 h were excluded, gram stain showed pneumococci in 63% of sputum specimens, and culture results were positive in 86%. for patients who had received no antibiotics, the gram stain was read as being consistent with pneumococci in 80% of cases, and sputum culture results were positive in 93%. although there are favorable reports of the utility of gram stain [118] , a meta-analysis showed a low yield, considering the number of patients with adequate specimens and definitive results [119] . recent data show that an adequate specimen with a predominant morphotype on gram stain was found in only 14% of 1669 hospitalized patients with cap [120] . higher psi scores did not predict higher yield. however, a positive gram stain was highly predictive of a subsequent positive culture result. the benefit of a sputum gram stain is, therefore, 2-fold. first, it broadens initial empirical coverage for less common etiologies, such as infection with s. aureus or gram-negative organisms. this indication is probably the most important, because it will lead to less inappropriate antibiotic therapy. second, it can validate the subsequent sputum culture results. forty percent or more of patients are unable to produce any sputum or to produce sputum in a timely manner [108, 120] . the yield of cultures is substantially higher with endotracheal aspirates, bronchoscopic sampling, or transthoracic needle aspirates [120] [121] [122] [123] [124] [125] [126] , although specimens obtained after initiation of antibiotic therapy are unreliable and must be interpreted carefully [120, 127, 128] . interpretation is improved with quantitative cultures of respiratory secretions from any source (sputum, tracheal aspirations, and bronchoscopic aspirations) or by interpretation based on semiquantitative culture results [122, 123, 129] . because of the significant influence on diagnostic yield and cost effectiveness, careful attention to the details of specimen handling and processing are critical if sputum cultures are obtained. because the best specimens are collected and processed before antibiotics are given, the time to consider obtaining expectorated sputum specimens from patients with factors listed in table 5 is before initiation of antibiotic therapy. once again, the best indication for more extensive respiratory tract cultures is severe cap. gram stain and culture of endotracheal aspirates from intubated patients with cap produce different results than expectorated sputum from non-icu patients [76, 120] . many of the pathogens in the broader microbiological spectrum of severe cap are unaffected by a single dose of antibiotics, unlike s. pneumoniae. in addition, an endotracheal aspirate does not require patient cooperation, is clearly a lower respiratory tract sample, and is less likely to be contaminated by oropharyngeal colonizers. nosocomial tracheal colonization is not an issue if the sample is obtained soon after intubation. therefore, culture and gram stain of endotracheal aspirates are recommended for patients intubated for severe cap. in addition to routine cultures, a specific request for culture of respiratory secretions on buffered charcoal yeast extract agar to isolate legionella species may be useful in this subset of patients with severe cap in areas where legionella is endemic, as well as in patients with a recent travel history [130] . the fact that a respiratory tract culture result is negative does not mean that it has no value. failure to detect s. aureus or gram-negative bacilli in good-quality specimens is strong evidence against the presence of these pathogens. growth inhibition by antibiotics is lower with these pathogens than with s. pneumoniae, but specimens obtained after initiation of antibiotic therapy are harder to interpret, with the possibility of colonization. necrotizing or cavitary pneumonia is a risk for ca-mrsa infection, and sputum samples should be obtained in all cases. negative gram stain and culture results should be adequate to withhold or stop treatment for mrsa infection. severe copd and alcoholism are major risk factors for infection with p. aeruginosa and other gram-negative pathogens [131] . once again, gram stain and culture of an adequate sputum specimen are usually adequate to exclude the need for empirical coverage of these pathogens. a sputum culture in patients with suspected legionnaires disease is important, because the identification of legionella species implies the possibility of an environmental source to which other susceptible individuals may be exposed. localized community outbreaks of legionnaires disease might be recognized by clinicians or local health departments because у2 patients might be admitted to the same hospital. however, outbreaks of legionnaires disease associated with hotels or cruise ships [132] [133] [134] are rarely detected by individual clinicians, because travelers typically disperse from the source of infection before developing symptoms. therefore, a travel history should be actively sought from patients with cap, and legionella testing should be performed for those who have traveled in the 2 weeks before the onset of symptoms. urinary antigen tests may be adequate to diagnose and treat an individual, but efforts to obtain a sputum specimen for culture are still indicated to facilitate epidemiologic tracking. the availability of a culture isolate of legionella dramatically improves the likelihood that an environmental source of legionella can be identified and remediated [135] [136] [137] . the yield of sputum culture is increased to 43%-57% when associated with a positive urinary antigen test result [138, 139] . attempts to obtain a sample for sputum culture from a patient with a positive pneumococcal urinary antigen test result may be indicated for similar reasons. patients with a productive cough and positive urinary antigen test results have positive sputum culture results in as many as 40%-80% of cases [140] [141] [142] [143] . in these cases, not only can sensitivity testing confirm the appropriate choice for the individual patient, but important data regarding local community antibiotic resistance rates can also be acquired. other cultures. patients with pleural effusions 15 cm in height on a lateral upright chest radiograph [111] should undergo thoracentesis to yield material for gram stain and culture for aerobic and anaerobic bacteria. the yield with pleural fluid cultures is low, but the impact on management decisions is substantial, in terms of both antibiotic choice and the need for drainage. nonbronchoscopic bronchoalveolar lavage (bal) in the ed has been studied in a small, randomized trial of intubated patients with cap [144] . a high percentage (87%) of nonbronchoscopic bal culture results were positive, even in some patients who had already received their first dose of antibiotics. unfortunately, tracheal aspirates were obtained from only a third of patients in the control group, but they all were culture positive. therefore, it is unclear that endotracheal aspirates are inferior to nonbronchoscopic bal. the use of bronchoscopic bal, protected specimen brushing, or transthoracic lung aspiration has not been prospectively studied for initial management of patients with cap [123] . the best indications are for immunocompromised patients with cap or for patients with cap in whom therapy failed [101, 145] . antigen tests. urinary antigen tests are commercially available and have been cleared by the us food and drug administration (fda) for detection of s. pneumoniae and l. pneumophila serogroup 1 [138, 140, [146] [147] [148] [149] . urinary antigen testing appears to have a higher diagnostic yield in patients with more severe illness [139, 140] . for pneumococcal pneumonia, the principal advantages of antigen tests are rapidity (∼15 min), simplicity, reasonable specificity in adults, and the ability to detect pneumococcal pneumonia after antibiotic therapy has been started. studies in adults show a sensitivity of 50%-80% and a specificity of 190% [146, 149, 150] . this is an attractive test for detecting pneumococcal pneumonia when samples for culture cannot be obtained in a timely fashion or when antibiotic therapy has already been initiated. serial specimens from patients with known bacteremia were still positive for pneumococcal urinary antigen in 83% of cases after 3 days of therapy [147] . comparisons with gram stain show that these 2 rapidly available tests often do not overlap, with only 28% concordance (25 of 88) among patients when results of either test were positive [140] . only ∼50% of binax pneumococcal urinary antigen-positive patients can be diagnosed by conventional methods [140, 150] . disadvantages include cost (approximately $30 per specimen), although this is offset by increased diagnosis-related group-based reimbursement for coding for pneumococcal pneumonia, and the lack of an organism for in vitro susceptibility tests. falsepositive results have been seen in children with chronic respiratory diseases who are colonized with s. pneumoniae [151] and in patients with an episode of cap within the previous 3 months [152] , but they do not appear to be a significant problem in colonized patients with copd [140, 152] . for legionella, several urinary antigen assays are available, but all detect only l. pneumophila serogroup 1. although this particular serogroup accounts for 80%-95% of communityacquired cases of legionnaires disease [138, 153] in many areas of north america, other species and serogroups predominate in specific locales [154, 155] . prior studies of culture-proven legionnaires disease indicate a sensitivity of 70%-90% and a specificity of nearly 99% for detection of l. pneumophila serogroup 1. the urine is positive for antigen on day 1 of illness and continues to be positive for weeks [138, 150] . the major issue with urinary bacterial antigen detection is whether the tests allow narrowing of empirical antibiotic therapy to a single specific agent. the recommended empirical antibiotic regimens will cover both of these microorganisms. results of a small observational study suggest that therapy with a macrolide alone is adequate for hospitalized patients with cap who test positive for l. pneumophila urinary antigen [156] . further research is needed in this area. in contrast, rapid antigen detection tests for influenza, which can also provide an etiologic diagnosis within 15-30 min, can lead to consideration of antiviral therapy. test performance varies according to the test used, sample type, duration of illness, and patient age. most show a sensitivity of 50%-70% in adults and a specificity approaching 100% [157] [158] [159] . advantages include the high specificity, the ability of some assays to distinguish between influenza a and b, the rapidity with which the results can be obtained, the possibly reduced use of antibacterial agents, and the utility of establishing this diagnosis for epidemiologic purposes, especially in hospitalized patients who may require infection control precautions. disadvantages include cost (approximately $30 per specimen), high rates of false-negative test results, false-positive assays with adenovirus infections, and the fact that the sensitivity is not superior to physician judgment among patients with typical symptoms during an influenza epidemic [157, 158, 160] . direct fluorescent antibody tests are available for influenza and rsv and require ∼2 h. for influenza virus, the sensitivity is better than with the point-of-care tests (85%-95%). they will detect animal subtypes such as h5n1 and, thus, may be preferred for hospitalized patients [161, 162] . for rsv, direct fluorescent antibody tests are so insensitive (sensitivity, 20%-30%) in adults that they are rarely of value [163] . acute-phase serologic testing. the standard for diagnosis of infection with most atypical pathogens, including chlamydophila pneumoniae, mycoplasma pneumoniae, and legionella species other than l. pneumophila, relies on acute-and convalescent-phase serologic testing. most studies use a microimmunofluorescence serologic test, but this test shows poor reproducibility [164] . management of patients on the basis of a single acute-phase titer is unreliable [165] , and initial antibiotic therapy will be completed before the earliest time point to check a convalescent-phase specimen. a new pcr test (bd probetec et legionella pneumophila; becton dickinson) that will detect all serotypes of l. pneumophila in sputum is now cleared by the fda, but extensive published clinical experience is lacking. most pcr reagents for other respiratory pathogens (except mycobacterium species) are "home grown," with requirements for use based on compliance with nccls criteria for analytical validity [166] . despite the increasing use of these tests for atypical pathogens [167, 168] , a 2001 review by the centers for disease control and prevention (cdc) of diagnostic assays for detection of c. pneumoniae indicated that, of the 18 pcr reagents, only 4 satisfied the criteria for a validated test [166] . the diagnostic criteria defined in this review are particularly important for use in prospective studies of cap, because most prior reports used liberal criteria, which resulted in exaggerated rates. for sars, several pcr assays have been developed, but these tests are inadequate because of high rates of false-negative assays in early stages of infection [169, 170] . a major goal of therapy is eradication of the infecting organism, with resultant resolution of clinical disease. as such, antimicrobials are a mainstay of treatment. appropriate drug selection is dependent on the causative pathogen and its antibiotic susceptibility. acute pneumonia may be caused by a wide variety of pathogens (table 6) . however, until more accurate and rapid diagnostic methods are available, the initial treatment for most patients will remain empirical. recommendations for therapy (table 7) apply to most cases; however, physicians should consider specific risk factors for each patient (table 8) . a syndromic approach to therapy (under the assumption that an etiology correlates with the presenting clinical manifestations) is not specific enough to reliably predict the etiology of cap [172] [173] [174] . even if a microbial etiology is identified, debate continues with regard to pathogen-specific treatment, because recent studies suggest coinfection by atypical pathogens (such as c. pneumoniae, legionella species, and viruses) and more traditional bacteria [120, 175] . however, the importance of treating multiple infecting organisms has not been firmly established. the majority of antibiotics released in the past several decades have an fda indication for cap, making the choice of antibiotics potentially overwhelming. selection of antimicrobial regimens for empirical therapy is based on prediction of the most likely pathogen(s) and knowledge of local susceptibility patterns. recommendations are generally for a class of antibiotics rather than a specific drug, unless outcome data clearly favor one drug. because overall efficacy remains good for many classes of agents, the more potent drugs are given preference because of their benefit in decreasing the risk of selection for antibiotic resistance. other factors for consideration of specific antimicrobials include pharmacokinetics/pharmacodynamics, compliance, safety, and cost. although cap may be caused by a myriad of pathogens, a limited number of agents are responsible for most cases. the emergence of newly recognized pathogens, such as the novel sars-associated coronavirus [170] , continually increases the challenge for appropriate management. table 6 lists the most common causes of cap, in decreasing order of frequency of occurrence and stratified for severity of illness as judged by site of care (ambulatory vs. hospitalized). s. pneumoniae is the most frequently isolated pathogen. other bacterial causes include nontypeable haemophilus influenzae and moraxella catarrhalis, generally in patients who have underlying bronchopulmonary disease, and s. aureus, especially during an influenza outbreak. risks for infection with enterobacteriaceae species and p. aeruginosa as etiologies for cap are chronic oral steroid administration or severe underlying bronchopulmonary disease, alcoholism, and frequent antibiotic therapy [79, 131] , whereas recent hospitalization would define cases as hcap. less common causes of pneumonia include, but are by no means limited to, streptococcus pyogenes, neisseria meningitidis, pasteurella multocida, and h. influenzae type b. the "atypical" organisms, so called because they are not detectable on gram stain or cultivatable on standard bacteriologic media, include m. pneumoniae, c. pneumoniae, legionella species, and respiratory viruses. with the exception of legionella species, these microorganisms are common causes of pneumonia, especially among outpatients. however, these pathogens are not often identified in clinical practice because, with a few exceptions, such as l. pneumophila and influenza virus, no specific, rapid, or standardized tests for their detection exist. although influenza remains the predominant viral cause of cap in adults, other commonly recognized viruses include rsv [107] , adenovirus, and parainfluenza virus, as well as less common viruses, including human metapneumovirus, herpes simplex virus, varicella-zoster virus, sars-associated coronavirus, and measles virus. in a recent study of immunocompetent adult patients admitted to the hospital with cap, 18% had evidence of a viral etiology, and, in 9%, a respiratory virus was the only pathogen identified [176] . studies that include outpatients find viral pneumonia rates as high as 36% [167] . the frequency of other etiologic agents-for example, m. tuberculosis, chlamydophila psittaci (psittacosis), coxiella burnetii (q fever), francisella tularensis (tularemia), bordetella pertussis (whooping cough), and endemic fungi (histoplasma capsulatum, coccidioides immitis, cryptococcus neoformans, and blastomyces hominis)-is largely determined by the epidemiologic setting (table 8) but rarely exceeds 2%-3% total [113, 177] . the exception may be endemic fungi in the appropriate geographic distribution [100] . the need for specific anaerobic coverage for cap is generally overestimated. anaerobic bacteria cannot be detected by diagnostic techniques in current use. anaerobic coverage is clearly indicated only in the classic aspiration pleuropulmonary syndrome in patients with a history of loss of consciousness as a result of alcohol/drug overdose or after seizures in patients with concomitant gingival disease or esophogeal motility disorders. antibiotic trials have not demonstrated a need to specifically treat these organisms in the majority of cap cases. smallvolume aspiration at the time of intubation should be adequately handled by standard empirical severe cap treatment [178] and by the high oxygen tension provided by mechanical ventilation. resistance to commonly used antibiotics for cap presents another major consideration in choosing empirical therapy. resistance patterns clearly vary by geography. local antibiotic prescribing patterns are a likely explanation [179] [180] [181] . however, clonal spread of resistant strains is well documented. therefore, antibiotic recommendations must be modified on the basis of local susceptibility patterns. the most reliable source is state/provincial or municipal health department regional data, if available. local hospital antibiograms are generally the most accessible source of data but may suffer from small numbers of isolates. drug-resistant s. pneumoniae (drsp). the emergence of drug-resistant pneumococcal isolates is well documented. the incidence of resistance appears to have stabilized somewhat in the past few years. resistance to penicillin and cephalosporins may even be decreasing, whereas macrolide resistance continues to increase [179, 182] . however, the clinical relevance of drsp for pneumonia is uncertain, and few well-controlled studies have examined the impact of in vitro resistance on clinical outcomes of cap. published studies are limited by small sample sizes, biases inherent in observational design, and the relative infrequency of isolates exhibiting high-level resistance [183] [184] [185] . current levels of b-lactam resistance do not generally result in cap treatment failures when appropriate agents (i.e., amoxicillin, ceftriaxone, or cefotaxime) and doses are used, even in the presence of bacteremia [112, 186] . the available data suggest that the clinically relevant level of penicillin resistance is a mic of at least 4 mg/l [3] . one report suggested that, if cefuroxime is used to treat pneumococcal bacteremia when the organism is resistant in vitro, the outcome is worse than with other therapies [112] . other discordant therapies, including penicillin, did not have an impact on mortality. data exist suggesting that resistance to macrolides [187] [188] [189] and older fluoroquinolones (ciprofloxacin and levofloxacin) [180, 190, 191] results in clinical failure. to date, no failures have been reported for the newer fluoroquinolones (moxifloxacin and gemifloxacin). risk factors for infection with b-lactam-resistant s. pneumoniae include age !2 years or 165 years, b-lactam therapy within the previous 3 months, alcoholism, medical comorbidities, immunosuppressive illness or therapy, and exposure to a child in a day care center [112, [192] [193] [194] . although the relative predictive value of these risk factors is unclear, recent treatment with antimicrobials is likely the most significant. recent therapy or repeated courses of therapy with b-lactams, macrolides, or fluoroquinolones are risk factors for pneumococcal resistance to the same class of antibiotic [181, 193, 195, 196] . one study found that use of either a b-lactam or macrolide within the previous 6 months predicted an increased likelihood that, if pneumococcal bacteremia is present, the organism would be penicillin resistant [196] . other studies have shown that repeated use of fluoroquinolones predicts an increased risk of infection with fluoroquinolone-resistant pneumococci [195, 197] . whether this risk applies equally to all fluoroquinolones or is more of a concern for less active antipneumococcal agents (levofloxacin and ciprofloxacin) than for more active agents (moxifloxacin and gemifloxacin) is uncertain [190, 197, 198] . recommendations for the use of highly active agents in patients at risk for infection with drsp is, therefore, based only in part on efficacy considerations; it is also based on a desire to prevent more resistance from emerging by employing the most potent regimen possible. although increasing the doses of certain agents (penicillins, cephalosporins, levofloxacin) may lead to adequate outcomes in the majority of cases, switching to more potent agents may lead to stabilization or even an overall decrease in resistance rates [179, 180] . ca-mrsa. recently, an increasing incidence of pneumonia due to ca-mrsa has been observed [199, 200] . ca-mrsa appears in 2 patterns: the typical hospital-acquired strain [80] and, recently, strains that are epidemiologically, genotypically, and phenotypically distinct from hospital-acquired strains [201, 202] . many of the former may represent hcap, because these earlier studies did not differentiate this group from typical cap. the latter are resistant to fewer antimicrobials than are hospitalacquired mrsa strains and often contain a novel type iv sccmec gene. in addition, most contain the gene for panton-valentine leukocidin [200, 202] , a toxin associated with clinical features of necrotizing pneumonia, shock, and respiratory failure, as well as formation of abscesses and empyemas. the large majority of cases published to date have been skin infections in children. in a large study of ca-mrsa in 3 communities, 2% of ca-mrsa infections were pneumonia [203] . however, pneumonia in both adults [204] and children has been reported, often associated with preceding influenza. this strain should also be suspected in patients who present with cavitary infiltrates without risk factors for anaerobic aspiration pneu-monia (gingivitis and a risk for loss of consciousness, such as seizures or alcohol abuse, or esophogeal motility disorders). diagnosis is usually straightforward, with high yields from sputum and blood cultures in this characteristic clinical scenario. ca-mrsa cap remains rare in most communities but is expected to be an emerging problem in cap treatment. outpatient treatment. the following regimens are recommended for outpatient treatment on the basis of the listed clinical risks. the most common pathogens identified from recent studies of mild (ambulatory) cap were s. pneumoniae, m. pneumoniae, c. pneumoniae, and h. influenzae [177, 205] . mycoplasma infection was most common among patients !50 years of age without significant comorbid conditions or abnormal vital signs, whereas s. pneumoniae was the most common pathogen among older patients and among those with significant underlying disease. hemophilus infection was found in 5%mostly in patients with comorbidities. the importance of ther-apy for mycoplasma infection and chlamydophila infection in mild cap has been the subject of debate, because many infections are self-limiting [206, 207] . nevertheless, studies from the 1960s of children indicate that treatment of mild m. pneumoniae cap reduces the morbidity of pneumonia and shortens the duration of symptoms [208] . the evidence to support specific treatment of these microorganisms in adults is lacking. macrolides have long been commonly prescribed for treatment of outpatients with cap in the united states, because of their activity against s. pneumoniae and the atypical pathogens. this class includes the erythromycin-type agents (including dirithromycin), clarithromycin, and the azalide azithromycin. although the least expensive, erythromycin is not often used now, because of gastrointestinal intolerance and lack of activity against h. influenzae. because of h. influenzae, azithromycin is preferred for outpatients with comorbidities such as copd. numerous randomized clinical trials have documented the efficacy of clarithromycin and azithromycin as monotherapy for outpatient cap, although several studies have demonstrated that clinical failure can occur with a resistant isolate. when such patients were hospitalized and treated with a b-lactam and a macrolide, however, all survived and generally recovered without significant complications [188, 189] . most of these patients had risk factors for which therapy with a macrolide alone is not recommended in the present guidelines. thus, for patients with a significant risk of drsp infection, monotherapy with a macrolide is not recommended. doxycycline is included as a cost-effective alternative on the basis of in vitro data indicating effectiveness equivalent to that of erythromycin for pneumococcal isolates. the use of fluoroquinolones to treat ambulatory patients with cap without comorbid conditions, risk factors for drsp, or recent antimicrobial use is discouraged because of concern that widespread use may lead to the development of fluoroquinolone resistance [185] . however, the fraction of total fluoroquinolone use specifically for cap is extremely small and unlikely to lead to increased resistance by itself. more concerning is a recent study suggesting that many outpatients given a fluoroquinolone may not have even required an antibiotic, that the dose and duration of treatment were often incorrect, and that another agent often should have been used as firstline therapy. this usage pattern may promote the rapid development of resistance to fluoroquinolones [209] . comorbidities or recent antimicrobial therapy increase the likelihood of infection with drsp and enteric gram-negative bacteria. for such patients, recommended empirical therapeutic options include (1) a respiratory fluoroquinolone (moxifloxacin, gemifloxacin, or levofloxacin [750 mg daily]) or (2) combination therapy with a b-lactam effective against s. pneumoniae plus a macrolide (doxycycline as an alternative). on the basis of present pharmacodynamic principles, high-dose amox-icillin (amoxicillin [1 g 3 times daily] or amoxicillin-clavulanate [2 g 2 times daily]) should target 193% of s. pneumoniae and is the preferred b-lactam. ceftriaxone is an alternative to highdose amoxicillin when parenteral therapy is feasible. selected oral cephalosporins (cefpodoxime and cefuroxime) can be used as alternatives [210] , but these are less active in vitro than highdose amoxicillin or ceftriaxone. agents in the same class as the patient had been receiving previously should not be used to treat patients with recent antibiotic exposure. telithromycin is the first of the ketolide antibiotics, derived from the macrolide family, and is active against s. pneumoniae that is resistant to other antimicrobials commonly used for cap (including penicillin, macrolides, and fluoroquinolones). several cap trials suggest that telithromycin is equivalent to comparators (including amoxicillin, clarithromycin, and trovafloxacin) [211] [212] [213] [214] . there have also been recent postmarketing reports of life-threatening hepatotoxicity [215] . at present, the committee is awaiting further evaluation of the safety of this drug by the fda before making its final recommendation. inpatient, non-icu treatment. the following regimens are recommended for hospital ward treatment. level i evidence) 19. a b-lactam plus a macrolide (strong recommendation; level i evidence) (preferred b-lactam agents include cefotaxime, ceftriaxone, and ampicillin; ertapenem for selected patients; with doxycycline [level iii evidence] as an alternative to the macrolide. a respiratory fluoroquinolone should be used for penicillin-allergic patients.) the recommendations of combination treatment with a blactam plus a macrolide or monotherapy with a fluoroquinolone were based on retrospective studies demonstrating a significant reduction in mortality compared with that associated with administration of a cephalosporin alone [216] [217] [218] [219] . multiple prospective randomized trials have demonstrated that either regimen results in high cure rates. the major discriminating factor between the 2 regimens is the patient's prior antibiotic exposure (within the past 3 months). preferred b-lactams are those effective against s. pneumoniae and other common, nonatypical pathogens without being overly broad spectrum. in january 2002, the clinical laboratory standards institute (formerly the nccls) increased the mic breakpoints for cefotaxime and ceftriaxone for nonmeningeal s. pneumoniae infections. these new breakpoints acknowledge that nonmeningeal infections caused by strains formerly considered to be intermediately susceptible, or even resistant, can be treated successfully with usual doses of these b-lactams [112, 186, 220] . two randomized, double-blind studies showed ertapenem to be equivalent to ceftriaxone [221, 222] . it also has excellent activity against anaerobic organisms, drsp, and most enterobacteriaceae species (including extended-spectrum b-lactamase producers, but not p. aeruginosa). ertapenem may be useful in treating patients with risks for infection with these pathogens and for patients who have recently received antibiotic therapy. however, clinical experience with this agent is limited. other "antipneumococcal, antipseudomonal" b-lactam agents are appropriate when resistant pathogens, such as pseudomonas, are likely to be present. doxycycline can be used as an alternative to a macrolide on the basis of scant data for treatment of legionella infections [171, 223, 224] . two randomized, double-blind studies of adults hospitalized for cap have demonstrated that parenteral azithromycin alone was as effective, with improved tolerability, as intravenous cefuroxime, with or without intravenous erythromycin [225, 226] . in another study, mortality and readmission rates were similar, but the mean los was shorter among patients receiving azithromycin alone than among those receiving other guideline-recommended therapy [227] . none of the 10 patients with erythromycin-resistant s. pneumoniae infections died or was transferred to the icu, including 6 who received azithromycin alone. another study showed that those receiving a macrolide alone had the lowest 30-day mortality but were the least ill [219] . such patients were younger and were more likely to be in lower-risk groups. these studies suggest that therapy with azithromycin alone can be considered for carefully selected patients with cap with nonsevere disease (patients admitted primarily for reasons other than cap) and no risk factors for infection with drsp or gramnegative pathogens. however, the emergence of high rates of macrolide resistance in many areas of the country suggests that this therapy cannot be routinely recommended. initial therapy should be given intravenously for most admitted patients, but some without risk factors for severe pneumonia could receive oral therapy, especially with highly bioavailable agents such as fluoroquinolones. when an intravenous b-lactam is combined with coverage for atypical pathogens, oral therapy with a macrolide or doxycycline is appropriate for selected patients without severe pneumonia risk factors [228] . inpatient, icu treatment. the following regimen is the minimal recommended treatment for patients admitted to the icu. 20 . a b-lactam (cefotaxime, ceftriaxone, or ampicillin-sulbactam) plus either azithromycin (level ii evidence) or a fluoroquinolone (level i evidence) (strong recommendation) (for penicillin-allergic patients, a respiratory fluoroquinolone and aztreonam are recommended.) a single randomized controlled trial of treatment for severe cap is available. patients with shock were excluded; however, among the patients with mechanical ventilation, treatment with a fluoroquinolone alone resulted in a trend toward inferior outcome [229] . because septic shock and mechanical ventilation are the clearest reasons for icu admission, the majority of icu patients would still require combination therapy. icu patients are routinely excluded from other trials; therefore, recommendations are extrapolated from nonsevere cases, in conjunction with case series and retrospective analyses of cohorts with severe cap. for all patients admitted to the icu, coverage for s. pneumoniae and legionella species should be ensured [78, 230] by using a potent antipneumococcal b-lactam and either a macrolide or a fluoroquinolone. therapy with a respiratory fluoroquinolone alone is not established for severe cap [229] , and, if the patient has concomitant pneumococcal meningitis, the efficacy of fluoroquinolone monotherapy is uncertain. in addition, 2 prospective observational studies [231, 232] and 3 retrospective analyses [233] [234] [235] have found that combination therapy for bacteremic pneumococcal pneumonia is associated with lower mortality than monotherapy. the mechanism of this benefit is unclear but was principally found in the patients with the most severe illness and has not been demonstrated in nonbacteremic pneumococcal cap studies. therefore, combination empirical therapy is recommended for at least 48 h or until results of diagnostic tests are known. in critically ill patients with cap, a large number of microorganisms other than s. pneumoniae and legionella species must be considered. a review of 9 studies that included 890 patients with cap who were admitted to the icu demonstrates that the most common pathogens in the icu population were (in descending order of frequency) s. pneumoniae, legionella species, h. influenzae, enterobacteriaceae species, s. aureus, and pseudomonas species [171] . the atypical pathogens responsible for severe cap may vary over time but can account collectively for у20% of severe pneumonia episodes. the dominant atypical pathogen in severe cap is legionella [230] , but some diagnostic bias probably accounts for this finding [78] . the recommended standard empirical regimen should routinely cover the 3 most common pathogens that cause severe cap, all of the atypical pathogens, and most of the relevant enterobacteriaceae species. treatment of mrsa or p. aeruginosa infection is the main reason to modify the standard empirical regimen. the following are additions or modifications to the basic empirical regimen recommended above if these pathogens are suspected. pseudomonal cap requires combination treatment to prevent inappropriate initial therapy, just as pseudomonas nosocomial pneumonia does [131] . once susceptibilities are known, treatment can be adjusted accordingly. alternative regimens are provided for patients who may have recently received an oral fluoroquinolone, in whom the aminoglycoside-containing regimen would be preferred. a consistent gram stain of tracheal aspirate, sputum, or blood is the best indication for pseudomonas coverage. other, easier-to-treat gram-negative microorganisms may ultimately be proven to be the causative pathogen, but empirical coverage of pseudomonas species until culture results are known is least likely to be associated with inappropriate therapy. other clinical risk factors for infection with pseudomonas species include structural lung diseases, such as bronchiectasis, or repeated exacerbations of severe copd leading to frequent steroid and/or antibiotic use, as well as prior antibiotic therapy [131] . these patients do not necessarily require icu admission for cap [236] , so pseudomonas infection remains a concern for them even if they are only hospitalized on a general ward. the major risk factor for infection with other serious gram-negative pathogens, such as klebsiella pneumoniae or acinetobacter species, is chronic alcoholism. (moderate recommendation; level iii evidence.) the best indicator of s. aureus infection is the presence of gram-positive cocci in clusters in a tracheal aspirate or in an adequate sputum sample. the same findings on preliminary results of blood cultures are not as reliable, because of the significant risk of contamination [95] . clinical risk factors for s. aureus cap include end-stage renal disease, injection drug abuse, prior influenza, and prior antibiotic therapy (especially with fluoroquinolones [237] ). for methicillin-sensitive s. aureus, the empirical combination therapy recommended above, which includes a b-lactam and sometimes a respiratory fluoroquinolone, should be adequate until susceptibility results are available and specific therapy with a penicillinase-resistant semisynthetic penicillin or first-generation cephalosporin can be initiated. both also offer additional coverage for drsp. neither linezolid [241] nor vancomycin [238] is an optimal drug for methicillin-sensitive s. aureus. although methicillin-resistant strains of s. aureus are still the minority, the excess mortality associated with inappropriate an-tibiotic therapy [80] would suggest that empirical coverage should be considered when ca-mrsa is a concern. the most effective therapy has yet to be defined. the majority of ca-mrsa strains are more susceptible in vitro to non-b-lactam antimicrobials, including trimethoprim-sulfamethoxazole (tmp-smx) and fluoroquinolones, than are hospital-acquired strains. previous experience with tmp-smx in other types of severe infections (endocarditis and septic thrombophlebitis) suggests that tmp-smx is inferior to vancomycin [239] . further experience and study of the adequacy of tmp-smx for ca-mrsa cap is clearly needed. vancomycin has never been specifically studied for cap, and linezolid has been found to be better than ceftriaxone for bacteremic s. pneumoniae in a nonblinded study [240] and superior to vancomycin in retrospective analysis of studies involving nosocomial mrsa pneumonia [241] . newer agents for mrsa have recently become available, and others are anticipated. of the presently available agents, daptomycin should not be used for cap, and no data on pneumonia are available for tigecycline. a concern with ca-mrsa is necrotizing pneumonia associated with production of panton-valentine leukocidin and other toxins. vancomycin clearly does not decrease toxin production, and the effect of tmp-smx and fluoroquinolones on toxin production is unclear. addition of clindamycin or use of linezolid, both of which have been shown to affect toxin production in a laboratory setting [242] , may warrant their consideration for treatment of these necrotizing pneumonias [204] . unfortunately, the emergence of resistance during therapy with clindamycin has been reported (especially in erythromycinresistant strains), and vancomycin would still be needed for bacterial killing. clinicians should be aware of epidemiologic conditions and/ or risk factors that may suggest that alternative or specific additional antibiotics should be considered. these conditions and specific pathogens, with preferred treatment, are listed in tables 8 and 9. pathogen-directed therapy 23 . once the etiology of cap has been identified on the basis of reliable microbiological methods, antimicrobial therapy should be directed at that pathogen. (moderate recommendation; level iii evidence.) treatment options may be simplified (table 9) if the etiologic agent is established or strongly suspected. diagnostic procedures that identify a specific etiology within 24-72 h can still be useful for guiding continued therapy. this information is often available at the time of consideration for a switch from parenteral to oral therapy and may be used to direct specific oral antimicrobial choices. if, for example, an appropriate culture reveals penicillin-susceptible s. pneumoniae, a narrowspectrum agent (such as penicillin or amoxicillin) may be used. this will, hopefully, reduce the selective pressure for resistance. the major issue with pathogen-specific therapy is management of bacteremic s. pneumoniae cap. the implications of the observational finding that dual therapy was associated with reduced mortality in bacteremic pneumococcal pneumonia [231] [232] [233] [234] [235] are uncertain. one explanation for the reduced mortality may be the presence of undiagnosed coinfection with an atypical pathogen; although reported to occur in 18%-38% of cap cases in some studies [73, 175] , much lower rates of undiagnosed coinfection are found in general [171] and specifically in severe cases [78] . an alternative explanation is the immunomodulatory effects of macrolides [244, 245] . it is important to note that these studies evaluated the effects of initial empirical therapy before the results of blood cultures were known and did not examine effects of pathogen-specific therapy after the results of blood cultures were available. the benefit of combination therapy was also most pronounced in the more severely ill patients [233, 234] . therefore, discontinuation of combination therapy after results of cultures are known is most likely safe in non-icu patients. oseltamivir or zanamivir is recommended for influenza a. (strong recommendation; level i evidence.) 25. use of oseltamivir and zanamivir is not recommended for patients with uncomplicated influenza with symptoms for 148 h (level i evidence), but these drugs may be used to reduce viral shedding in hospitalized patients or for influenza pneumonia. (moderate recommendation; level iii evidence.) studies that demonstrate that treatment of influenza is effective only if instituted within 48 h of the onset of symptoms have been performed only in uncomplicated cases [246] [247] [248] [249] . the impact of such treatment on patients who are hospitalized with influenza pneumonia or a bacterial pneumonia complicating influenza is unclear. in hospitalized adults with influenza, a minority of whom had radiographically documented pneumonia, no obvious benefit was found in one retrospective study of amantadine treatment [250] . treatment of antigen-or culture-positive patients with influenza with antivirals in addition to antibiotics is warranted, even if the radiographic infiltrate is caused by a subsequent bacterial superinfection. because of the longer period of persistent positivity after infection, the appropriate treatment for patients diagnosed with only 1 of the rapid diagnostic tests is unclear. because such patients often have recoverable virus (median duration of 4 days) after hos-pitalization, antiviral treatment seems reasonable from an infection-control standpoint alone. because of its broad influenza spectrum, low risk of resistance emergence, and lack of bronchospasm risk, oseltamivir is an appropriate choice for hospitalized patients. the neuraminidase inhibitors are effective against both influenza a and b viruses, whereas the m2 inhibitors, amantadine, and rimantadine are active only against influenza a [251] . in addition, viruses recently circulating in the united states and canada are often resistant to the m2 inhibitors on the basis of antiviral testing [252, 253] . therefore, neither amantadine nor rimantadine should be used for treatment or chemoprophylaxis of influenza a in the united states until susceptibility to these antiviral medications has been reestablished among circulating influenza a viruses [249] . early treatment of influenza in ambulatory adults with inhaled zanamivir or oral oseltamivir appears to reduce the likelihood of lower respiratory tract complications [254] [255] [256] . the use of influenza antiviral medications appears to reduce the likelihood of respiratory tract complications, as reflected by reduced usage rates of antibacterial agents in ambulatory patients with influenza. although clearly important in outpatient pneumonia, this experience may also apply to patients hospitalized primarily for influenza. parenteral acyclovir is indicated for treatment of varicellazoster virus infection [257] recent human infections caused by avian influenza a (h5n1) in vietnam, thailand, cambodia, china, indonesia, egypt, and turkey raise the possibility of a pandemic in the near future. the severity of h5n1 infection in humans distinguishes it from that caused by routine seasonal influenza. respiratory failure requiring hospitalization and intensive care has been seen in the majority of the 1140 recognized cases, and mortality is ∼50% [258, 259] . if a pandemic occurs, deaths will result from primary influenza pneumonia with or without secondary bacterial pneumonia. this section highlights issues for consideration, recognizing that treatment recommendations will likely change as the pandemic progresses. more specific guidance can be found on the idsa, ats, cdc, and who web sites as the key features of the pandemic become clearer. additional guidance is available at http://www.pandemicflu.gov. the who has delineated 6 phases of an influenza pandemic, defined by increasing levels of risk and public health response [260] . during the current pandemic alert phase (phase 3: cases of novel influenza infection without sustained person-to-person transmission), testing should be focused on confirming all suspected cases in areas where h5n1 infection has been documented in poultry and on detecting the arrival of the pandemic strain in unaffected countries. early clinical features of h5n1 infection include persistent fever, cough, and respiratory difficulty progressing over 3-5 days, as well as lymphopenia on admission to the hospital [258, 259, 261] . exposure to sick and dying poultry in an area with known or suspected h5n1 activity has been reported by most patients, although the recognition of poultry outbreaks has sometimes followed the recognition of human cases [261] . rapid bedside tests to detect influenza a have been used as screening tools for avian influenza in some settings. throat swabs tested by rt-pcr have been the most sensitive for confirming h5n1 infection to date, but nasopharyngeal swabs, washes, and aspirates; bal fluid; lung and other tissues; and stool have yielded positive results by rt-pcr and viral culture with varying sensitivity. convalescent-phase serum can be tested by microneutralization for antibodies to h5 antigen in a small number of international reference laboratories. specimens from suspected cases of h5n1 infection should be sent to public health laboratories with appropriate biocontainment facilities; the case should be discussed with health department officials to arrange the transfer of specimens and to initiate an epidemiologic evaluation. during later phases of an ongoing pandemic, testing may be necessary for many more patients, so that appropriate treatment and infection control decisions can be made, and to assist in defining the extent of the pandemic. recommendations for such testing will evolve on the basis of the features of the pandemic, and guidance should be sought from the cdc and who web sites (http://www.cdc.gov and http://www.who.int). patients with confirmed or suspected h5n1 influenza should be treated with oseltamivir. most h5n1 isolates since 2004 have been susceptible to the neuraminidase inhibitors oseltamivir and zanamivir and resistant to the adamantanes (amantidine and rimantidine) [262, 263] . the current recommendation is for a 5-day course of treatment at the standard dosage of 75 mg 2 times daily. in addition, droplet precautions should be used for patients with suspected h5n1 influenza, and they should be placed in respiratory isolation until that etiology is ruled out. health care personnel should wear n-95 (or higher) respirators during medical procedures that have a high likelihood of generating infectious respiratory aerosols. bacterial superinfections, particularly pneumonia, are important complications of influenza pneumonia. the bacterial etiologies of cap after influenza infection have included s. pneumoniae, s. aureus, h. influenzae, and group a streptococci. legionella, chlamydophila, and mycoplasma species are not important causes of secondary bacterial pneumonia after influenza. appropriate agents would therefore include cefotaxime, ceftriaxone, and respiratory fluoroquinolones. treatment with vancomycin, linezolid, or other agents directed against ca-mrsa should be limited to patients with confirmed infection or a compatible clinical presentation (shock and necrotizing pneumonia). because shortages of antibacterials and antivirals are anticipated during a pandemic, the appropriate use of diagnostic tests will be even more important to help target antibacterial therapy whenever possible, especially for patients admitted to the hospital. time to first antibiotic dose for cap has recently received significant attention from a quality-of-care perspective. this emphasis is based on 2 retrospective studies of medicare beneficiaries that demonstrated statistically significantly lower mortality among patients who received early antibiotic therapy [109, 264] . the initial study suggested a breakpoint of 8 h [264] , whereas the subsequent analysis found that 4 h was associated with lower mortality [109] . studies that document the time to first antibiotic dose do not consistently demonstrate this difference, although none had as large a patient population. most importantly, prospective trials of care by protocol have not demonstrated a survival benefit to increasing the percentage of patients with cap who receive antibiotics within the first 4-8 h [22, 65] . early antibiotic administration does not appear to shorten the time to clinical stability, either [265] , although time of first dose does appear to correlate with los [266, 267] . a problem of internal consistency is also present, because, in both studies [109, 264] , patients who received antibiotics in the first 2 h after presentation actually did worse than those who retemperature р37.8؇c heart rate р100 beats/min respiratory rate р24 breaths/min systolic blood pressure у90 mm hg arterial oxygen saturation у90% or po 2 у60 mm hg on room air ability to maintain oral intake a normal mental status a note. criteria are from [268, 274, 294] . po 2 , oxygen partial pressure. a important for discharge or oral switch decision but not necessarily for determination of nonresponse. ceived antibiotics 2-4 h after presentation. for these and other reasons, the committee did not feel that a specific time window for delivery of the first antibiotic dose should be recommended. however, the committee does feel that therapy should be administered as soon as possible after the diagnosis is considered likely. conversely, a delay in antibiotic therapy has adverse consequences in many infections. for critically ill, hemodynamically unstable patients, early antibiotic therapy should be encouraged, although no prospective data support this recommendation. delay in beginning antibiotic treatment during the transition from the ed is not uncommon. especially with the frequent use of once-daily antibiotics for cap, timing and communication issues may result in patients not receiving antibiotics for 18 h after hospital admission. the committee felt that the best and most practical resolution to this issue was that the initial dose be given in the ed [22] . data from the medicare database indicated that antibiotic treatment before hospital admission was also associated with lower mortality [109] . given that there are even more concerns regarding timing of the first dose of antibiotic when the patient is directly admitted to a busy inpatient unit, provision of the first dose in the physician's office may be best if the recommended oral or intramuscular antibiotics are available in the office. with the use of a potent, highly bioavailable antibiotic, the ability to eat and drink is the major consideration for switching from intravenous to oral antibiotic therapy for non-icu patients. initially, ramirez et al. [268] defined a set of criteria for an early switch from intravenous to oral therapy (table 10). in general, as many as two-thirds of all patients have clinical improvement and meet criteria for a therapy switch in the first 3 days, and most non-icu patients meet these criteria by day 7. subsequent studies have suggested that even more liberal criteria are adequate for the switch to oral therapy. an alternative approach is to change from intravenous to oral therapy at a predetermined time, regardless of the clinical response [269] . one study population with nonsevere illness was randomized to receive either oral therapy alone or intravenous therapy, with the switch occurring after 72 h without fever. the study population with severe illness was randomized to receive either intravenous therapy with a switch to oral therapy after 2 days or a full 10-day course of intravenous antibiotics. time to resolution of symptoms for the patients with nonsevere illness was similar with either regimen. among patients with more severe illness, the rapid switch to oral therapy had the same rate of treatment failure and the same time to resolution of symptoms as prolonged intravenous therapy. the rapid-switch group required fewer inpatient days (6 vs. 11) , although this was likely partially a result of the protocol, but the patients also had fewer adverse events. the need to keep patients in the hospital once clinical stability is achieved has been questioned, even though physicians commonly choose to observe patients receiving oral therapy for у1 day. even in the presence of pneumococcal bacteremia, a switch to oral therapy can be safely done once clinical stability is achieved and prolonged intravenous therapy is not needed [270] . such patients generally take longer (approximately half a day) to become clinically stable than do nonbacteremic patients. the benefits of in-hospital observation after a switch to oral therapy are limited and add to the cost of care [32] . discharge should be considered when the patient is a candidate for oral therapy and when there is no need to treat any comorbid illness, no need for further diagnostic testing, and no unmet social needs [32, 271, 272] . although it is clear that clinically stable patients can be safely switched to oral therapy and discharged, the need to wait for all of the features of clinical stability to be present before a patient is discharged is uncertain. for example, not all investigators have found it necessary to have the white blood cell count improve. using the definition for clinical stability in table 10, halm et al. [273] found that 19.1% of 680 patients were discharged from the hospital with у1 instability. death or readmission occurred in 10.5% of patients with no instability on discharge, in 13.7% of patients with 1 instability, and in 46.2% with у2 instabilities. in general, patients in higher psi classes take longer to reach clinical stability than do patients in lower risk classes [274] . this finding may reflect the fact that elderly patients with multiple comorbidities often recover more slowly. arrangements for appropriate follow-up care, including rehabilitation, should therefore be initiated early for these patients. in general, when switching to oral antibiotics, either the same agent as the intravenous antibiotic or the same drug class should be used. switching to a different class of agents simply because of its high bioavailability (such as a fluoroquinolone) is probably not necessary for a responding patient. for patients who received intravenous b-lactam-macrolide combination therapy, a switch to a macrolide alone appears to be safe for those who do not have drsp or gram-negative enteric pathogens isolated [275] . most patients with cap have been treated for 7-10 days or longer, but few well-controlled studies have evaluated the optimal duration of therapy for patients with cap, managed in or out of the hospital. available data on short-course treatment do not suggest any difference in outcome with appropriate therapy in either inpatients or outpatients [276] . duration is also difficult to define in a uniform fashion, because some antibiotics (such as azithromycin) are administered for a short time yet have a long half-life at respiratory sites of infection. in trials of antibiotic therapy for cap, azithromycin has been used for 3-5 days as oral therapy for outpatients, with some reports of single-dose therapy for patients with atypical pathogen infections [276] [277] [278] . results with azithromycin should not be extrapolated to other drugs with significantly shorter half-lives. the ketolide telithromycin has been used for 5-7 days to treat outpatients, including some with pneumococcal bacteremia or psi classes уiii [211] . in a recent study, highdose (750 mg) levofloxacin therapy for 5 days was equally successful and resulted in more afebrile patients by day 3 than did the 500-mg dose for 7-10 days (49.1% vs. 38.5%; p p ) [276] . on the basis of these studies, 5 days appears to be .03 the minimal overall duration of therapy documented to be effective in usual forms of cap. as is discussed above, most patients become clinically stable within 3-7 days, so longer durations of therapy are rarely necessary. patients with persistent clinical instability are often readmitted to the hospital and may not be candidates for shortduration therapy. short-duration therapy may be suboptimal for patients with bacteremic s. aureus pneumonia (because of the risk of associated endocarditis and deep-seated infection), for those with meningitis or endocarditis complicating pneumonia, and for those infected with other, less common pathogens (e.g., burkholderia pseudomallei or endemic fungi). an 8-day course of therapy for nosocomial p. aeruginosa pneumonia led to relapse more commonly than did a 15-day course of therapy [279] . whether the same results would be applicable to cap cases is unclear, but the presence of cavities or other signs of tissue necrosis may warrant prolonged treatment. studies of duration of therapy have focused on patients receiving empirical treatment, and reliable data defining treatment duration after an initially ineffective regimen are lacking. drotrecogin alfa activated is the first immunomodulatory therapy approved for severe sepsis. in the united states, the fda recommended the use of drotrecogin alfa activated for patients at high risk of death. the high-risk criterion suggested by the fda was an acute physiologic and chronic health assessment (apache) ii score у25, based on a subgroup analysis of the overall study. however, the survival advantage (absolute risk reduction, 9.8%) of drotrecogin alfa activated treatment of patients in the cap subgroup was equivalent to that in the subgroup with apache ii scores у25 [92, 280, 281] . the greatest reduction in the mortality rate was for s. pneumoniae infection (relative risk, 0.56; 95% ci, 0.35-0.88) [282] . subsequent data have suggested that the benefit appears to be greatest when the treatment is given as early in the hospital admission as possible. in the subgroup with severe cap caused by a pathogen other than s. pneumoniae and treated with appropriate antibiotics, there was no evidence that drotrecogin alfa activated affected mortality. although the benefit of drotrecogin alfa activated is clearly greatest for patients with cap who have high apache ii scores, this criterion alone may not be adequate to select appropriate patients. an apache ii score у25 was selected by a subgroup analysis of the entire study cohort and may not be similarly calibrated in a cap-only cohort. two-organ failure, the criterion suggested for drotrecogin alfa activated use by the european regulatory agency, did not influence the mortality benefit for patients with cap [92] . therefore, in addition to patients with septic shock, other patients with severe cap could be considered for treatment with drotrecogin alfa activated. those with sepsis-induced leukopenia are at extremely high risk of death and ards and are, therefore, potential candidates. conversely, the benefit of drotrecogin alfa activated is not as clear when respiratory failure is caused more by exacerbation of underlying lung disease rather than by the pneumonia itself. other minor criteria for severe cap proposed above are similar to organ failure criteria used in many sepsis trials. consideration of treatment with drotrecogin alfa activated is appropriate, but the strength of the recommendation is only level ii. 35 . hypotensive, fluid-resuscitated patients with severe cap should be screened for occult adrenal insufficiency. (moderate recommendation; level ii evidence.) a large, multicenter trial has suggested that stress-dose (200-300 mg of hydrocortisone per day or equivalent) steroid treatment improves outcomes of vasopressor-dependent patients with septic shock who do not have an appropriate cortisol response to stimulation [283] . once again, patients with cap made up a significant fraction of patients entered into the trial. in addition, 3 small pilot studies have suggested that there is a benefit to corticosteroid therapy even for patients with severe cap who are not in shock [284] [285] [286] . the small sample size and baseline differences between groups compromise the conclusions. although the criteria for steroid replacement therapy remain controversial, the frequency of intermittent steroid treatment in patients at risk for severe cap, such as those with severe copd, suggests that screening of patients with severe cap is appropriate with replacement if inadequate cortisol levels are documented. if corticosteroids are used, close attention to tight glucose control is required [287] . 36 patients who do not require immediate intubation but who have either hypoxemia or respiratory distress should receive a trial of niv [114, 288, 289] . patients with underlying copd are most likely to benefit. patients with cap who were ran-domized to receive niv had a 125% absolute risk reduction for the need for intubation [114] . the use of niv may also improve intermediate-term mortality. inability to expectorate may limit the use of niv [290] , but intermittent application of niv may allow for its use in patients with productive cough unless sputum production is excessive. prompt recognition of a failed niv trial is critically important, because most studies demonstrate worse outcomes for patients who require intubation after a prolonged niv trial [288, 290] . within the first 1-2 h of niv, failure to improve respiratory rate and oxygenation [114, 289, 290] or failure to decrease carbon dioxide partial pressure (pco 2 ) in patients with initial hypercarbia [114] predicts niv failure and warrants prompt intubation. niv provides no benefit for patients with ards [289] , which may be nearly indistinguishable from cap among patients with bilateral alveolar infiltrates. patients with cap who have severe hypoxemia (pao 2 /fio 2 ratio, !150) are also poor candidates for niv [290] . 37. low-tidal-volume ventilation (6 cm 3 /kg of ideal body weight) should be used for patients undergoing ventilation who have diffuse bilateral pneumonia or ards. (strong recommendation; level i evidence.) distinguishing between diffuse bilateral pneumonia and ards is difficult, but it may not be an important distinction. results of the ardsnet trial suggest that the use of low-tidalvolume ventilation provides a survival advantage [291] . pneumonia, principally cap, was the most common cause of ards in that trial, and the benefit of the low-tidal-volume ventilatory strategy appeared to be equivalent in the population with pneumonia compared with the entire cohort. the absolute risk reduction for mortality in the pneumonia cohort was 11%, indicating that, in order to avoid 1 death, 9 patients must be treated [292] . other aspects of the management of severe sepsis and septic shock in patients with cap do not appear to be significantly different from those for patients with other sources of infection. recommendations for these aspects of care are reviewed elsewhere [293] . because of the limitations of diagnostic testing, the majority of cap is still treated empirically. critical to empirical therapy is an understanding of the management of patients who do not follow the normal response pattern. although difficult to define, nonresponse is not uncommon. overall, 6%-15% of hospitalized patients with cap do not respond to the initial antibiotic treatment [81, 84, 101, 294] . the incidence of treatment failure among patients with cap who are not hospitalized is not well known, because population-based studies are required. almirall et al. [295] described an overall hospitalization rate of 60% in a population-based study, but the rate of failure among the 30% of patients who initially presented to their primary care physician was not provided. the frequency of prior antibiotic therapy among medicare patients admitted to the hospital with cap is 24%-40% [95, 109] , but the percentage who received prior antibiotic therapy for the acute episode of pneumonia itself versus other indications is unclear. for patients initially admitted to the icu, the risk of failure to respond is already high; as many as 40% will experience deterioration even after initial stabilization in the icu [101] . mortality among nonresponding patients is increased several-fold in comparison with that among responding patients [296] . overall mortality rates as high as 49% have been reported for an entire population of nonresponding hospitalized patients with cap [76, 84, 101] , and the mortality rate reported in one study of early failure was 27% [81] . apache ii score was not the only factor independently associated with mortality in the nonresponding group, suggesting that the excess mortality may be due to factors other than severity of illness at presentation [101] . 38. the use of a systematic classification of possible causes of failure to respond, based on time of onset and type of failure (table 11) , is recommended. (moderate recommendation; level ii evidence.) the term "nonresponding pneumonia" is used to define a situation in which an inadequate clinical response is present despite antibiotic treatment. lack of a clear-cut and validated definition in the literature makes nonresponse difficult to study. lack of response also varies according to the site of treatment. lack of response in outpatients is very different from that in patients admitted to the icu. the time of evaluation is also important. persistent fever after the first day of treatment differs significantly from fever persisting (or recurring) at day 7 of treatment. table 11 provides a construct for evaluating nonresponse to antibiotic treatment of cap, based on several studies addressing this issue [76, 81, 84, 101] . two patterns of unacceptable response are seen in hospitalized patients [101] . the first is progressive pneumonia or actual clinical deterioration, with acute respiratory failure requiring ventilatory support and/or septic shock, usually occurring within the first 72 h of hospital admission. as is noted above, as many as 45% of patients with cap who ultimately require icu admission are initially admitted to a non-icu setting and are transferred because of deterioration [75] . deterioration and development of respira-tory failure or hypotension 172 h after initial treatment is often related to intercurrent complications, deterioration in underlying disease, or development of nosocomial superinfection. the second pattern is that of persistent or nonresponding pneumonia. nonresponse can be defined as absence of or delay in achieving clinical stability, using the criteria in table 10 [274, 294] . when these criteria were used, the median time to achieve clinical stability was 3 days for all patients, but a quarter of patients took у6 days to meet all of these criteria for stability [274] . stricter definitions for each of the criteria and higher psi scores were associated with longer times to achieve clinical stability. conversely, subsequent transfer to the icu after achieving this degree of clinical stability occurred in !1% of [297] . given these results, concern regarding nonresponse should be tempered before 72 h of therapy. antibiotic changes during this period should be considered only for patients with deterioration or in whom new culture data or epidemiologic clues suggest alternative etiologies. finally, nonresolving or slow-resolving pneumonia has been used to refer to the conditions of patients who present with persistence of pulmonary infiltrates 130 days after initial pneumonia-like syndrome [298] . as many as 20% of these patients will be found to have diseases other than cap when carefully evaluated [295] . two studies have evaluated the risk factors for a lack of response in multivariate analyses [81, 84] , including those amenable to medical intervention. use of fluoroquinolones was independently associated with a better response in one study [84] , whereas discordant antimicrobial therapy was associated with early failure [81] . in table 12, the different risk and protective factors and their respective odds ratios are summarized. specific causes that may be responsible for a lack of response in cap have been classified by arancibia et al. [101] (table 11) . this classification may be useful for clinicians as a systematic approach to diagnose the potential causes of nonresponse in cap. although in the original study only 8 (16%) of 49 cases could not be classified [101] , a subsequent prospective multicenter trial found that the cause of failure could not be determined in 44% [84] . management of nonresponding cap. nonresponse to antibiotics in cap will generally result in у1 of 3 clinical responses: (1) transfer of the patient to a higher level of care, (2) further diagnostic testing, and (3) escalation or change in treatment. issues regarding hospital admission and icu transfer are discussed above. an inadequate host response, rather than inappropriate antibiotic therapy or unexpected microorganisms, is the most common cause of apparent antibiotic failure when guidelinerecommended therapy is used. decisions regarding further diagnostic testing and antibiotic change/escalation are intimately intertwined and need to be discussed in tandem. information regarding the utility of extensive microbiological testing in cases of nonresponding cap is mainly retrospective and therefore affected by selection bias. a systematic diagnostic approach, which included invasive, noninvasive, and imaging procedures, in a series of nonresponding patients with cap obtained a specific diagnosis in 73% [101] . in a different study, mortality among patients with microbiologically guided versus empirical antibiotic changes was not improved (mortality rate, 67% vs. 64%, respectively) [76] . however, no antibiotic changes were based solely on sputum smears, suggesting that invasive cultures or nonculture methods may be needed. mismatch between the susceptibility of a common causative organism, infection with a pathogen not covered by the usual empirical regimen, and nosocomial superinfection pneumonia are major causes of apparent antibiotic failure. therefore, the first response to nonresponse or deterioration is to reevaluate the initial microbiological results. culture or sensitivity data not available at admission may now make the cause of clinical failure obvious. in addition, a further history of any risk factors for infection with unusual microorganisms (table 8) should be taken if not done previously. viruses are relatively neglected as a cause of infection in adults but may account for 10%-20% of cases [299] . other family members or coworkers may have developed viral symptoms in the interval since the patient was admitted, increasing suspicion of this cause. the evaluation of nonresponse is severely hampered if a microbiological diagnosis was not made on initial presentation. if cultures were not obtained, clinical decisions are much more difficult than if the adequate cultures were obtained but negative. risk factors for nonresponse or deterioration (table 12) , therefore, figure prominently in the list of situations in which more aggressive initial diagnostic testing is warranted (table 5) . blood cultures should be repeated for deterioration or progressive pneumonia. deteriorating patients have many of the risk factors for bacteremia, and blood cultures are still high yield even in the face of prior antibiotic therapy [95] . positive blood culture results in the face of what should be adequate antibiotic therapy should increase the suspicion of either antibiotic-resistant isolates or metastatic sites, such as endocarditis or arthritis. despite the high frequency of infectious pulmonary causes of nonresponse, the diagnostic utility of respiratory tract cultures is less clear. caution in the interpretation of sputum or tracheal aspirate cultures, especially of gram-negative bacilli, is warranted because early colonization, rather than superinfection with resistant bacteria, is not uncommon in specimens obtained after initiation of antibiotic treatment. once again, the absence of multidrug-resistant pathogens, such as mrsa or pseudomonas, is strong evidence that they are not the cause of nonresponse. an etiology was determined by bronchoscopy in 44% of patients with cap, mainly in those not responding to therapy [300] . despite the potential benefit suggested by these results, and in contrast to ventilator-associated pneumonia [301, 302] , no randomized study has compared the utility of invasive versus noninvasive strategies in the cap population with nonresponse. rapid urinary antigen tests for s. pneumoniae and l. pneumophila remain positive for days after initiation of antibiotic therapy [147, 152] and, therefore, may be high-yield tests in this group. a urinary antigen test result that is positive for l. pneumophila has several clinical implications, including that coverage for legionella should be added if not started empirically [81] . this finding may be a partial explanation for the finding that fluoroquinolones are associated with a lower incidence of nonresponse [84] . if a patient has persistent fever, the faster response to fluoroquinolones in legionella cap warrants consideration of switching coverage from a macrolide [303] . stopping the b-lactam component of combination therapy to exclude drug fever is probably also safe [156] . because one of the major explanations for nonresponse is poor host immunity rather than incorrect antibiotics, a positive pneumococcal antigen test result would at least clarify the probable original pathogen and turn attention to other causes of failure. in addition, a positive pneumococcal antigen test result would also help with interpretation of subsequent sputum/tracheal aspirate cultures, which may indicate early superinfection. nonresponse may also be mimicked by concomitant or subsequent extrapulmonary infection, such as intravascular catheter, urinary, abdominal, and skin infections, particularly in icu patients. appropriate cultures of these sites should be considered for patients with nonresponse to cap therapy. in addition to microbiological diagnostic procedures, several other tests appear to be valuable for selected patients with nonresponse: • chest ct. in addition to ruling out pulmonary emboli, a ct scan can disclose other reasons for antibiotic failure, including pleural effusions, lung abscess, or central airway obstruction. the pattern of opacities may also suggest alternative noninfectious disease, such as bronchiolitis obliterans organizing pneumonia. • thoracentesis. empyema and parapneumonic effusions are important causes of nonresponse [81, 101] , and thoracentesis should be performed whenever significant pleural fluid is present. • bronchoscopy with bal and transbronchial biopsies. if the differential of nonresponse includes noninfectious pneumonia mimics, bronchoscopy will provide more diagnostic information than routine microbiological cultures. bal may reveal noninfectious entities, such as pulmonary hemorrhage or acute eosinophilic pneumonia, or hints of infectious diseases, such as lymphocytic rather than neutrophilic alveolitis pointing toward virus or chlamydophila infection. transbronchial biopsies can also yield a specific diagnosis. antibiotic management of nonresponse in cap has not been studied. the overwhelming majority of cases of apparent nonresponse are due to the severity of illness at presentation or a delay in treatment response related to host factors. other than the use of combination therapy for severe bacteremic pneumococcal pneumonia [112, 231, 233, 234] , there is no documentation that additional antibiotics for early deterioration lead to a better outcome. the presence of risk factors for potentially untreated microorganisms may warrant temporary empirical broadening of the antibiotic regimen until results of diagnostic tests are available. vaccines targeting pneumococcal disease and influenza remain the mainstay for preventing cap. pneumococcal polysaccharide vaccine and inactivated influenza vaccine are recommended for all older adults and for younger persons with medical conditions that place them at high risk for pneumonia morbidity and mortality (table 13) [304, 305] . the new live attenuated influenza vaccine is recommended for healthy persons 5-49 years of age, including health care workers [304] . postlicensure epidemiologic studies have documented the effectiveness of pneumococcal polysaccharide vaccines for prevention of invasive infection (bacteremia and meningitis) among elderly individuals and younger adults with certain chronic medical conditions [306] [307] [308] [309] . the overall effectiveness against invasive pneumococcal disease among persons у65 years of age is 44%-75% [306, 308, 310] , although efficacy may decrease with advancing age [308] . the effectiveness of the vaccine against pneumococcal disease in immunocompromised persons is less clear, and results of studies evaluating its effectiveness against pneumonia without bacteremia have been mixed. the vaccine has been shown to be cost effective for general populations of adults 50-64 years of age and у65 years of age [311, 312] . a second dose of pneumococcal polysaccharide vaccine after a у5-year interval has been shown to be safe, with only slightly more local reactions than are seen after the first dose [313] . because the safety of a third dose has not been demonstrated, current guidelines do not suggest repeated revaccination. the pneumococcal conjugate vaccine is under investigation for use in adults but is currently only licensed for use in young children [314, 315] . however, its use in children !5 years of age has dramatically reduced invasive pneumococcal bacteremia among adults as well [314, 316] . the effectiveness of influenza vaccines depends on host factors and on how closely the antigens in the vaccine are matched with the circulating strain of influenza. a systematic review demonstrates that influenza vaccine effectively prevents pneumonia, hospitalization, and death [317, 318] . a recent large observational study of adults у65 years of age found that vaccination against influenza was associated with a reduction in the risk of hospitalization for cardiac disease (19% reduction), cerebrovascular disease (16%-23% reduction), and pneumonia or influenza (29%-32% reduction) and a reduction in the risk of death from all causes (48%-50% reduction) [319] . in longterm-care facilities, vaccination of health care workers with influenza vaccine is an important preventive health measure [318, 320, 321] . because the main virulence factors of influenza virus, a neuraminidase and hemagglutinin, adapt quickly to selective pressures, new vaccine formulations are created each year on the basis of the strains expected to be circulating, and annual revaccination is needed for optimal protection. 43 many people who should receive either influenza or pneumococcal polysaccharide vaccine have not received them. according to a 2003 survey, only 69% of adults у65 years of age had received influenza vaccine in the past year, and only 64% had ever received pneumococcal polysaccharide vaccine [322] . coverage levels are lower for younger persons with vaccine indications. among adults 18-64 years of age with diabetes, 49% had received influenza vaccine, and 37% had ever received pneumococcal vaccine [323] . studies of vaccine delivery methods indicate that the use of standing orders is the best way to improve vaccination coverage in office, hospital, or long-term care settings [324] . hospitalization of at-risk patients represents an underutilized opportunity to assess vaccination status and to either provide or recommend immunization. ideally, patients should be vaccinated before developing pneumonia; therefore, admissions for illnesses other than respiratory tract infections would be an appropriate focus. however, admission for pneumonia is an important trigger for assessing the need for immunization. the actual immunization may be better provided at the time of outpatient follow-up, especially with the emphasis on early discharge of patients with cap. patients with an acute fever should not be vaccinated until their fever has resolved. confusion of a febrile reaction to immunization with recurrent/superinfection pneumonia is a risk. however, immunization at discharge for pneumonia is warranted for patients for whom outpatient follow-up is unreliable, and such vaccinations have been safely given to many patients. the best time for influenza vaccination in north america is october and november, although vaccination in december and later is recommended for those who were not vaccinated earlier. influenza and pneumococcal vaccines can be given at the same time in different arms. chemoprophylaxis can be used as an adjunct to vaccination for prevention and control of influenza. oseltamivir and zanamivir are both approved for prophylaxis; amantadine and rimantadine have fda indications for chemoprophylaxis against influenza a infection, but these agents are currently not recommended because of the frequency of resistance among strains circulating in the united states and canada [252, 253] . developing an adequate immune response to the inactivated influenza vaccine takes ∼2 weeks in adults; chemoprophylaxis may be useful during this period for those with household exposure to influenza, those who live or work in institutions with an influenza outbreak, or those who are at high risk for influenza complications in the setting of a community outbreak [325, 326] . chemoprophylaxis also may be useful for persons with contraindications to influenza vaccine or as an adjunct to vaccination for those who may not respond well to influenza vaccine (e.g., persons with hiv infection) [325, 326] . the use of influenza antiviral medications for treatment or chemoprophylaxis should not affect the response to the inactivated vaccine. because it is unknown whether administering influenza antiviral medications affects the performance of the new live attenuated intranasal vaccine, this vaccine should not be used in conjunction with antiviral agents. other types of vaccination can be considered. pertussis is a rare cause of pneumonia itself. however, pneumonia is one of the major complications of pertussis. concern over waning immunity has led the acip to emphasize adult immunization for pertussis [327] . one-time vaccination with the new tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine-adsorbed (tdap) product, adacel (sanofi pasteur)is recommended for adults 19-64 years of age. for most adults, the vaccine should be given in place of their next routine tetanus-diphtheria booster; adults with close contact with infants !12 months of age and health care workers should receive the vaccine as soon as possible, with an interval as short as 2 years after their most recent tetanus/diphtheria booster. 46 smoking is associated with a substantial risk of pneumococcal bacteremia; one report showed that smoking was the strongest of multiple risks for invasive pneumococcal disease in immunocompetent nonelderly adults [328] . smoking has also been identified as a risk for legionella infection [329] . smoking cessation should be attempted when smokers are hospitalized; this is particularly important and relevant when these patients are hospitalized for pneumonia. materials for clinicians and patients to assist with smoking cessation are available online from the us surgeon general (http://www.surgeongeneral.gov/tobacco), the centers for disease control and prevention (http://www.cdc.gov/ tobacco), and the american cancer society (http://www .cancer.org). the most successful approaches to quitting include some combination of nicotine replacement and/or bupropion, a method to change habits, and emotional support. given the increased risk of pneumonia, the committee felt that persons unwilling to stop smoking should be given the pneumococcal polysaccharide vaccine, although this is not currently an aciprecommended indication. 48 . cases of pneumonia that are of public health concern should be reported immediately to the state or local health department. (strong recommendation; level iii evidence.) public health interventions are important for preventing some forms of pneumonia. notifying the state or local health department about a condition of interest is the first step to getting public health professionals involved. rules and regulations regarding which diseases are reportable differ between states. for pneumonia, most states require reporting for legionnaires disease, sars, and psittacosis, so that an investigation can determine whether others may be at risk and whether control measures are necessary. for legionnaires disease, reporting of cases has helped to identify common-source outbreaks caused by environmental contamination [130] . for sars, close observation and, in some cases, quarantine of close contacts have been critical for controlling transmission [330] . in addition, any time avian influenza (h5n1) or a possible terrorism agent (e.g., plague, tularemia, or anthrax) is being considered as the etiology of pneumonia, the case should be reported immediately, even before a definitive diagnosis is obtained. in addition, pneumonia cases that are caused by pathogens not thought to be endemic to the area should be reported, even if those conditions are not typically on the list of reportable conditions, because control strategies might be possible. for other respiratory diseases, episodes that are suspected of being part of an outbreak or cluster should be reported. for pneumococcal disease and influenza, outbreaks can occur in crowded settings of susceptible hosts, such as homeless shelters, nursing homes, and jails. in these settings, prophylaxis, vaccination, and infection control methods are used to control further transmission [331] . for mycoplasma, antibiotic prophylaxis has been used in schools and institutions to control outbreaks [332] . 49 . respiratory hygiene measures, including the use of hand hygiene and masks or tissues for patients with cough, should be used in outpatient settings and eds as a means to reduce the spread of respiratory infections. (strong recommendation; level iii evidence.) in part because of the emergence of sars, improved respiratory hygiene measures ("respiratory hygiene" or "cough etiquette") have been promoted as a means for reducing transmission of respiratory infections in outpatient clinics and eds [333] . key components of respiratory hygiene include encouraging patients to alert providers when they present for a visit and have symptoms of a respiratory infection; the use of hand hygiene measures, such as alcohol-based hand gels; and the use of masks or tissues to cover the mouth for patients with respiratory illnesses. in a survey of the us population, the use of masks in outpatient settings was viewed as an acceptable means for reducing the spread of respiratory infections [334] . for hospitalized patients, infection control recommendations typically are pathogen specific. for more details on the use of personal protective equipment and other measures to prevent transmission within health care settings, refer to the healthcare infection control practices advisory committee [335] . performance indicators are tools to help guideline users measure both the extent and the effects of implementation of guidelines. such tools or measures can be indicators of the process itself, outcomes, or both. deviations from the recommendations are expected in a proportion of cases, and compliance in 80%-95% of cases is generally appropriate, depending on the indicator. four specific performance indicators have been selected for the cap guidelines, 3 of which focus on treatment issues and 1 of which deals with prevention: • initial empirical treatment of cap should be consistent with guideline recommendations. data exist that support the role of cap guidelines and that have demonstrated reductions in cost, los, and mortality when the guidelines are followed. reasons for deviation from the guidelines should be clearly documented in the medical record. • the first treatment dose for patients who are to be admitted to the hospital should be given in the ed. unlike in prior guidelines, a specific time frame is not being recommended. initiation of treatment would be expected within 6-8 h of presentation whenever the admission diagnosis is likely cap. a rush to treatment without a diagnosis of cap can, however, result in the inappropriate use of antibiotics with a concomitant increase in costs, adverse drug events, increased antibiotic selection pressure, and, possibly, increased antibiotic resistance. consideration should be given to monitoring the number of patients who receive empirical antibiotics in the ed but are admitted to the hospital without an infectious diagnosis. • mortality data for all patients with cap admitted to wards, icus, or high-level monitoring units should be collected. although tools to predict mortality and severity of illness exist-such as the psi and curb-65 criteria, respectivelynone is foolproof. overall mortality rates for all patients with cap admitted to the hospital, including general medical wards, should be monitored and compared with severity-adjusted norms. in addition, careful attention should be paid to the percentage of patients with severe cap, as defined in this document, who are admitted initially to a non-icu or a high-level monitoring unit and to their mortality rate. • it is important to determine what percentage of at-risk patients in one's practice actually receive immunization for influenza or pneumococcal infection. prevention of infection is clearly more desirable than having to treat established infection, but it is clear that target groups are undervaccin-ated. trying to increase the number of protected individuals is a desirable end point and, therefore, a goal worth pursuing. this is particularly true for influenza, because the vaccine data are more compelling, but it is important to try to protect against pneumococcal infection as well. coverage of 90% of adults у65 years of age should be the target. 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prospective, randomized comparison of oseltamivir treatment with or without postexposure prophylaxis efficacy of an acellular pertussis vaccine among adolescents and adults cigarette smoking and invasive pneumococcal disease. active bacterial core surveillance team surveillance for legionnaires' disease: risk factors for morbidity and mortality sars in healthcare facilities an outbreak of multidrugresistant pneumococcal pneumonia and bacteremia among unvaccinated nursing home residents azithromycin prophylaxis during a hospital outbreak of mycoplasma pneumoniae pneumonia respiratory hygiene/cough etiquette in health-care settings experiences with influenza-like illness and attitudes regarding influenza prevention-united states, 2003-04 influenza season guideline for isolation precautions in hospitals. the hospital infection control practices advisory committee the committee wishes to express its gratitude to robert balk, christian brun-buisson, ali el-sohl, alan fein, donald e. low, constantine manthous, thomas j. marrie, joseph f. plouffe, and david a. talan, for their thoughtful review of an earlier version of the guidelines. supplement sponsorship. this article was published as part of a supplement entitled "infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults," sponsored by the infectious diseases society of america. key: cord-353116-7t1prfkr authors: bhargava, ashish; fukushima, elisa akagi; levine, miriam; zhao, wei; tanveer, farah; szpunar, susanna m; saravolatz, louis title: predictors for severe covid-19 infection date: 2020-05-30 journal: clin infect dis doi: 10.1093/cid/ciaa674 sha: doc_id: 353116 cord_uid: 7t1prfkr background: covid-19 is a pandemic disease caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2). predictors for severe covid-19 infection have not been well defined. determination of risk factors for severe infection would enable identifying patients who may benefit from aggressive supportive care and early intervention. methods: we conducted a retrospective observational study of 197 patients with confirmed covid-19 infection admitted to a tertiary academic medical center. results: of 197 hospitalized patients, the mean (sd) age of the cohort was 60.6 (16.2) years, 103 (52.3%) were male and 156 (82.1%) were black. severe covid-19 infection was noted in 74 (37.6%) patients, requiring intubation. patients aged above 60 were significantly more likely to have severe infection. patients with severe infection were significantly more likely to have diabetes, renal disease, chronic pulmonary disease and had significantly higher white blood cell counts, lower lymphocyte counts, and increased c-reactive protein (crp) compared to patients with non-severe infection. in multivariable logistic regression analysis, risk factors for severe infection included pre-existing renal disease (odds ratio [or], 7.4; 95% ci 2.5-22.0), oxygen requirement at hospitalization (or, 2.9; 95% ci, 1.3-6.7), acute renal injury (or, 2.7; 95% ci 1.3-5.6) and initial crp (or,1.006; 95% ci, 1.001-1.01). race, age and socioeconomic status were not identified as independent predictors. conclusions: acute or pre-existing renal disease, supplemental oxygen at the time of hospitalization and initial crp were independent predictors for the development of severe covid-19 infections. every 1 unit increase in crp increased the risk of severe disease by 0.06%. a c c e p t e d m a n u s c r i p t 5 in december 2019, the first pneumonia cases of unknown origins were identified in wuhan city, hubei province, china [1] . the pathogen was identified as a novel coronavirus (ncov), now called severe acute respiratory syndrome coronavirus (sars-cov), with the disease termed covid-19 [2] . because of its rapid spread, the world health organization has declared 2019-ncov as pandemic [3] . as of april 28, 2020, a total of 3, 090, 844 confirmed cases had been reported in at 184 countries [4] . sars-cov-2 infections have been described among asymptomatic (who never developed symptoms) as well as pre-symptomatic patients (who are not yet symptomatic) [5] [6] [7] [8] [9] [10] . the clinical spectrum from the largest cohort of symptomatic covid-19 patients from china ranged from mild to critically ill cases [11] . age was described as a strong risk factor for severe disease, with the highest case fatalities occurring in those 80 years and older [11] [12] [13] . preliminary data from the united states (u.s.) also suggested that adverse outcomes were most frequent among persons 85 years of age and older, but it also recognized that severe infections could occur in adults of any age group [14, 15] . comorbid conditions of hypertension, diabetes, chronic lung and renal disease were also associated with severe infections and adverse outcomes [16] [17] [18] . medications such as non-steroidal antiinflammatory drugs (nsaids), angiotensin-converting enzyme (ace) inhibitors or angiotensin receptor blockers (arbs) were suggested to increase the severity of infection [19] , but currently there are no data to suggest a link between these medications and adverse outcomes. the determination of risk factors for severity in covid-19 infection would enable identification of high-risk patients who may benefit from close monitoring, aggressive supportive care and early intervention. a c c e p t e d m a n u s c r i p t 6 to address this question, we collected clinical data from a cohort of hospitalized patients with the aim of identifying predictors for developing severe covid-19 infections. obesity and severe obesity were defined according to cdc definitions [22] . fever was defined as an axillary temperature of 37.5°c or higher. lymphocytopenia was defined as a lymphocyte count of less than 1500 cells per cubic millimeter. thrombocytopenia was defined as a platelet count of less than 150,000 per cubic millimeter. acute renal injury or elevated creatinine on admission was defined as increase in serum creatinine by ≥0.3mg/dl (≥26.5 micromol/l) within 48 hours or increase in serum creatinine to ≥1.5 times baseline, which is known or presumed to have occurred within the prior seven days [23] . patients with pre-existing renal disease were on dialysis, had a history of renal transplant, had uremic syndrome, or had a creatinine > 3mg/dl in prior admissions. using the nine-digit zip code, the area deprivation rank for each individual patient was also obtained. the area deprivation index (adi) is a measurement of healthcare deprivation based upon where a person lives; it correlates with socioeconomic status [24] . a higher adi means more deprived. on a national level the adis are ranked from 1 to 100 (1 = least deprived, 100 = most deprived) and on a state level it is from 1 to 10 (1 = least deprived, 10 = most deprived). a c c e p t e d m a n u s c r i p t 8 statistical analysis was performed using spss v. 26.0 (armonk, ny). descriptive statistics were generated to characterize the study population. continuous variables were described as the mean with standard deviation or median with interquartile range. univariable analysis was done using student's t-test, analysis of variance followed by multiple pairwise comparisons using the bonferroni correction of the p-value, the mann-whitney u test and chi-squared analysis. variables that were found to be significant (p <0.05) predictors of severity were then entered a multivariable logistic regression model using a forward likelihood ratio algorithm. when two variables were measuring the same underlying factor, the variable with the highest univariable measure of association was used in the model. results from the regression are reported as odds ratios with 95% confidence intervals. all there was no significant association found between race and severity (p -0.4). to examine this further, we also assessed the association between the median state adi rank with both race and severity of disease. although there was a significant association between race and median state adi rank (white median state rank 6.0 (iqr: 2.8,8) vs. black median rank 9.0 (iqr: 7,9.75)), (p -<0.0001), there was no association between median state adi rank and severity of disease. the most common symptoms at the onset of illness in the studied cohort were cough (141 including higher white blood cell counts, lower lymphocyte and platelet counts, and increased c-reactive protein (crp) levels compared with those patients with non-severe infection. although patients with severe infection had significantly elevated procalcitonin levels, which raises concern for the presence of secondary bacterial infection, these patients also had acute renal injury on admission which may have caused the elevated procalcitonin. has become pandemic. while little has been reported regarding the predictors for severe covid-19 infections, much is known regarding the risk factors and predictors for mortality [11, 25] . in our study we report pre-existing renal disease, supplemental oxygen requirement at admission, acute renal insufficiency, and initial crp value as independent predictors of severe covid-19 infections. it is also interesting that acute renal insufficiency (whether in patients with chronic kidney disease or normal baseline renal function) was associated with adverse outcome. sars-cov-2 is strongly suspected to use angiotensin converting enzyme 2 (ace2) as its receptor, and ace2 binding affinity has been shown to be one of the most important determinants of sars-cov infectivity [28] . perhaps acute renal insufficiency reflects more efficient binding of sars-cov-2 to ace2 given the location of ace2 expression. interestingly, ace inhibitors and angiotensin ii type-1 receptor blockers (arbs), increase ace2 expression, yet our analysis did not detect an association between ace or arb use, or hypertension or diabetes, and disease severity. persons taking ace or arb at home but presenting with renal insufficiency generally have those medications held upon admission. while sars-cov-2 enters cells by binding to ace2, ace2 also reduces inflammation [19] . if one hypothesizes that ace2 expression decreases inflammation, and that withholding drugs that increase its expression was done mainly in patients with acute renal insufficiency, perhaps a proinflammatory reaction to drug withdrawal could contribute to unfavorable outcome in such patients. the need for supplemental oxygen for baseline hypoxia was an independent factor for severe disease in our study. a recently published study showed oxygen saturation below 90% despite oxygen supplementation was a powerful predictor for fatal outcome [29] . given that ace2 is expressed in lung epithelium, hypoxia may represent more avid binding to sars-cov-2 in those hosts. interestingly, ace2 is also expressed by endothelial cells, which represent one third of lung cells [30] . the endothelium functions to promote vasodilation, fibrinolysis, and anti-aggregation; thus, endothelial damage may lead to a hypercoagulable state [31] . accumulation of coagulation factors in lungs can drive ards through activation of a c c e p t e d m a n u s c r i p t 13 protease activated receptors. microvascular permeability from endothelial injury can also facilitate viral invasion [31] . thus, direct effect of viral invasion and indirect effects thorough endothelial damage lead to severe hypoxia. initial c-reactive protein (crp) level also associated with severe infection. every 1 unit increase in crp increased the risk of severe disease by 0.06%. crp is a homopentameric acute-phase inflammatory protein. baseline crp values are influenced by age, gender, smoking status, weight, lipid levels, and blood pressure, and by genetics [32] . recent studies have reported that cases of sever covid-19 exhibit increased plasma levels of interleukin (il) 2, il6, il7, il10, granulocyte colony-stimulating factor (gcsf), tumor necrosis factor (tnf) alpha, and others [33] . il-6 is the main inducer of crp gene expression, with il-1 and tnf-alpha also playing a role [32] . elevated crp may reflect severe disease as an indirect marker of elevated il-6 and tnf-alpha. crp not only reflects inflammation, it also enhances the immune response. crp can be irreversibly dissociated into monomeric subunits termed monomeric or modified crp (mcrp) at either high concentrations of urea or elevated temperatures in the absence of calcium, and mcrp promotes monocyte chemotaxis and recruitment of circulating leukocytes to areas of inflammation. modified crp also binds immunoglobulin g (igg) fc receptors in an interaction leading to release of proinflammatory cytokines [32] . thus, elevated crp at admission may both reflect significant inflammation and itself drive further inflammation. and given that elevated levels of urea promote formation of mcrp, it fits that acute and chronic kidney disease may be associated with adverse outcomes. in our data, having a known sick contact was associated with lower risk of severe disease. perhaps some of those patients with a sick contact knew they were at risk of a c c e p t e d m a n u s c r i p t 14 exposure and therefore were already attempting to minimize that risk through hand-hygiene, masks, or physical isolation or using separate bathrooms, thus decreasing the potential amount of virus to which they were exposed. this would, however, only account for patients where someone else was symptomatic or exposed soon enough for the patient to be able to take precautions. patients with known sick contacts may also have presented sooner due to a heightened suspicion of having contracted covid-19, and thus received care more rapidly [34] . recall bias may also have contributed. one other potential explanation is that the sickest patients were more confused or were intubated rapidly and therefore could not provide a history of sick contact, which would have impacted the recording of a sick contact in the electronic medical record and thus our results. patients over 60 years old were significantly more likely to have severe infection. in multivariable analysis, however, age, was not found to be an independent predicting factor for the severe infection. this may reflect that the occurrence of kidney disease tends to be higher in older people and kidney disease was the stronger predictor in the model. older age has been significantly associated with death in previous studies [35, 36] . this might be related due to less robust immune responses as older age has been linked with declined of comorbid conditions including significant elevation in creatinine on admission than those not admitted to icu [13] . thus, age in our studied cohort might have been a confounding factor to elevated serum creatinine and c-reactive protein. during the covid-19 pandemic, racial and ethnic minorities especially blacks, have been reported to be severely or disproportionately impacted. our study did not show a disparity in severity by race, so we also investigated the relationship by adi (as a proxy for socioeconomic status). no association was found between adi and severity or between race and severity after controlling for adi. our study has several limitations. this was a single institution study among all the admitted patients which makes generalization of interpretations difficult. because of the retrospective nature of the study design, all variables in the studied patients were not available. therefore, the role of some of these variables in predicting severity of the infection could have been underestimated. last but not the least, the small sample size of our study and a predominantly black and overweight/obese cohort could have limited the generalizability of interpretation for some of the findings (for eg: race). nonetheless our study did involve a population of black patients in the detroit 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coronavirus disease 2019 in china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china severe outcomes among patients with coronavirus disease 2019 (covid-19) -united states characteristics and outcomes of 21 critically ill patients with covid-19 in washington state clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study novel coronavirus pneumonia emergency response epidemiology t covid-19 pathophysiology: a review fibrinolytic abnormalities in acute respiratory distress syndrome (ards) and versatility of thrombolytic drugs to treat covid-19 role of c-reactive protein at sites of inflammation and infection transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients journey of a thai taxi driver and novel coronavirus risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study abbreviations: n: number, or: odds ratio, ci: confidence interval, acei: angiotensin converting enzyme inhibitor, arbs: angiotensin ii receptor blockers a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-349556-k312qkvh authors: roldán-santiago, ernesto; benito-berlinches, amparo; martínez-garcía, laura; quereda, carmen; rodríguez-martín, eulalia; pérez-elías, pilar; lópez-pintor, jose maría; walo-delgado, p e; moreno-zamora, ana; fernández-velasco, jose ignacio; garcía-abellás, patricia; ballester-gonzález, rubén; villar, luisa m; pérez-elías, maría jesús title: sars-cov-2 spreads to lymph nodes and strongly expands cd4+ t(emra) cells in a patient with mild covid-19 date: 2020-09-18 journal: clin infect dis doi: 10.1093/cid/ciaa1422 sha: doc_id: 349556 cord_uid: k312qkvh a woman with mild covid-19 developed cervical adenopathy, being diagnosed of epstein−barr virus infectious mononucleosis. after a fnap we demonstrate that sars-cov-2 is found in lymph nodes (lns) even in mild disease along with a strong expansion of terminally differentiated effector memory cd4+t-cells , a cell population that is practically absent in ln. m a n u s c r i p t introduction a novel coronavirus (severe acute respiratory syndrome-coronavirus-2; sars-cov-2) has caused a global pandemic of respiratory illness called coronavirus infectious disease 2019 . most frequent covid-19 symptoms include mild, self-limiting respiratory tract illness up to severe progressive pneumonia, multiorgan failure, and death. 1 although lymphadenopathy is a common manifestation in many viral diseases, radiology analysis has thus far concluded that lymphadenopathies are characteristically absent in most sars-cov-2 patients who were investigated using computerized tomography (ct). 2 to date, has not been reported in superficial, palpable lymph nodes (ln). the underlying reason for the frequent lymphocytopenia observed in patients with severe disease remains unclear because it is typically not feasible to sample ln . 3 we report the case of a young woman who experienced, mild classical covid-19 symptoms. after 3 weeks, she had a torpid evolution with reappearance of fever and cervical node enlargement, with singular immunophenotypic ln findings. on first days of march 2020, 21-year-old caucasian woman experienced symptoms of fever, malaise, frontal headache, and cough. she was monitored by phone, and suspected covid-19 was diagnosed. twenty-five days later, our patient contacted again with her family caregiver because slight persistent fever, odynophagia, and right cervical node enlargement. antibiotics were prescribed without improvement. six days later, she attended our hospital emergency room. she had no relevant medical history. on physical examination it was observed 12 breaths/minute and arterial oxygen saturation of 98%. the right and left a c c e p t e d m a n u s c r i p t 4 cervical submandibular regions showed palpable ln (diameter, 2−3 cm and <1 cm, respectively), which were not painful or adhered to deep tissue. a blood test including hemogram, biochemistry, and serologies (supplementary material table 1 ) and an x-ray showed normal results, except for slightly elevated c-reactive protein (6.6 mg/l).. rt-pcr nasopharyngeal sars-cov-2 swab was positive. purulent tonsillitis was noted next days, with associated development of additional new lymphadenopathy and enlargement of existing lymph nodes. to rule out a malignant etiology or the possible involvement of sars-cov-2, a fine needle aspiration (fna) puncture was performed after informed consent. ymptoms persisted for >10 days and then progressively improved. by first of may, most symptoms had resolved, and the ln decreased in size. the nucleic acids were extracted from the samples (serum, node fna, and nasopharyngeal swab) using nuclisens easy-mag platform (biomériux, boston, massachusetts (ma), usa). for sars-cov-2 detection, the taqman 2019-ncov assay kit (thermo fisher, waltham, ma usa) was used, amplifying orf1ab, s and n proteins, sequences unique to sars-cov-2. serological or molecular tests for the diagnosis of ebv, cmv and neurotropic viruses were performed with commercially available kits. fna was performed with four passes using a 25g needle attached to an aspiration device (cameco enebyberg, sweden). ish was performed using the bond ebv probe ish kit (leica biosystems, wetzlar, germany), according to the manufacturer's instructions, in formalinfixed and paraffin-embedded cell blocks. a c c e p t e d m a n u s c r i p t 5 all conjugated antibodies were obtained from bd biosciences (san jose, ca, usa). antibodies were added to peripheral blood (pb) or fna samples and incubated for 20 min. samples were acquired using a facscanto ii flow cytometer (bd biosciences). all tests have negative controls. table 1 ). rt-pcr of a fna diluted right cervical node sample showed a positive result for sars-cov-2 (cycle threshold for sar-cov-2n gene=37). a c c e p t e d m a n u s c r i p t 6 we analyzed cd4+ and cd8+ t-cell naive and memory phenotypes in matching ln and pb samples from our patient. results were compared with patients who were diagnosed previously with reactive adenopathy related to ebv or other infectious diseases. each subset was gated for cd45ra and cd197 (ccr7) antigen expression. functional subsets were defined as naive cd45ra+cd197+, central memory (t cm ) cd45ra-cd197+, effector memory (t em ) cd45ra-cd197-, and cd45ra+ effector memory (t emra ) cd45ra+cd197-. naive or central memory cells, which are the two main cd4+ subsets that are usually detected in normal or reactive ln that are or are not infected by ebv (fig. 1b) , switched almost completely to effector memory and especially to t emra t-cells (fig. 1a) . the latter population was virtually absent in normal or reactive lymphoid tissues. however, these changes were much less pronounced in ln cd8+ functional subsets (fig. 1c−d) . a matched blood sample from our patient was analyzed. t emra cd4+ t-cells were practically absent, with all four functional subsets distributed in a similar manner to other viral reactive processes (fig. 1f ) or healthy donors (data not shown). this study shows, for the first time, that sars-cov-2 infects from the initial infection site (respiratory tract) to the locoregional ln, such as cervical ln, even in mild disease. additionally, changes in the t lymphocyte immune profile that were observed in the ln are distinct from that observed in pb. several reports reveal that only 6% of patients who were admitted to hospital for covid-19 had lymphadenopathies. 4 feng et al. 5 showed that sars-cov-2 directly infects macrophages in lns and the spleen from six autopsies using immunofluorescent staining and electron microscopy. consistent with these results, one could hypothesize that sars-cov-2 belongs a c c e p t e d m a n u s c r i p t 7 to a group of viruses that does not normally induce extensive adenopathies, although its detection in ln necropsies from covid-19 patients is the rule rather than the exception. additionally, palpable lymphadenopathies have not been reported in covid-19 patients. however, we present a case of a palpable adenopathy that was caused by co-infection with sars-cov-2 and ebv. the findings strongly suggest that the enlarged ln was a consequence of ebv rather than sars-cov-2 infection, but this co-infection was an excellent opportunity to assess the presence of coronavirus in lns from patients with mild symptoms. our patient was laboratory-confirmed positive for the sars-cov-2 virus using quantitative rt-pcr on an fna sample. this case suggests that virus reaches lns , regardless of disease severity.the other relevant finding of this study is an unexpected expansion of cd4+ t emra in the patient's cervical ln. to date, sars-cov-2-induced cellular immunity has been poorly characterized in anatomical compartments other than pb or lung. the first data regarding changes in lymphocyte pb populations in patients with severe symptoms showed t-lymphopenia, an increase in naive cd4+ t-cells, and a decrease in memory cd4+ t-cells. 6 however, as mentioned previously, we observed a large increase in the percentage of cd4+ t emra cells in ln, a cell subset virtually absent in ln from healthy 7 or infected individuals, including ebv infection. 8 in this case report, these results had to be interpreted based on ebv pathogenesis, since it should not be forgotten that most ebv-specific cd8+ or cd4+ t cells during symptomatic primary infection have a cd45ra-/cd45ro+ phenotype in pb and lymphoid tissues. 9 ebv does not significantly affect t-cell subset distribution in lymphoid organs. 8 in contrast to ebv infection, we observed a remarkable redistribution of cd4+ t-cells in a cervical ln that was co-infected with sars-cov-2 and ebv, including the almost total disappearance of naive and central memory cd4+ t-cells and a very intense expansion of cd4+ effector memory cells. these striking differences make it unlikely that ebv contributes directly to the described cd4+ t emra phenotype. however, we cannot exclude that an unknown interplay between a c c e p t e d m a n u s c r i p t 8 active ebv infection and sars-cov-2 may affect the huge ln cd4+ t emra lymphocyte expansion. the magnitude of the expansion is so great that it could result, at least partially, from bystander activation. 10 in contrast to cd4+ cells, our results showed that the percentages of ln cd8+ t-cell subsets remained essentially unchanged compared to normal lymphoid tissue. this finding may initially seem surprising because cd8+ cells are usually the main cell population involved in the control of viral infection. an increasing amount of evidence suggests that an increase in cd8+ t-cells with an activated phenotype in the blood is related to sars-cov-2 clearance, 11 and that a high cd8+ t-cell count in the lungs is associated with better control of covid-19 progression. 12 a c c e p t e d m a n u s c r i p t m a n u s c r i p t clinical features of patients infected with 2019 novel coronavirus in wuhan, china ct imaging features of 2019 novel coronavirus (2019-ncov) lymphopenia predicts disease severity of covid-19: a descriptive and predictive study radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes for the modena covid-19 working group sars-cov-2, the virus that causes covid-19: cytometry and the new challenge for global health distribution and compartmentalization of human circulating and tissue-resident memory t cells subsets characterization of the cd4 t cell response to epstein-barr virus during primary and persistent infection tonsilar homing of epstein-barr virus-specific cd8+ t cells and the virus-host balance human cd4+ memory t-cells are preferential targets for bystander activation and apoptosis breadth of concomitant responses prior to patient recovery: a case report of non-severe covid-19 baseline pulmonary levels of cd8+ t cells and nk cells inversely correlate with expression of the sars-cov-2 entry receptor ace2 unique phenotypes and clonal expansions of human cd4 effector memory t cells re-expressing cd45ra key: cord-351589-32kd9vva authors: mang, sebastian; kaddu-mulindwa, dominic; metz, carlos; becker, andré; seiler, frederik; smola, sigrun; maßmann, alexander; becker, sören l; papan, cihan; bals, robert; lepper, philipp m; danziger, guy title: pneumocystis jirovecii pneumonia and sars-cov-2 co-infection in newly diagnosed hiv-1 infection date: 2020-07-01 journal: clin infect dis doi: 10.1093/cid/ciaa906 sha: doc_id: 351589 cord_uid: 32kd9vva nan m a n u s c r i p t 2 dear editor -it was recently suggested that excess risk of respiratory failure due to covid-19 may be lower than expected for people living with human immunodeficiency virus (plwh) [1] . we report the case of a 52-year-old male from our intensive care unit (icu) who developed acute respiratory failure due to covid-19, pneumocystis jirovecii pneumonia (pjp) and newly diagnosed hiv, stage 3 [2] . diagnosis of sars-cov-2 disguised the presence of pjp. on the 25th of april 2020, a 52-year-old gentleman presented at the emergency unit of a nearby hospital with fever of 40°c, cough, and shortness of breath. he deteriorated soon after, requiring endotracheal intubation. sars-cov-2 was detected from tracheal aspirate. in addition, bronchial aspirate samples grew with staphylococcus aureus, pseudomonas aeruginosa and acinetobacter dijkshoorniae. blood cultures grew vancomycin-resistant enterococcus faecium and staphylococcus epidermidis. hence, a broad antibiotic regimen containing meropenem and linezolid was initiated, yet the patient continued to have daily fevers up to 40°c without responding to antipyretics or antibiotics. he deteriorated further despite escalated pressure-controlled invasive ventilation and was finally transferred to our intensive care unit on 13 th may, 2020, for possible initiation of extracorporeal membrane oxygenation (ecmo). for evaluation of pulmonary covid-19 manifestation and in preparation for possible ecmo, computed tomography (ct) was performed on 13 th and 14 th may. chest ct showed bilateral ground glass opacities, consolidations, and crazy-paving pattern typical for covid-19 [3] , [4] . as a potential sign for subacute manifestation, airway changes, pleural changes, fibrosis, and nodules were present (figure 1 over the next two weeks, his state improved significantly. noradrenaline administration could be terminated three days after initiation of trimethoprim-sulfamethoxazole. antiretroviral therapy had no apparent side effects, especially creatinine clearance and liver function remained stable over the following weeks. cmv-copies declined to <450 u/ml two weeks after ganciclovir was started on 14 th may. under art the viral load declined to 2,800 copies/ml (equaling 2.11 log units) on 25 th may, 2020. hiv-1 genotyping revealed no relevant drug resistances. after convalescence and ability to swallow reliably, art was switched to a single tablet regime to maintain patient's compliance and to reduce the pill burden. a c c e p t e d m a n u s c r i p t 8 figure 1 why aren't people living with hiv at higher risk for developing severe coronavirus disease 2019 (covid-19) radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study. the lancet infectious diseases coronavirus disease 2019 (covid-19) ct findings: a systematic review and meta-analysis the authors are grateful to our patient for giving his consent for publication. we would like to acknowledge all colleagues from our department as well as other departments and labs at a c c e p t e d m a n u s c r i p t key: cord-353103-sdij1d90 authors: yao, xueting; ye, fei; zhang, miao; cui, cheng; huang, baoying; niu, peihua; liu, xu; zhao, li; dong, erdan; song, chunli; zhan, siyan; lu, roujian; li, haiyan; tan, wenjie; liu, dongyang title: in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) date: 2020-03-09 journal: clin infect dis doi: 10.1093/cid/ciaa237 sha: doc_id: 353103 cord_uid: sdij1d90 background: the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) first broke out in wuhan (china) and subsequently spread worldwide. chloroquine has been sporadically used in treating sars-cov-2 infection. hydroxychloroquine shares the same mechanism of action as chloroquine, but its more tolerable safety profile makes it the preferred drug to treat malaria and autoimmune conditions. we propose that the immunomodulatory effect of hydroxychloroquine also may be useful in controlling the cytokine storm that occurs late-phase in critically ill sars-cov-2 infected patients. currently, there is no evidence to support the use of hydroxychloroquine in sars-cov-2 infection. methods: the pharmacological activity of chloroquine and hydroxychloroquine was tested using sars-cov-2 infected vero cells. physiologically-based pharmacokinetic models (pbpk) were implemented for both drugs separately by integrating their in vitro data. using the pbpk models, hydroxychloroquine concentrations in lung fluid were simulated under 5 different dosing regimens to explore the most effective regimen whilst considering the drug’s safety profile. results: hydroxychloroquine (ec(50)=0.72 μm) was found to be more potent than chloroquine (ec(50)=5.47 μm) in vitro. based on pbpk models results, a loading dose of 400 mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of 200 mg given twice daily for 4 days is recommended for sars-cov-2 infection, as it reached three times the potency of chloroquine phosphate when given 500 mg twice daily 5 days in advance. conclusions: hydroxychloroquine was found to be more potent than chloroquine to inhibit sars-cov-2 in vitro. chloroquine is a widely used anti-malarial with immunomodulatory effects [1] [2] [3] [4] [5] . in a recent in vitro study chloroquine was found to inhibit the growth of sars-cov-2 in vitro [6] . this finding has been supported by clinical studies conducted in approximately one-hundred sars-cov-2 infected patients [7, 8] . hydroxychloroquine is an analog of chloroquine that has fewer concerns about drug-drug interactions. in the previous sars outbreak, hydroxychloroquine was reported to have anti-sars-cov activity in vitro [9] . this suggests that hydroxychloroquine may be a potential pharmacological agent for the treatment of covid-19 infection. however, to date, there is no clinical evidence to support the use of hydroxychloroquine as a treatment for sars-cov-2 infection. the molecular mechanism of action of chloroquine and hydroxychloroquine has not been fully elucidated. findings from previous studies have suggested that chloroquine and hydroxychloroquine may inhibit the coronavirus through a series of steps. firstly, the drugs can change the ph at the surface of the cell membrane and thus, inhibit the fusion of the virus to the cell membrane. it can also inhibit nucleic acid replication, 6 / 25 glycosylation of viral proteins, virus assembly, new virus particle transport, virus release and other processes to achieve its antiviral effects [10] . a reliable estimation of hydroxychloroquine and chloroquine concentrations in the lung, the target tissue, may be used for guiding dose recommendations. physiologically-based pharmacokinetic (pbpk) models are a mathematical modelling technique that can predict drug concentrations in human tissues in silico by integrating physiological and drug disposition parameters. pbpk models are widely used in drug development to help identify whether a clinical trial is warranted as well as help guide the use of drugs based on predictions from well-validated models [11, 12] . in this study we aimed to: (i) investigate the antiviral and prophylactic activity of hydroxychloroquine and chloroquine in vitro, (ii) build a pbpk model for hydroxychloroquine and chloroquine using data from literature, and, (iii) predict drug concentrations under different dosing regimens using the developed pbpk models. chloroquine phosphate and hydroxychloroquine sulfate were purchased from beijing innochem science & technology co, ltd. the lyophilized powder was diluted in double distilled water to 10 mm. hydroxychloroquine sulfate was readily soluble in water. chloroquine phosphate was dissolved by shaking the solution at room temperature for 2 hours. the chloroquine and hydroxychloroquine solutions were 7 / 25 filtered through a 0.22 μm membrane and were then stored at −80°c. the clinically isolated sars-cov-2 virus strain, c-tan-ncov wuhan strain 01, was propagated in vero cells. the vero cells were derived from the african green monkey kidney and were grown in dulbecco's modified eagle medium (dmem) (sigma aldrich, boston, ma, usa) supplemented with 5% fetal bovine serum (logan, ut, usa). the cells were maintained in a humidified atmosphere with 5% co2 at 37°c. the culture medium was replaced each day. the anti-sars-cov-2 activity of chloroquine and hydroxychloroquine was investigated in vitro. cells were seeded into 96-well plates at a density of 1×10 4 cells/well and were grown for 24 hours. the in vitro experiment was divided into two sections, named: (i) the treatment study and (ii) the prophylactic study. drug pretreatment study: vero cells were pretreated with chloroquine or hydroxychloroquine for 2 hours and then, were removed from the drug-containing 8 / 25 medium. the virus-containing medium was then added to the infected vero cells (as described for the treatment study) for 2 hours. following this, the virus-containing medium was removed and replaced with fresh medium that did not contain drugs or viruses. the supernatant was collected, and, the rna was extracted and analyzed by relative quantification using rt-pcr (methods described in a previously published study) [13, 14] . all experiments were conducted in triplicates. the relative expression was estimated using the 2 -△△ ct method. a sigmoidal concentration-response function, y=bottom + (top-bottom)/ (1+10^((logec50-x)*hillslope)), was fit to the data using nonlinear regression. the 9 / 25 ec50 values were calculated using prism (graphpad software, san diego, ca, usa). the pbpk models for chloroquine and hydroxychloroquine were developed using simcyp simulator (version 18). the chloroquine compound file was provided by simcyp limited (a certara company, blades enterprise centre, sheffield, uk) and the hydroxychloroquine compound file was self-built. physical and chemical parameters were obtained from the literature [15] . pharmacokinetic parameters, such as liver intrinsic clearance, fa and ka, were determined from clinical data [16] . these data are summarized in supplement 1. the lung to blood concentration ratio for chloroquine and hydroxychloroquine (obtained from animal studies) was used to predict the drug concentration in the lungs [17, 18] . published chloroquine and hydroxychloroquine clinical trial data were used to validate the developed pbpk models (details summarized in supplement 2) [16, [19] [20] [21] [22] [23] . data obtained from the literature in graphical form were extracted using plot digitizer (version 2.26, getdata). pharmacokinetic parameters that could not be sourced from the literature were estimated using extracted data in phoenix (version 8.6, certara company). concentration-time profiles were simulated under different published clinical trial 10 / 25 protocols using the developed pbpk models for hydroxychloroquine and chloroquine [16, [19] [20] [21] [22] [23] . the simcyp "healthy volunteer", "chinese healthy volunteer" and "pediatric" virtual populations were used in the simulations as the clinical trials were conducted in caucasian, chinese and children populations, respectively. simulated exposure data was compared to observed data. the criterion to determine model accuracy was based on whether the observed data fell within the 90% confidence interval of the predicted values. the ratio of predicted pharmacokinetic (pk) parameters (e.g. cmax and auc) to observed values was used to evaluate model performance. the predicted values were considered reasonable if the ratio of predicted to observed data was within a 2-fold range (0.5≤ratio≤2.0). the exposure of chloroquine and hydroxychloroquine in the lungs, plasma and blood were simulated under different dosing regimens (shown in table 1 ) using the validated pbpk models. a correction factor for chloroquine base and hydroxychloroquine base was input into the model simulations. chloroquine phosphate 500 mg is equivalent to 300 mg of chloroquine base and 200 mg of hydroxychloroquine sulfate is equivalent to 155 mg of hydroxychloroquine base. the "chinese healthy volunteers" virtual population provided in simcyp was used for the simulations. all simulations were performed with 10 trials and 10 subjects per trial. virtual subjects were aged between 20 to 50 years of age, and, 50% of the subjects were male and 50% female. the pbpk models were used to predict the lung tissue concentrations of chloroquine and hydroxychloroquine under different dosing regimens ( table 1 ). the lung trough concentration on days 1, 3, 5 and 10 were adjusted by the plasma unbound fraction (fu,plasma) to obtain the free lung trough concentration. the ratio of the free lung trough concentration to the in vitro ec50 values (rltec) was calculated and the results tabulated. in a recent clinical trial 500 mg of chloroquine phosphate given twice daily was shown to be effective on study day 5 (rltec, day5). this dosing regimen for chloroquine was used as the target for dose optimization for hydroxychloroquine (i.e., the rltec of hydroxychloroquine should not be lower than the rltec, day5 of chloroquine at any time). results from the in vitro study showed that both chloroquine and hydroxychloroquine have good antiviral activity. chloroquine and hydroxychloroquine were found to decrease the viral replication in a concentration-dependent manner. the ec50 values for chloroquine were 23.90 and 5.47 μm at 24 and 48 hours, respectively (figure 1a) . ec50 values for hydroxychloroquine were 6.14 and 0.72 μm at 24 and 48 hours, respectively (figure 1b) . hydroxychloroquine exhibited a superior in vitro antiviral effect in comparison to 12 / 25 chloroquine when drug was added prior to the viral challenge. the ec50 values for chloroquine were >100 and 18.01 μm at 24 and 48 hours, respectively. ec50 values for hydroxychloroquine were 6.25 and 5.85 μm at 24 and 48 hours, respectively. it was noted that with longer incubation times the ec50 values for chloroquine and hydroxychloroquine tended to decrease. the inhibitory effect of chloroquine was poor. this was particularly evident at 24 hours, whereby, even at the highest concentration of chloroquine used in the study, the inhibition rate did not exceed 50% (figure 1c and 1d). the predicted and observed plasma/blood concentration time profiles for chloroquine and hydroxychloroquine is shown in figure 2 . intravenous data was used to understand the distribution and elimination phase of the two drugs, and, oral administration data was used to understand the intracorporal absorption process. most of the observed data fell within the 90% prediction interval. the ratio of predicted to observed pk parameters (cmax and auc) were within the range of 0.5 to 2.0 (details summarized in supplement 2), indicating that the prediction accuracy of the developed pbpk models was acceptable and could be used to simulate the different dosing scenarios. the simulated lung, blood and plasma concentration time profiles for chloroquine and 13 / 25 hydroxychloroquine under the different dosing regimens is shown in figure 3 . it can be seen that the lung, blood and plasma concentrations of chloroquine increased slowly after the first dose was given and was yet to reach steady state on day 10. the simulated chloroquine concentration in lung tissue was much higher than in plasma, where the lung to plasma ratio increased with time and reached a ratio of approximately 400. the projected lung, blood and plasma concentrations of hydroxychloroquine rapidly increased and reached steady state following the initial loading dose and subsequent maintenance doses (figure 3b to 3f). the free lung trough concentrations were also projected from the simulations. the ratio of free lung trough concentration to ec50 (rltec) under the different dosing regimens is shown in table 1 . the rltec values of hydroxychloroquine were found to be higher than the rltec values of chloroquine on days 1, 3, 5 and 10. this suggests that hydroxychloroquine may achieve ideal clinical efficacy under the simulated dosing regimens. the rltec on day 1 was notably higher for hydroxychloroquine than for chloroquine. this is likely to be due to the loading dose of hydroxychloroquine given, thus enabling a faster clinical effect. there was no significant difference between the once and twice daily maintenance dosing regimens (regimen c and d, respectively) when 14 / 25 used from day 2 to day 10; hence, the once daily dosing regimen may be preferred to improve patient compliance. despite regimen f being a 5 day treatment regimen, the lung trough concentrations were still above the target concentration on day 10. however, if the treatment duration of regimen f was extended to 10 days (i.e. regimen e) it resulted in a higher drug concentration on day 10. overall, regimen f may be the best regimen whilst considering both efficacy, safety and patient compliance. in this study, hydroxychloroquine exhibited better in vitro anti-sars-cov-2 activity than chloroquine. this was demonstrated by the ec50 values for hydroxychloroquine always being smaller than the ec50 values for chloroquine, indicating that hydroxychloroquine has a more potent antiviral activity (shown in figure 1 ). in the study by wang et al [6] , chloroquine was shown to have an inhibitory effect on sars-cov-2 with an ec50 value of 1.13 μm after a 48 hour incubation time. these findings are comparable with our in vitro chloroquine results of an ec50 value of 5.47 μm. in addition, an unpublished clinical trial has demonstrated the therapeutic effect of chloroquine in sars-cov-2 infected patients. on the basis of hydroxychloroquine's superior antiviral and prophylactic activity, as well as its more tolerable safety profile in comparison to chloroquine, we believe that hydroxychloroquine may be a promising drug for the treatment of sars-cov-2 infection [24] . in our study we noted that the ec50 values for hydroxychloroquine and chloroquine decreased with longer incubation times. this suggests that incubation time may influence the drug's antiviral activity. both hydroxychloroquine and chloroquine have been reported to accumulate in cells [25] . it is possible that a longer incubation time may provide more time for the drug to accumulate to higher intracellular concentrations and ultimately exhibit a better antiviral effect [26] . another possible explanation is that the drug-induced cytotoxicity may take time to develop, and hence, the drug effect may increase with time [27] . the pbpk model for hydroxychloroquine and chloroquine was validated with in vivo pk data from humans, rats and mice. the model was able to reasonably predict drug concentrations in human lung tissue. a permeability rate limiting model was implemented in the pbpk model to mimic the characteristics of both drugs. in addition, a high lung to plasma partition coefficient ratio (kp ratio) was used to imitate the drugs' high accumulation in lung tissue. the kp ratio for humans was assumed to be same as the ratio for rats because there was no human data available. this assumption may be reasonable as the transportation of both drugs is completely via passive diffusion (i.e. no transporters are involved). in some patients it has been reported that their immune response to the sars-cov-2 virus results in the increase of cytokines il-6 and il-10 [13, 28] . this may progress to a cytokine storm, followed by multi-organ failure and potentially death. both hydroxychloroquine and chloroquine have immunomodulatory effects and can 16 / 25 suppress the increase of immune factors [29, 30] . bearing this in mind, it is possible that early treatment with either of the drugs may help prevent the progression of the disease to a critical, life-threatening state. in critically ill sars-cov-2 infected patients the use of corticosteroids may be harmful [31] . whilst, the use of immunosuppressants (e.g. tocilizumab) are not ideal either as it can suppress the immune system and lead to an increased risk of infection [32] . in this setting, hydroxychloroquine may be an ideal drug to treat sars-cov-2 infection as it can inhibit the virus via its antiviral effects and help mediate the cytokine storm via its immunomodulatory effects. based on work conducted in our lab, we recommend the concomitant use of low dose hydroxychloroquine with an anti-inflammatory drug to help mitigate the cytokine storm in critically ill sars-cov-2 patients. several clinical trials are currently investigating the use of hydroxychloroquine to treat sars-cov-2 infection. however, it is worth noting that the dosing regimens used in these trials are mainly based on previous clinical experience, raising the concern that adverse effects may occur in study participants (supplement 3). in this study, an optimized dosing regimen was designed for hydroxychloroquine to have a high loading dose and low maintenance dose based on it's unique pharmacokinetics (i.e. high accumulation in cells and long elimination half-life). using pbpk modelling and simulation techniques the optimal dosing regimen for hydroxychloroquine was evaluated in in silico. the simulation results demonstrated that regimen f was able to achieve treatment efficacy as well as have a good safety profile, even considering 17 / 25 possible underestimation of drug efficacy to some extent. however, future clinical trials evaluating this regimen are required before it can be readily used in the clinic. the combination of the in vitro antiviral activity data and predicted drug concentrations in this study are being used to support the design of dosing regimens used in a future clinical trial. figure 1 . the antiviral activities of chloroquine and hydroxychloroquine for treatment or prophylactic treatment against sars-cov-2 in vitro. the antiviral activities of chloroquine and hydroxychloroquine for therapeutic and prophylactic use were tested on the vero cells infected with sars-cov-2 clinically isolate strain. (a) and (b): for treatment group, chloroquine and hydroxychloroquine were added medium after the vero cells infected and cells were incubated with medium contained drugs for 24 or 48 hours. (c) and (d): for prophylactic treatment group, the vero cells were pre-treated with chloroquine and hydroxychloroquine for 2 hours, and then viruses were added to medium to infect cells. after that, the medium was removed and replaced with fresh medium without drugs or viruses and cells were incubated for 24 or 48 hours. the viral yield in the cell supernatant was quantified by rt-pcr. chloroquine and hydroxychloroquine as inhibitors of human immunodeficiency virus (hiv-1) activity. curr pharm des in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro inhibition of human influenza a virus replication by chloroquine fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro efficacy of chloroquine and lopinavir/ ritonavir in mild/general novel coronavirus (covid-19) infections: a prospective, open-label, multicenter randomized controlled clinical study ministry of science and technology of china: chloroquine phosphate is effective in the treatment of novel coronavirus pneumonia design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities mechanism of action of hydroxychloroquine as an antirheumatic drug. semin arthritis rheum physiologically based pharmacokinetic analyses-format and content guidance for industry predictive performance of physiologically-based pharmacokinetic models in predicting drug-drug interactions involving enzyme modulation clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china hydroxychloroquine: a physiologically-based pharmacokinetic model in the context of cancer-related autophagy modulation bioavailability of hydroxychloroquine tablets in healthy volunteers kinetics of the distribution and elimination of chloroquine in the rat animal toxicity and pharmacokinetics of hydroxychloroquine sulfate hydroxychloroquine sulfate tablets in chinese healthy volunteers by lc-ms/ms. rheumatol ther a dose-ranging study of the pharmacokinetics of hydroxy-chloroquine following intravenous administration to healthy volunteers single dose disposition of chloroquine in kwashiorkor and normal children--evidence for decreased absorption in kwashiorkor disposition of chloroquine in man after single intravenous and oral doses prevention of chloroquine absorption by activated charcoal pharmacokinetics of hydroxychloroquine and chloroquine during treatment of rheumatic diseases endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture adefovir and tenofovir exhibit long-lasting anti-hepatitis b virus activity in cell culture chloroquine synergizes sunitinib cytotoxicity via modulating autophagic, apoptotic and angiogenic machineries mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology effects of chloroquine on viral infections: an old drug against today's diseases on the use of corticosteroids for 2019-ncov pneumonia systematic review and meta-analysis of serious infections with tofacitinib and biologic disease-modifying antirheumatic drug treatment in rheumatoid arthritis clinical trials we want to thank dr. lisa almond and luke guilliatt from simcyp limited for providing the chloroquine compound file. c.s., h.l., and d.l. have patents pending for anti-microbial infection pharmoceutical composition and its application. all other authors have no potential conflicts. key: cord-336563-hwemigk7 authors: bhimraj, adarsh; morgan, rebecca l; shumaker, amy hirsch; lavergne, valery; baden, lindsey; cheng, vincent chi-chung; edwards, kathryn m; gandhi, rajesh; muller, william j; o’horo, john c; shoham, shmuel; murad, m hassan; mustafa, reem a; sultan, shahnaz; falck-ytter, yngve title: infectious diseases society of america guidelines on the treatment and management of patients with covid-19 date: 2020-04-27 journal: clin infect dis doi: 10.1093/cid/ciaa478 sha: doc_id: 336563 cord_uid: hwemigk7 background: there are many pharmacologic therapies that are being used or considered for treatment of covid-19. there is a need for frequently updated practice guidelines on their use, based on critical evaluation of rapidly emerging literature. objective: develop evidence-based rapid guidelines intended to support patients, clinicians and other health-care professionals in their decisions about treatment and management of patients with covid-19. methods: idsa formed a multidisciplinary guideline panel of infectious disease clinicians, pharmacists, and methodologists with varied areas of expertise. process followed a rapid recommendation checklist. the panel prioritized questions and outcomes. then a systematic review of the peer-reviewed and grey literature was conducted. the grading of recommendations assessment, development and evaluation (grade) approach was used to assess the certainty of evidence and make recommendations. results: the idsa guideline panel agreed on 7 treatment recommendations and provided narrative summaries of other treatments undergoing evaluations. conclusions: the panel expressed the overarching goal that patients be recruited into ongoing trials, which would provide much needed evidence on the efficacy and safety of various therapies for covid-19, given that we could not make a determination whether the benefits outweigh harms for most treatments. a c c e p t e d m a n u s c r i p t it is important to realize that guidelines cannot always account for individual variation among patients. they are assessments of current scientific and clinical information provided as an educational service; are not continually updated and may not reflect the most recent evidence (new evidence may emerge between the time information is developed and when it is published or read); should not be considered inclusive of all proper treatments methods of care, or as a statement of the standard of care; do not mandate any particular course of medical care; and are not intended to supplant physician judgment with respect to particular patients or special clinical situations. whether and the extent to which to follow guidelines is voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances. while idsa makes every effort to present accurate, complete, and reliable information, these guidelines are presented "as is" without any warranty, either express or implied. idsa (and its officers, directors, members, employees, and agents) assume no responsibility for any loss, damage, or claim with respect to any liabilities, including direct, special, indirect, or consequential damages, incurred in connection with these guidelines or reliance on the information presented. the guidelines represent the proprietary and copyrighted property of idsa. copyright 2020 infectious diseases society of america. all rights reserved. no part of these guidelines may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of idsa. permission is granted to physicians and health care providers solely to copy and use the guidelines in their professional practices and clinical decision-making. no license or permission is granted to any person or entity, and prior written authorization by idsa is required, to sell, distribute, or modify the guidelines, or to make derivative works of or incorporate the guidelines into any product, including but not limited to clinical decision support software or any other software product. except for the permission granted above, any person or entity desiring to use the guidelines in any way must contact idsa for approval in accordance with the terms and conditions of third-party use, in particular any use of the guidelines in any software product. executive summary covid-19 is a pandemic with a rapidly increasing incidence of infections and deaths. many pharmacologic therapies are being used or considered for treatment. given the rapidity of emerging literature, idsa identified the need to develop living, frequently updated evidence-based guidelines to support patients, clinicians and other health-care professionals in their decisions about treatment and management of patients with covid-19. summarized below are the recommendations with comments related to the clinical practice guideline for the treatment and management of covid-19. a detailed description of background, methods, evidence summary and rationale that support each recommendation, and research needs can be found online in the full text. in brief, per grade methodology, recommendations are labeled m a n u s c r i p t as "strong" or "conditional". the word "recommend" indicates strong recommendations and "suggest" indicates conditional recommendations. in situations where promising interventions were judged to have insufficient evidence of benefit to support their use and with potential appreciable harms or costs, the expert panel recommended their use in the context of a clinical trial. the guideline panel used the word "only" in recommendations about therapeutic agents with higher uncertainty and/or more potential for harm. these recommendations acknowledge the current "knowledge gap" and aim at avoiding premature favorable recommendations for potentially ineffective or harmful interventions. the panel expressed the overarching goal that patients be recruited into ongoing trials, which would provide much needed evidence on the efficacy and safety of various therapies for covid-19. the panel determined that when an explicit trade-off between the highly uncertain benefits and the known putative harms of these therapeutic agents were considered, a net positive benefit was not reached and could possibly be negative (risk of excess harm). the panel acknowledges that enrolling patients in rcts might not be feasible for many frontline providers due to limited access and infrastructure. should lack of access to clinical trials exist, we encourage setting up local or collaborative registries to systematically evaluate the efficacy and safety of drugs to contribute to the knowledge base. each clinician can play a role in advancing our understanding of this disease through a local registry or other data collection efforts. m a n u s c r i p t background the first cases of coronavirus disease 2019 (covid-19) were reported from wuhan, china in early december 2019 [1] , now known to be caused by a novel beta-coronavirus, named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). within a span of months covid 19 has become pandemic due to its transmissibility, spreading across continents with the number of cases and deaths rising daily [2] . although most infected individuals exhibit a mild illness (80%+), 14% have serious and 5% have critical illness. approximately 10% will require hospital admission due to covid-19 pneumonia, of which approximately 10% will require icu care, including invasive ventilation due to acute respiratory distress syndrome (ards) [3] . while mortality appears to be more common in older individuals and those with comorbidities, such as chronic lung disease, cardiovascular disease, hypertension and diabetes, young people with no comorbidities also appear to be at risk for critical illness including multi-organ failure and death. there has been an expanding number of studies rapidly published online and in academic journals; however, some of these may be of limited quality and are pre-published without sufficient peerreview. critical appraisal of the existing studies is needed to determine if the existing evidence is sufficient to support currently proposed management strategies. given the rapid global spread of sars cov-2 and the difficulty for the overburdened front-line providers and policymakers to stay up to date on emerging literature, idsa has recognized the necessity of developing a rapid guideline for the treatment of covid-19. the guideline panel used a methodologically rigorous process for evaluating the best available evidence and providing treatment recommendations. two additional guidelines on diagnostic testing and infection prevention are also under development. these guidelines will be frequently updated as substantive literature becomes available and will be accessible on an easy to navigate web and device interface at http://www.idsociety.org/covid19guidelines. the guideline panel considered making a recommendation for therapeutic agents that were currently available for use. the panel chose not to give a recommendation for remdesivir as it is mostly available in the context of ongoing trials with limited availability for compassionate use and expanded access use. we anticipate data from rcts for remdesivir will be soon available; at that point, we will update the guidelines to provide formal recommendations on its use. there are several ongoing trials evaluating therapeutic agents for the treatment of covid-19. as data becomes available from these trials and if there is a preponderance of evidence to suggest the use of a therapeutic agent even in the context of clinical trials is no longer warranted it will be removed from future updates of the guideline (and the removal will be noted in the updated a c c e p t e d m a n u s c r i p t guidelines). if there is emerging evidence on the efficacy or safety of a therapeutic agent not mentioned in the current version of the guideline it will be included in future updates of the guideline. these recommendations are intended to inform patients, clinicians, and other health professionals by providing the latest available evidence. this guideline was developed using the grade approach for evidence assessment. in addition, given the need for an urgent response to a major public health crisis, the methodological approach was modified according to the gin/mcmaster checklist for the development of rapid recommendations [4] . the panel was composed of nine members including front line clinicians, infectious diseases specialists who are members of the idsa, the hiv medical association (hivma), the society for healthcare epidemiology of america (shea), and the pediatric infectious diseases society (pids). they represented the disciplines of public health, pharmacology, pediatrics, medical microbiology, preventive care, critical care, as well as hepatology, nephrology and gastroenterology. the evidence foundation provided technical support and guideline methodologists for the development of this guideline. the conflict of interest (coi) review group included two representatives from idsa who were responsible for reviewing, evaluating and approving all disclosures. all members of the expert panel complied with the coi process for reviewing and managing conflicts of interest, which required disclosure of any financial, intellectual, or other interest that might be construed as constituting an actual, potential, or apparent conflict, regardless of relevancy to the guideline topic. the assessment of disclosed relationships for possible coi was based on the relative weight of the financial relationship (i.e., monetary amount) and the relevance of the relationship (i.e., the degree to which an association might reasonably be interpreted by an independent observer as related to the topic or recommendation of consideration). the coi review group ensured that the majority of the panel and chair was without potential relevant (related to the topic) conflicts. the chair and all members of the technical team were determined to be unconflicted. m a n u s c r i p t clinical questions were developed into a pico format (population, intervention, comparison, outcomes) [5] prior to the first panel meeting. panel members prioritized questions with available evidence that met the minimum acceptable criteria (i.e., the body of evidence reported on at least a case-series design, case reports were excluded). panel members prioritized patient-important outcomes such as mortality, development of ards (need for non-invasive or invasive ventilation) and clinical improvement (such as disease-oriented outcomes inferred by radiological findings or virologic cure), and severe adverse events leading to treatment discontinuation. additional drug specific harms were evaluated when clinically relevant, including possible drug-drug reactions, if applicable. the nice highly-sensitive search was reviewed by the methodologist in consultation with the technical team information specialist and was determined to have high sensitivity [6] . an additional term, covid, was added to the search strategy used in addition to the treatment terms identified in the pico questions (supplementary table s1 ). ovid medline and embase were searched from 2019 through april 4, 2020. horizon scans were performed daily during the evidence assessment and recommendation process to locate additional grey literature and manuscript pre-prints. reference lists and literature suggested by panelists were reviewed for inclusion. no restrictions were placed on language or study type. two reviewers independently screened titles and abstracts, as well as eligible full-text studies. when acceptable randomized controlled trials of effectiveness were found, no additional non-randomized studies or non-comparative evidence (i.e., single arm case series) were sought. evidence from single arm studies reporting on non-comparative rates of outcomes of interest were included if a historical control event rate could be estimated from the literature. reviewers extracted relevant information into a standardized data extraction form. for several interventions, no direct evidence was available other than case reports or mechanistic considerations. the panel either decided to include plausible indirect evidence and make a recommendation (e.g., from studies of sars-cov) or to provide a short narrative discussion of the intervention. m a n u s c r i p t data extracted from the available evidence included: mortality, clinical progression or improvement as reported in the studies, virologic clearance, and adverse events. where applicable, data were pooled using random effects model (fixed effects model for 2 or less trials or pooling of rates) using revman or openmeta [7] . cochrane risk of bias tools for randomized clinical trials (rcts) and observational studies and modified domains were used in assessing confounding bias, selection bias, and misclassification bias [8] . the certainty of evidence was assessed using the grading of recommendations assessment, development and evaluation (grade) approach [9] . within grade, the body of evidence across each outcome is assessed for domains that may reduce or increase one's certainty in the evidence. factors that may reduce one's certainty include risk of bias (study limitations), inconsistency (unexplained heterogeneity across study findings), indirectness (applicability or generalizability to the research question), imprecision (the confidence in the estimate of an effect to support a particular decision) or publication bias (selective publication of studies). one's certainty in the evidence may be strengthened if the following considerations are present: large or very large magnitude of effect, evidence of a dose-response gradient, or opposing residual confounding. grade summary of findings tables were developed in gradepro guideline development tool [10] . the panel considered core elements of the grade evidence in the decision process, including certainty of evidence and balance between desirable and undesirable effects. additional domains were acknowledged where applicable (feasibility, resource use, acceptability). for all recommendations, the expert panelists reached consensus. voting rules were agreed on prior to the panel meetings for situations when consensus could not be reached. as per grade methodology, recommendations are labeled as "strong" or "conditional". the words "we recommend" indicate strong recommendations and "we suggest" indicate conditional recommendations. figure 1 provides the suggested interpretation of strong and weak recommendations for patients, clinicians, and healthcare policymakers. for recommendations where the comparators are not formally stated, the comparison of interest is implicitly referred to as "not using the intervention". the guideline panel used the word "only" in recommendations about therapeutic agents with higher uncertainty and/or more potential for harm. for example, the toxicity of hydroxychloroquine/chloroquine plus azithromycin (recommendation 2) carries particular risk for patients in the outpatient setting who may not be enrolled in a trial and therefore may have inadequate monitoring; or, for recommendation 6, there is concern related to tocilizumab shortages for other indications and potential toxicity of worsening infection/drug interactions. these recommendations acknowledge the current "knowledge gap" and aim at avoiding premature favorable recommendations for their use and to avoid encouraging the rapid m a n u s c r i p t diffusion of potentially ineffective or harmful interventions. detailed suggestions about the specific research questions that should be addressed are found in the table (see supplementary table s2 ). m a n u s c r i p t regular, frequent screening of the literature will take place to determine the need for revisions based on the likelihood that any new data will have an impact on the recommendations. if necessary, the entire expert panel will be reconvened to discuss potential changes. two rcts of patients with confirmed covid-19 with mild pneumonia (e.g., positive ct scan without oxygen requirement) or non-severe infection admitted to the hospital treated with hydroxychloroquine (hcq) reported on mortality at 14 days, clinical progression (radiological progression on ct scan), clinical improvement, failure of virologic clearance (pcr), and adverse events (both) [11, 12] (table 1 ). in addition, we identified four publications describing three trials of combination treatment with hcq plus azithromycin (az) among hospitalized patients with covid-19 reporting on the outcomes of mortality, failure of virologic clearance (assessed with pcr test), and adverse events (i.e., significant qt prolongation leading to treatment discontinuation) [13] [14] [15] [16] (table 2) . the currently available best evidence failed to demonstrate or to exclude a beneficial effect of hcq on clinical progression of covid-19 (as inferred by radiological findings; rr: 0.61; 95% ci: 0.26, 1.43; see figure s2 ), or on viral clearance by pcr tests (rr: 2.00; 95% ci: 0.02, 20.00; see figure s3 ), although a somewhat higher proportion in the hcq group experienced clinical improvement (rr: 1.47; 95% ci 1.02, 2.11). however, the certainty in the evidence was rated as very low mainly due to small sample sizes (sparse data), co-interventions, and risk of bias due to methodological limitations. in addition, the selected outcomes should be considered indirect, as important patient outcomes (e.g., mortality, rate of progression to ards and need for mechanical ventilation) were unavailable. m a n u s c r i p t studies evaluating the addition of azithromycin to hcq provided indirect comparisons of failure of virologic clearance to historical controls. the observed risk of mortality among patients receiving hcq+az during hospital stay was 3.4% (6/175 patients). however, an estimated mortality rate in an untreated cohort was not provided in the manuscript. when compared to a lack of viral clearance in historical controls (100% virologic failure), 12 symptomatic patients were compared at day 5 or 6 from a separate hospital in france. patients receiving treatment with hcq+az experienced numerically fewer cases of virologic failure (43% pooled virologic failure; 29/71 patients) (figure s3 ). there is very low certainty in this comparison of treatment effect mainly due to very high-risk selection bias, making any claims of effectiveness highly uncertain. in addition, relying on intermediary outcomes, such as viral clearance to determine patient-important outcomes (including a reduction in development of pneumonia, hospital or icu admission, or need for intubation) add another layer of imprecision. two studies described significant qt prolongation in 10 of 95 treated patients, either resulting in an qt increase to over 500 ms or discontinuation of the hcq/az treatment, illustrating the high risk for clinically relevant arrhythmias for this treatment [15, 16] . in addition, several case reports of qt prolongation related to hydroxychloroquine have also been published [17] [18] [19] [20] . in another prospective cohort study in 224 covid-19 uninfected patients with sle who received either chloroquine or hydroxychloroquine for routine care, gastrointestinal side effects occurred in 7% of patients [21] . several case reports have been published citing the risk of a prolonged qt prolongation, torsades de pointes, and ventricular tachycardia in patients receiving azithromycin alone. in a large cohort study, patients taking a five-day course of azithromycin had an increased risk of sudden cardiac death with a hazard ratio of 2.71 (1.58-4.64) vs. 0.85 (0.45-1.60), compared to patients receiving no antibiotic or amoxicillin, respectively [22] . given the cumulative effect on cardiac conduction seen with hydroxychloroquine and azithromycin, if this combination was to be used in the context of a clinical trial, baseline and follow-up ecg monitoring would be indicated, as well as careful surveillance for other concomitant medications known to prolong the qt interval. renal clearance accounts for 15-25% of total clearance of hydroxychloroquine, however dose adjustments are not recommended according to package labeling. chloroquine and hydroxychloroquine are metabolized by cytochrome p450 isoenzymes 2c8, 2d6, and 3a4 [23] , therefore inhibitors and inducers of these enzymes may result in altered pharmacokinetics of these agents. providers are encouraged to visit resources such as the newly created website, https://www.covid19-druginteractions.org/ to aid in the evaluation and management of drug interactions with current and emerging investigational agents for covid-19. azithromycin is low risk for cytochrome p450 interactions [24] ; however additional pharmacologic adverse events including gastrointestinal effects and qt prolongation need to be carefully considered particularly in the outpatient setting where frequent ecg monitoring is not feasible. a c c e p t e d m a n u s c r i p t the panel agreed that the overall certainty of evidence was very low due to concerns with risk of bias, inconsistency, indirectness, imprecision, and publication bias. the guideline panel recommends that, because of uncertainty regarding its risks and benefits, the use of hcq should be in the context of a clinical trial. because of the potential for toxicity, the panel recommends that the hcq+az combination only be used in the context of a clinical trial. this recommendation does not address the use of azithromycin for secondary bacterial pneumonia in patients with covid-19. additional randomized controlled trials and prospective outcome registries are needed to inform research for treatment with hcq alone or in combination with azithromycin for patients with covid-19 (table s2 . best practices/suggestions for research of treatments for patients with covid-19). one rct and two case studies reported on treatment with combination lopinavir/ritonavir for hospitalized patients with covid-19 [25] [26] [27] (table 3) . cao et al. randomized 199 hospitalized patients with severe covid-19 to receive treatment with lopinavir/ritonavir in addition to standard of care (n=99) or standard of care alone (n=100) for 14 days. the trial reported on the following outcomes: mortality, failure of clinical improvement (measured using a 7-point scale or hospital discharge), and adverse events leading to treatment discontinuation. based on a modified intention to treat analysis, treatment with lopinavir/ritonavir failed to show or exclude a beneficial effect on mortality (rr: 0.67; 95% ci: 0.38, 1.17), although failure of clinical improvement was lower in the lopinavir group (rr: 0.78; 95% ci: 0.63, 0.97; itt analysis). nearly 14% of lopinavir/ritonavir recipients were unable to complete the full 14-day course of administration due primarily to gastrointestinal adverse events, including anorexia, nausea, abdominal discomfort, or diarrhea, as well as two serious adverse episodes of acute gastritis. two recipients also had self-limited skin eruptions. the risk of hepatic injury, pancreatitis, severe cutaneous eruptions, qt prolongation, and the potential for multiple drug interactions due to cyp3a inhibition, are all well documented with this drug combination. the panel elected to inform their decision based on the rct [27] . the panel determined the certainty of evidence to be very low due to concerns with risk of bias (lack of blinding) and imprecision. in the randomized clinical trial conducted by cao et al, the group that received lopinavir/ritonavir and the group that did not had similar rates of viral decay. this finding suggests that lopinavir/ritonavir is not having a measurable antiviral effect, its purported mechanism of action. the guideline panel recommends the use of lopinavir/ritonavir only in the context of a clinical trial. additional clinical trials or prospective outcome registries are needed to inform research for treatment with lopinavir/ritonavir and other hiv-1 protease inhibitors for patients with covid-19 (supplementary table s2) . the panel determined that due to the limitation of direct covid-19 data, indirect evidence from the 2003 sars outbreak and from mers would also be considered. a systematic review [30] reported on 15 studies, 13 of which were inconclusive to any benefits of corticosteroids. one rct reported that sars-cov-1 viral loads showed delayed viral clearance associated with corticosteroid use. the same review also reported on a subset of ards patients (three trials). one small rct in 24 patients using a lower dose methylprednisolone for two days showed possible improvement of ards; however, two larger trials showed little or no effect in critically ill patients with pulmonary failure. the authors concluded that despite widespread use of corticosteroids during the sars outbreak, conclusive evidence of benefit was lacking and that administering steroids early in the disease process before viral replication is controlled may lead to a delay in viral clearance. the panel deemed the certainty of the direct evidence as very low owing to concerns with risk of bias, inconsistency, and imprecision. the panel based their decision to conditionally recommend against the use of corticosteroids among patients admitted to the hospital on the indirect findings from the systematic review on sars-cov. m a n u s c r i p t as covid-19is a self-limited viral illness in most cases, a small subset of patients progresses from covid-19 pneumonia to develop ards. based on limited data from other coronaviruses, there is no clear benefit and potential harm from corticosteroids. carefully designed rcts and prospective outcome registries are needed to determine the dose, route, timing, and duration of such treatment on the prevention of clinical deterioration and to better understand the potential harms associated with its use. if a person is on a steroid (inhaled or systemic) for another indication (e.g., asthma), the steroid should be continued. m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t studies reporting on the pathogenesis of sars and mers-cov suggest a release of proinflammatory cytokines including interleukins-6 (il-6) [31] during the clinical illness. our search identified one study [31] that reported on 21 severe or critical patients with covid-19 treated with tocilizumab, an il-6 blocker ( table 6 ). this study had no control group. to estimate a control group rate in patients who did not get treatment with tocilizumab, xu et al. described findings from yang 2020, which suggested a baseline mortality rate of 60% in critical patients and 11% in severe patients admitted to the icu [32] . we estimate that the patients in xu 2020 (21 patients, 4 critical and 17 severe) would have a baseline mortality risk of 20% as matched in severity. therefore, treatment with tocilizumab may have reduced mortality since there were no deaths reported out of 21 patients. however, this conclusion remains highly uncertain given the lack of a contemporaneous control or adjustments for confounding factors. out of 21 patients, 19 were discharged from the hospital suggesting a 9.5% rate of failure of clinical improvement in the ct scan findings. xu et al. reported no serious adverse events [31] . however, patients receiving tocilizumab are often at an increased risk of serious infections (bacterial, viral, invasive fungal infections, and tuberculosis) and hepatitis b reactivation [33] . cases of anaphylaxis, severe allergic reactions, severe liver damage and hepatic failure, and intestinal perforation have been reported after tocilizumab administration in patients without covid-19. tocilizumab is not metabolized by the cytochrome p450 isoenzyme system, however elevated il-6 levels seen in inflammatory states have been shown to inhibit these enzymes, thereby slowing the metabolism of drugs through these pathways. as the 3a4 pathway is responsible for metabolism of many commonly used medications, administration of il-6 inhibitors like tocilizumab may result in enhanced metabolism in drugs utilizing the cytochrome p450 system [34, 35] . the panel determined that the overall certainty of the evidence was very low due to concerns of high risk of bias due to confounding, indirectness, and imprecision. m a n u s c r i p t the guideline panel recommended tocilizumab only in the context of a clinical trial. additional clinical trials are needed to inform research on the effectiveness of treatment with tocilizumab for patients with covid-19 (supplementary table s2 ). m a n u s c r i p t a c c e p t e d m a n u s c r i p t our search identified two case series of a total of 15 patients reporting on the outcomes of mortality, failure of clinical improvement (as inferred by need for continued mechanical ventilation), and treatment related adverse events among hospitalized patients with covid-19 (table 7 ) [36, 37] . all five patients in shen 2020 were mechanically ventilated at time of treatment compared with three out of 10 patients in the duan et al study. duan 2020 included a comparison of the 10 treated patients to 10 historical control patients matched on age, gender, and severity of illness. both studies lacked adjustments for critical confounders including co-treatments, baseline characteristics, disease severity, and timing of plasma delivery. compared with a 30% mortality rate in the historical control (3/10), no deaths were reported among patients receiving covid-19 convalescent plasma. out of eight patients across both studies on mechanical ventilation at time of treatment, 50% (n=4) were extubated at time of data collection. among 10 patients, no serious adverse reactions or safety events were recorded following covid-19 convalescent transfusion. the panel agreed on the overall certainty of evidence as very low due to concerns with risk of bias and imprecision. continuation of mechanical ventilation was used as a surrogate for failure of clinical improvement; however, the panel recognized the importance of the timeframe for extubation when associating it to plasma transfusion. given the limited information provided about time of extubation, the panel recognized an additional knowledge gap with the assessment of this outcome. the guideline panel recommends covid-19 convalescent plasma in the context of a clinical trial. additional clinical trials are needed to inform research for treatment with covid-19 convalescent plasma for patients with covid-19 (supplementary table s2 ). m a n u s c r i p t in addition to the clinical questions addressed above, the panel identified several treatments currently undergoing evaluation for which additional data are needed to formulate recommendations. narrative summaries for these treatments are provided below. in-vitro antiviral activity of darunavir against sars-cov-2 showed no activity at clinically relevant concentrations. three randomized, open-label clinical trials are currently listed on clinicaltrials.gov evaluating darunavir/cobicistat as a potential therapeutic option for covid-19. janssen, the manufacturer of darunavir/cobcistat has reported that one of these trials [38] has concluded that darunavir/cobicstat plus conventional treatments was not effective in achieving viral clearance at day seven post randomization, m a n u s c r i p t compared to conventional treatments alone. clinical outcomes of this trial including rate of critical illness and mortality 14 days after randomization, have not been reported to date. lopinavir-ritonavir is a combination of protease inhibitors for the treatment of hiv infection. lopinavir-ritonavir has been shown to have in-vitro antiviral activity against beta-coronaviruses such as sars-cov, and mers-cov [39] [40] [41] [42] . since lopinavir-ritonavir is not specifically designed for treatment of coronavirus, lopinavir-ritonavir alone may not demonstrate a difference from placebo in reducing viral load when treatment was initiated at a median of 13 days after symptoms onset [41] . in an open label treatment trial, lopinavir-ritonavir with ribavirin reduced the mortality and requirement of intensive care support of hospitalized sars patients compared with historical control [41] . many interferons, especially interferon beta have been shown to have modest in-vitro antiviral activity against sars-cov and mers-cov [39, 40] . lopinavir-ritonavir or interferon beta-1b has been shown to reduce viral load of mers-cov and improve lung pathology in a nonhuman primate model of common marmoset [42] . lopinavir/ritonavir and interferon-β1b alone or in combination are being evaluated in clinical trials. there is a long history of using convalescent plasma as treatment for infectious diseases, including severe viral lower respiratory tract infections [43] . individuals who have recovered from sars-cov-2 infection may generate neutralizing antibodies [44, 45] that could have application to prevention of infection in certain settings, such as individuals with underlying conditions predisposing to severe disease and those with high-risk exposure. monoclonal antibodies against other respiratory viruses have been shown to be protective against hospitalization in specific high-risk populations [46, 47] and animal models have suggested utility in prophylaxis against sars coronavirus infection [48] . there are some risks associated with the use of convalescent plasma like transfusion-related acute lung injury or a theoretical risk of antibody-dependent enhancement of infection (ade). ade can occur in several viral diseases and involves an enhancement of disease in the presence of certain antibodies [49] . a trial from patients recovered from sars-cov-2 infection for use as prophylaxis in adults with a high -risk exposure is expected to begin recruiting shortly [50] . there are only in vitro data available on the activity of ribavirin on sars-cov-2 currently. the ec50 (half maximal effective concentrations) was significantly higher than for chloroquine and remdesivir, so it appears less potent in vitro compared to these agents [51] . there are limited clinical studies in sars-cov-1 and mers-cov infections. in a systematic review of ribavirin treatment in patients infected with sars-cov-1, 26 studies were classified as inconclusive, and four showed possible harm [30] . in a retrospective observational study in patients with mers-cov infection, the combination of ribavirin and interferon, compared to no antiviral treatment, was not associated with improvement in the 90-day mortality or more rapid mers-cov rna clearance [52] . m a n u s c r i p t oseltamivir oseltamivir is a neuraminidase inhibitor used for prophylaxis and treatment of influenza. given its specificity for an enzyme not found on coronaviruses, it is unclear what the mechanism of action would be against covid-19. however, this has been used in combinations of antiviral therapy in wuhan [53] and continues to be explored as a therapeutic option as part of combination regimens. two trials evaluating combination regimens are underway in wuhan [54, 55] as well as a trial in thailand proposing different combinations [56] . none of the trials or case reports have examined oseltamivir as monotherapy. intravenous immunoglobulin (ivig) has been used as an adjuvant to treat a variety of pathogens either as a pooled product or in a concentrated more pathogen focused (hyperimmune) form. as the community from which a given batch of ivig is derived from includes increasing numbers of individuals who have recovered from sars-cov-2, the possibility of protective antibodies being present in the pooled product is increased. however, the potential utility of ivig for the treatment of sars-cov-2 is unknown at this time. its use has been reported in a few patients with covid-19 [57] , but studies are needed to determine if there may be a role for ivig in the treatment of sars-cov-2. remdesivir (gs-5734) is a broad-spectrum antiviral nucleotide prodrug with potent in vitro activity against a range of rna viruses including ebola virus, marburg, mers-cov, sars-cov, respiratory syncytial virus, nipah virus, and hendra virus [58] [59] [60] . the mechanism of action of remdesivir is premature termination of viral rna transcription [60] . its use improved disease outcomes and reduced viral loads in sars-cov-infected mice [59] . the efficacy of prophylactic and therapeutic remdesivir was tested in a rhesus macaque model of mers-cov infection [61] . prophylactic remdesivir treatment initiated 24 hours prior to inoculation completely prevented mers-cov-induced clinical disease, strongly inhibited mers-cov replication in respiratory tissues, and prevented the formation of lung lesions [61] . therapeutic remdesivir treatment initiated 12 hours postinoculation reduced clinical signs, virus replication in the lungs, and decreased the presence and severity of lung lesions. a recent case series of 53 patients with severe covid-19 pneumonia who received remdesivir under a compassionate-use protocol reported clinical improvement in 68% after a median follow-up of 18 days, with 13% mortality and a generally acceptable toxicity profile [62] . however, there was no comparison group of similar patients who received standard care at the participating institutions. because rcts for remdesivir have not been completed, formalized recommendations will be made once the entire body of evidence for remdesivir is available. m a n u s c r i p t should nsaids be stopped in patients infected with covid-19? the role of nsaids in the management of sars-cov2 has been discussed widely. recent anecdotal reports and subsequent warnings from health officials have suggested against the use of nsaids in the care of patients with covid-19; however, neither fda, ema, or who have identified evidence linking nsaids to covid-related clinical deterioration. human coronaviruses, including sars cov-2, use ace2 to bind to human targets and gain entry into target cells [63] . it has been theorized that nsaids, due to upregulation in ace2 in human target cells, may lead to a more severe course of covid-19 in those taking nsaids. while no causal evidence of adverse outcomes with nsaids in the management of covid-19 have been published, there are well known risks of nonsteroidal anti-inflammatory agents including cardiovascular, gastrointestinal and renal adverse events [64, 65] . in the setting of bacterial pneumonia, nsaids may impair recruitment of polymorphonuclear cells, resulting in a delayed inflammatory response and resolution of infection, however a causal relationship has not been established [66, 67] . rcts are needed to better understand the safety of nsaids in the management of patients with covid-19. one rct is currently underway to evaluate the role of naproxen in those critically ill with covid-19 [68] . should ace and arb's for hypertension be stopped in patients infected with covid19? angiotensin converting enzyme 2 (ace2) is the receptor for sars cov-2 on human cells. because angiotensinconverting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) may increase ace2 expression, the possibility has been raised that these drugs may increase the likelihood of acquiring sars-cov-2 or may exacerbate the course of covid-19. to date, however, there are no clinical data to support this hypothetical concern. for this reason, the american heart association, the heart failure society of america and the american college of cardiology all recommend that ace inhibitors or arbs be continued in people who have an indication for these medications [69] . during epidemics like the current covid-19 pandemic, when there are no clinically proven treatments, the tendency is to use drugs based on in vitro antiviral activity, or on anti-inflammatory effects or based on limited observational studies. it is commendable that observational studies are done during an epidemic, but often they do not have concurrent controls, have a significant risk of bias, and use surrogate outcomes like viral clearance rather than patient-important outcomes. medications that were thought to be effective m a n u s c r i p t based on in vitro studies and observational studies for other diseases were later proven to be ineffective in clinical trials [70] . due to the understandable urgency in producing, synthesizing and disseminating data during the current pandemic, there has been a noticeable increase in fast track publication of studies. in addition to wellestablished concerns that may decrease our certainty in the available evidence, there may be additional issues that will ultimately influence the trustworthiness of that evidence, including: 1) circumvention of usual research steps (delay of irb approval [71] , inclusion of same patients in several studies); 2) limited peer-review process (the usual due diligence from editors and reviewers is side-stepped, potentially leading to unnoticed errors in data and calculations, incomplete reporting of methods and results, as well as underestimation of study limitations); 3) increased potential for publication bias (in the interest of showing promising data and in the race to achieve recognition, there may be added inclination to publish positive results and disregard negative ones). the extent and impact of these considerations remain currently uncertain but were acknowledged in the development of this guideline. despite these limitations, the recommendations were based on evidence from the best available clinical studies with patient-important endpoints. the panel determined that when an explicit trade-off between the highly uncertain benefits (e.g., the panel was unable to confirm that hcq increases viral cure or reduces mortality) and the known putative harms (qt prolongation and drug-drug interactions) were considered, a net positive benefit was not reached and could possibly be negative (risk of excess harm). the safety of drugs used for the treatment of covid-19, especially in patients with cardiovascular disease, immunosuppressive conditions, or those who are critically ill with multi-organ failure has also not been studied. drugs like azithromycin and hydroxychloroquine can cause qt prolongation and potentially lifethreatening arrhythmias. steroids and il-6 inhibitors can be immunosuppressive and potentially increase risk of secondary infections. steroids may produce long term side effect such as osteonecrosis [72] . given that the panel could not make a determination whether the benefits outweigh harms for these treatments it would be ethical and prudent to enroll patients with covid-19 in clinical trials, rather than use clinically unproven therapies [73] . there are multiple ongoing trials, some with adaptive designs, which potentially can quickly answer pressing questions on efficacy and safety of drugs in the treatment of patients with covid-19. we acknowledge that enrolling patients in rcts might not be feasible for many frontline providers due to limited access and infrastructure. should lack of access to clinical trials exist, we encourage setting up local or collaborative registries to systematically evaluate the efficacy and safety of drugs to contribute to the knowledge base. without such evaluations we often attribute success to drugs and failure to disease (covid-19) [70] . during such a pandemic, barriers to conducting studies and enrolling patients in trials for already overburdened front line providers should be minimized while ensuring the rights and safety of patients [74] . for clinical trials and observational studies, it is critical to determine a priori standardized & practical definitions of patient populations, clinical syndromes, disease severity and outcomes. observational and non-experimental studies can sometimes answer questions not addressed by trials, but there is still a need for standardized definitions. for clinical syndromes clearly distinguishing between asymptomatic carrier state, upper respiratory tract infection and lower respiratory tract infection is important. illness severity should be reasonably defined using readily available clinical criteria of end organ failure, like the degree of m a n u s c r i p t respiratory failure using sa02 or fi02:pa02 ratios for lower respiratory tract infection, as opposed to location-based severity determinations such as icu admission, which can lead to bias based on resource limitations (i.e., bed availability) or regional/institutional practice patterns [75] . for outcomes of prophylaxis trials, the primary endpoint should be prevention of infection and for therapeutic trials patient centered outcomes like reduction of mortality (both short term and long term) [76] . trials should also study treatments in high risk populations or special populations like immunosuppressed patients, people with hiv, patients with cardiovascular comorbidities and pregnant women. the panel expressed the overarching goal that patients be recruited into ongoing trials, which would provide much needed evidence on the efficacy and safety of various therapies for covid-19. this is a living guideline that will be frequently updated as new data emerges. updates and changes to the guideline will be posted to the idsa website. clinical characteristics of coronavirus disease 2019 in china world health organization. coronavirus disease 2019 (covid-19) situation report -75. geneva: world health organization characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention development of rapid guidelines: 3. gin-mcmaster guideline development checklist extension for rapid recommendations grade guidelines: 2. framing the question and deciding on important outcomes national institute for health and care excellence. scoping. interim process and methods for developing rapid guidelines on covid-19 (pmg35). london: national institute for health and care excellence closing the gap between methodologists and end-users: r as a computational back-end cochrane handbook for systematic reviews of interventions grade guidelines: 1. introduction-grade evidence profiles and summary of findings tables gradepro guideline development tool available at: gradepro.org efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease-19 (covid-19) hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: an observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection the qt interval in patients with sars-cov-2 infection treated with hydroxychloroquine/azithromycin suspected hydroxychloroquine-associated qt-interval prolongation in a patient with systemic lupus erythematosus chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia syncope in a patient being treated for hepatic and intestinal amoebiasis conduction disorder and qt prolongation secondary to long-term treatment with chloroquine discontinuation of antimalarial drugs in systemic lupus erythematosus azithromycin and the risk of cardiovascular death therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases drug interactions of clinical significance early, low-dose and short-term application of corticosteroid treatment in patients with severe covid-19 pneumonia: single-center experience from wuhan, china clinical features and progression of acute respiratory distress syndrome in coronavirus disease 2019 a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 medication patterns and disease progression among 165 patients with coronavirus disease 2019 (covid-19) in wuhan, china: a single-centered, retrospective, observational study risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease sars: systematic review of treatment effects effective treatment of severe covid-19 patients with tocilizumab clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study actemra® (tocilizumab) injection, for intravenous or subcutaneous use interleukin-6 and cytochrome-p450, reason for concern? a physiologically based pharmacokinetic modeling approach to predict disease-drug interactions: suppression of cyp3a by il-6 treatment of 5 critically ill patients with covid-19 with convalescent plasma the feasibility of convalescent plasma therapy in severe covid-19 patients: a pilot study efficacy and safety of darunavir and cobicistat for treatment of pneumonia caused by 2019-ncov (daco-ncov) in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov establishment and validation of a pseudovirus neutralization assay for sars-cov-2 the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically significant congenital heart disease human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets the convalescent sera option for containing covid-19 efficacy and safety human coronavirus immune plasma (hcip) vs. control (sars-cov-2 non-immune plasma) among adults exposed to covid-19 (cssc-001). available at remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a prospective/retrospective, randomized controlled clinical study of antiviral therapy in the 2019-ncov pneumonia a randomized, open, controlled clinical study to evaluate the efficacy of asc09f and ritonavir for 2019-ncov pneumonia various combination of protease inhibitors, oseltamivir, favipiravir, and hydroxychloroquine for treatment of covid19 : a randomized control trial (thdms-covid19) high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection compassionate use of remdesivir for patients with severe covid-19 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor vascular and upper gastrointestinal effects of nonsteroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials non-steroidal anti-inflammatory drug induced acute kidney injury in the community dwelling general population and people with chronic kidney disease: systematic review and meta-analysis nonsteroidal antiinflammatory drugs may affect the presentation and course of community-acquired pneumonia risks related to the use of non-steroidal anti-inflammatory drugs in community-acquired pneumonia in adult and pediatric patients efficacy of addition of naproxen in the treatment of critically ill patients hospitalized for covid-19 infection (enacovid) hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 treating covid-19-off-label drug use, compassionate use, and randomized clinical trials during pandemics editorial concern-possible reporting of the same patients with covid-19 in different reports steroid therapy and the risk of osteonecrosis in sars patients: a dose-response meta-analysis the ebola clinical trials: a precedent for research ethics in disasters clinical research ethics for critically ill patients: a pandemic proposal early observational research and registries during the 2009-2010 influenza a pandemic choosing outcomes for clinical trials: a pragmatic perspective key: cord-356084-621qzpqd authors: qu, jiuxin; wu, chi; li, xiaoyong; zhang, guobin; jiang, zhaofang; li, xiaohe; zhu, qing; liu, lei title: profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) date: 2020-04-27 journal: clin infect dis doi: 10.1093/cid/ciaa489 sha: doc_id: 356084 cord_uid: 621qzpqd we profiled the serological responses to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) nucleocapsid (n) protein and spike (s) glycoprotein. the majority of the patients developed robust antibody responses between 17 and 23 days after illness onset. delayed, but stronger antibody responses were observed in critical patients. a novel coronavirus (sars-cov-2) causing an outbreak of infectious pneumonia (covid-19) emerged in december 2019 [1, 2] . because there is currently no specific immunity in the population, humans of all ages and races are susceptible to sars-cov-2 infection. the world health organization (who) has declared sars-cov-2 a pandemic, and as of apr 18, 2020, a total of 2,160,207 confirmed covid-19 cases and 146,088 related deaths had been reported [3] . diagnosis relies on viral rna detection by rt-pcr using nasopharyngeal (np) swabs. considering the existence of asymptomatic transmission and false negative results of pcr caused by sampling mistakes or sometimes low viral shedding of the np [4] , improvement of covid-19 diagnostic assays are still needed. similar to sars-cov and mers-cov, the understanding of antibody responses specific to sars-cov-2 in patients will be helpful for diagnosis, seroepidemiologic surveys, and pathogenesis studies. in this study, we investigated the humoral immunity of hospitalized patients, analyzed the profile of igg and igm antibodies against the sars-cov-2 in 41 covid-19 patients between three and 43 days of their illness. between january 11, 2020 and february 10, 2020, 394 covid-19 patients were admitted to the third people's hospital of shenzhen. sars-cov-2 was confirmed by two repeated positive results from our hospital and local chinese centers for disease control and prevention using two different commercial rt-pcr kits approved by the national medical products administration (nmpa), according to the manufacturer's protocol. forty-one patients with preserved serial serum samples were included in this study. patients were classified using the following criteria: 1) mild cases: clinical symptoms were mild without manifestation of pneumonia on imaging; 2) moderate cases: fever, respiratory symptoms, and with radiological findings of pneumonia; 3) severe cases: meeting any one of the following criteria: respiratory distress, hypoxia (spo2≤93%), or abnormal blood gas analysis: (pao2<60mmhg, paco2>50 mmhg); 4) critical cases: meeting any one of the following criteria: respiratory failure requiring mechanical ventilation, shock, or other organ failure that m a n u s c r i p t 2 requires icu care. 41 patients were then divided into three groups: mild and moderate (15 patients), severe (16 patients), and critical (10 patients). a total of 347 serum specimens from these patients (5-31 samples from each patient) were collected between three and 43 days of disease onset for routine clinical testing. control sera were collected from 10 patients with influenza and 28 patients completing routine check-ups between february 4, 2020 and february 10, 2020 at our hospital. the control sera were tested for the presence of igg and igm simultaneously with covid-19 sera by the same method. the study was approved by the institutional review board of the third people's hospital of shenzhen (number 2020-0036). igg and igm antibodies against sars-cov-2 were measured using iflash-sars-cov-2 igg/igm chemiluminescent immunoassay kit (c86095g/c86095m, yhlo biotech, shenzhen). according to the instructions, the sensitivity and specificity of the kits was 90% and 95% for igg, 80% and 95% for igm. as a screening assay for covid-19 diagnosis, combined nucleocapsid (n) protein and spike (s) glycoprotein were used as coated antigens to increase the sensitivity. the levels of igg and igm antibodies were positively correlated with the relative luminescence unit (rlu), and were calculated as au/ml. briefly, the serum samples of both healthy patients and confirmed covid-19 patients were tested. according to the receiver operating curve (roc curve), the corresponding concentration point of auc (area under the roc curve) greater than 0.9 was defined as the cut-off point, and the level of this point was defined as 10au / ml. scatter plots were drawn to illustrate the cumultative proportion of patients with igg all controls enrolled in the study tested negative (table s4 ). basic demographic information of the study participants are described in table s1 . table s2 , and figure s1 ). the level of igg antibody reached the highest concentration on day 30, while the highest concentration of igm antibody appeared on day 18, but then began to decline. the trends in antibody production was analyzed among the three groups with different disease severities during the first six weeks after disease onset, as illustrated in figure 1c and 1d. for igg, the fitting curve of those in the critical group rose rapidly above the cut-off value from day seven and peaked on day 20, while the fitting curves of the non-critical groups rose gently from day five. although the igg level of those in the mild and moderate group was still rising on day 28, the igg response of the critical group was significantly stronger than that of non-critical groups within 4 weeks after illness onset (p=0.0001, table s3 ). for igm, the fitting curve of the critical group rose above the cut-off value on day 10, peaked on day 23, then began to decline. however, the igm levels of non-critical groups rose above the cut-off value as early as day five, peaked on day 16, then dereased. in the majority of the patients, there were antibody responses to sars-cov-2 during the first three weeks of the disease. the seroconversion time of igg antibody was earlier than that of igm antibody (table s2 and figure s1 ). the profile of antibodies against sars-cov-2 was comparable with previous findings of sars-cov infections [5, 6] . li et al., reported that both igg and igm antibody levels increased to detectable levels from the second week of illness in 20 sars-cov patients [5] . similarly, woo et al., also observed that the seroconversion time for igg was three days earlier than that for igm after the sars-cov infection [6] . the negative igm results in five patients were possibly caused by the window phase of antibody production, as serum specimens were collected between day three and day 13, thus longer surveillance is needed. on the other hand, park et al., reported that early antibody response was associated with reduced disease severity in mers-cov infections [7] . xu et al., revealed that imbalance of the immune system was a pathogenesis factor from the pathological finding of a covid-19 case [8] . here, compared to non-critical groups, we also observed delayed igg and igm antibody responses in the critical group ( figure 1c and 1d). moreover, the slope of the igg antibody response was steeper in the critical group (table s3) , which might be a hint of inflammatory storm. the intervention window might be the second week after illness onset for most patients. our study has several limitations. firstly, liu et al. found that acute lung injury in chinese macaques caused by sars-cov could be mediated by higher anti-spike igg [9] , and we detected high levels of igg antibody in critical patients. since we used combined n and s proteins as capture antigen to increase sensitivity of this assay, further studies are needed to separate the effects of specific anti-n and anti-s antibodies. secondly, we did not test the possible cross-reactivity of our in-house a c c e p t e d m a n u s c r i p t 5 assay with common human coronaviruses (e.g., hcov-oc43 or others), mers-cov, or sars-cov. no sars-cov or mers-cov infections had been reported by any of the patients in the study, and the infection rate of common hcov infections has been estimated to be as low as 0.8% in a previous study [10] . thus, even if the crossreactivity exists, it would have limited impact on the validity of these findings. a c c e p t e d m a n u s c r i p t 9 figure 1 clinical features of patients infected with 2019 novel coronavirus in wuhan clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. the lancet respiratory medicine who, <coronavirus disease 2019(covid-19) situation report-89.pdf> molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes profile of specific antibodies to the sars-associated coronavirus longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus kinetics of serologic responses to mers coronavirus infection in humans pathological findings of covid-19 associated with acute respiratory distress syndrome. the lancet respiratory medicine anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection viral etiology of community-acquired pneumonia among adolescents and adults with mild or moderate severity and its relation to age and severity potential conflicts of interest. the authors declare no conflict of interest. a c c e p t e d m a n u s c r i p t 7 key: cord-356316-ui11jr2h authors: patel, monita r; carroll, darin; ussery, emily; whitham, hilary; elkins, christopher a; noble-wang, judith; rasheed, james kamile; lu, xioayan; lindstrom, stephen; bowen, virginia; waller, jessica; armstrong, gregory; gerber, susan; brooks, john t title: performance of oropharyngeal swab testing compared to nasopharyngeal swab testing for diagnosis of covid-19 —united states, january-february 2020 date: 2020-06-16 journal: clin infect dis doi: 10.1093/cid/ciaa759 sha: doc_id: 356316 cord_uid: ui11jr2h among 146 nasopharyngeal (np) and oropharyngeal (op) swab pairs collected ≤7 days since illness onset, cdc real-time rt-pcr sars-cov-2 assay diagnostic results were 95.2% concordant. however, np swab ct values were lower (indicating more virus) in 66.7% of concordant-positive pairs, suggesting np swabs may more accurately detect amount of sars-cov-2. m a n u s c r i p t testing for sars-cov-2, the virus that causes coronavirus disease 2019 , remains critical to identify persons with covid-19 and implement clinical and public health interventions to reduce morbidity and mortality and prevent virus transmission. current us centers for disease control and prevention (cdc) guidelines identify nasopharyngeal (np) and oropharyngeal (op) swabs as acceptable upper respiratory specimens to test for presence of sars-cov-2 ribonucleic acid (rna) (1) . relative to np swabs, op swabs may be less challenging to collect, require less healthcare provider training or may logistically be the only option based on available supplies. some indirect evidence from testing for other respiratory illnesses and coronaviruses suggests op swabs may be less sensitive than np swabs (2, 3) . however, to date, limited published data exist about testing performance of op swab compared to np swab for sars-cov-2 rna (4). we analyzed data on op and np swabs tested for sars-cov-2 rna by cdc through march 3, 2020 a c c e p t e d m a n u s c r i p t we matched np and op swabs collected on the same date in the same person to create np-op swab pairs. in our main analysis, we analyzed pairs collected ≤7 days since of the reported illness onset date; if more than one pair was collected from a person in this timeframe, we included the earliest (first) pair in our primary analysis. as sensitivity analyses, we also examined first pairs collected >7 days since illness onset date and second follow-up pairs collected >7 days since illness onset (regardless of timing of first pair). we examined concordance/discordance in diagnostic results and ct value distributions between np and op swabs within pairs. we also calculated sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) with 95% confidence intervals (ci) for op swab, compared with np swab. np swab was selected as the comparator because during the testing timeframe, np swab was the preferred upper respiratory specimen per cdc guidelines; however, op swab was an acceptable alternative specimen for diagnostic testing (independent of np swab testing/result) (1). accordingly, we also calculated absolute sensitivities for op and np a c c e p t e d m a n u s c r i p t overall, among persons with specimens collected early in the illness course, sars-cov-2 rna diagnostic results were highly concordant between op and np swabs. despite this, among concordant-positive specimens, ct values were significantly lower among np swabs. these findings are partially aligned with a study from germany among persons tested ≤5 days since illness, which similarly did not find meaningful differences in sars-cov-2 rna detection between np and op swabs, but contrastingly did not find differences in viral loads between np and op swabs (6) . in our analysis, using np swab as the comparator, specificity and npv of op swab were high and sensitivity and ppv of op swab were moderate, but had wide cis that included low values. absolute sensitivity was only slightly lower for op swab compared to np swab. differences in ct values between np and op swab among concordantpositive pairs, did not ultimately impact most diagnostic results in our main analysis where ct values were relatively low and well under the cutoff value of 40 cycles. in contrast, in sensitivity analyses of np-op swab pairs collected >7 days since illness onset, sensitivity of op swab was comparatively low. current infectious diseases society of america guidelines specifically recommend collection of np, mid-turbinate or nasal swabs rather than op swabs alone for all symptomatic persons; our findings suggest this recommendation may be particularly relevant for persons later in the illness course and who may have lower amount of sars-cov-2 viral rna (7). the findings in this report are subject to at least four limitations. first, specimen collection procedures, including type and material of swab used, and specimen handling may also impact test performance. these data were not available to us, so sub-analyses, for instance by type of swab used, were not possible. second, missing and erroneous personal identification numbers or specimen collection dates limited our ability to match all potential np and op swab pairs and account for all specimen pairs within a unique person. if missing data or errors were a c c e p t e d m a n u s c r i p t not at random, this may have biased our results. third, the number of specimen pairs and the number of positive results were small and precluded our ability to estimate sensitivity and ppv with better precision. fourth, specimens were tested using the cdc 2019-ncov real-time rt pcr diagnostic panel currently approved under an emergency use authorization, and our findings, particularly those around ct values, may not be generalizable to other nucleic acid tests. consequently, our findings may not be fully generalizable to current specimen collection and testing circumstances and should thus be interpreted accordingly. together, our findings support cdc guidelines that identify np and op swabs as acceptable specimens for sars-cov-2 rna testing; but suggest that np swab may comparatively be a more sensitive specimen type for testing persons later in the illness course. regardless of type of specimen collected, in persons with a single negative sars-cov-2 rna test, signs and symptoms of covid-19, epidemiological links, and other risk factors may need to be considered to inform subsequent clinical management and public health interventions to prevent further transmission (5). m a n u s c r i p t interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19) identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays sars-cov-2 viral load in upper respiratory specimens of infected patients cdc. cdc 2019-ncov real-time rt-pcr diagnostic panel instructions for use ga: us department of health and human services, cdc virological assessment of hospitalized patients with covid-2019 guidelines on the diagnosis of covid-19 a c c e p t e d m a n u s c r i p t key: cord-348144-t0chpsuh authors: lucas, alexander h.; apicella, michael a.; taylor, christopher e. title: carbohydrate moieties as vaccine candidates date: 2005-09-01 journal: clin infect dis doi: 10.1086/432582 sha: doc_id: 348144 cord_uid: t0chpsuh carbohydrate epitopes or glycotopes are structurally diverse, occur in a variety of chemical contexts, and are present on the surfaces of cells in the body and on the surfaces of pathogens. these various structures and modes of presentation affect how they are perceived and processed by the body and dictate the outcome of the immune response directed against them. this review focuses on mechanisms of carbohydrate immunity, with an emphasis on carbohydrate vaccines that have been or are being developed for protection against encapsulated bacterial pathogens. we discuss the cellular basis of carbohydrate immunity, newly identified glycotope processing pathways and recognition capabilities, and the synthetic and microarray technologies that are being developed that will permit new experimental approaches to carbohydrate vaccine development and the exploration of the interaction of the immune system with self and nonself glycans. this article focuses primarily on mechanisms of carbohydrate immunity, with an emphasis on carbohydrate vaccines that have been or are being developed for prevention of diseases caused by encapsulated bacterial pathogens. data on the use of recent technological advances in immunology, genomics, and glycomics in vaccine development are also presented. it has long been known that bactericidal and/or opsonic antibodies directed against capsular polysaccharide (ps) glycotopes protect against invasive diseases caused by encapsulated bacteria, and, accordingly, vaccine development has focused on the elicitation of these antibody specificities. purified microbial ps vaccines have been in use for 140 years, but they have proven to be variably immunogenic and variably efficacious in protecting susceptible populations against invasive meningococcal, pneumococcal, and haemophilus influenzae type b (hib) diseases [1, 2] . when administered as purified pss, their effectiveness generally is limited by the modest nature of the immune response, the lack of a memory response, and the inability to stimulate responses in young children who are at the greatest risk for serious infections due to encapsulated organisms. the demonstration that infants could produce protective anticapsular antibody responses following immunization with hib psprotein conjugates represented a major advance in public health and laid the groundwork for the subsequent development of new conjugate vaccines [3] . an effective pneumococcal heptavalent capsular ps conjugate vaccine has been developed and is licensed in the united states for use in infants and children [4] . use of this vaccine has led to a significant reduction in serious pneumococcal disease caused by pneumococci expressing capsular serotypes contained within the vaccine. the vaccine has also been shown to reduce the incidence of streptococcus pneumoniae otitis media [5, 6] . studies from israel have indicated that this vaccine may impact favorably by reducing the number of antibiotic-resistant strains in the vaccinated population [7, 8] . however, emerging evidence demonstrates that serotype replacement with nonvaccine capsular types may be a future problem necessitating changes in the vaccine spectrum. an 11-valent pneumococcal ps conjugate vaccine is presently undergoing evaluation. meningococcal serogroup a ps is unusual because it is immunogenic in infants as young as 3 months of age, and administration of repeated doses elicits booster antibody responses. reimmunization at 2 and 6 years of age maintains a high degree of protection. older children and adults require only 1 injection. vaccines against group a meningococci have been highly effective in preventing disease in a number of regions. in finland, herd immunity to group a meningococci was induced by vaccinating approximately one-third of the population. use of vaccines has significantly reduced the prevalence of disease in china. household contact vaccination has proven to be highly effective in africa. however, attempts at preparing protein conjugates of group a meningococcal capsular ps have not increased immunogenicity. group b meningococcus is the major cause of serious meningococcal disease in the united states [9] . group b capsular ps is nonimmunogenic, presumably because of its identity with self-antigens [10] . the potential for induction of autoimmunity and the lack of immunogenicity have discouraged commercial development of ps-based group b vaccines. alternative approaches have focused on other antigens, such as the outer membrane vesicle. the outer membrane-vesicle vaccine has good efficacy in older children and adults; however, the data for infants are mixed [11] . following extensive testing, an omv vaccine prepared from an epidemic group b strain in new zealand has been provisionally licensed. this vaccine has been introduced into the population in an attempt to control the epidemic [12] . investigators at wyeth and oxford university, england, are studying detoxified meningococcal lipopolysaccharide as a conjugate vaccine. the inner core of the meningococcal lipopolysaccharide is relatively highly conserved, and the use of appropriate conjugation procedures can result in a broadly cross-reactive meningococcal lipopolysaccharide vaccine. the meningococcal group c ps conjugate vaccine has proven to be an excellent vaccine [13] . in the united kingdom, a decrease in the rate of group c disease was observed after vaccination with this conjugate. in children who were vaccinated during infancy at 2, 3, and 4 months of age, the effectiveness of the vaccine decreased to very low levels after 1 year [13] . there was a much smaller decline in efficacy in infants who were vaccinated at 5-11 months of age, suggesting that a booster dose administered at 13-15 months of age might protect against the loss of efficacy and is now recommended. follow-up studies also demonstrated that there was no evidence of a switch of meningococcal capsular serogroup from serogroup c to other serogroups caused by vaccine pressure. as noted above, with the exception of the group a meningococcal ps, children aged !2 years generally are unresponsive to vaccination with purified ps antigens. one early explanation for this anergy was that infants lacked the relevant ps-specific b cells. however, numerous studies have demonstrated that an insufficient antibody repertoire cannot account for the refractoriness of the infant to ps vaccination. infants can produce anti-ps antibodies after ps conjugate vaccination, and studies of the hib ps antibody repertoire have shown that infants can utilize the same variable (v) region genes as those used by adults [14] . despite intensive investigation, the mechanism underlying the unresponsiveness of the infant to ps vaccination has not been precisely identified. the mechanism is likely multifactorial and may involve lacking expression of sufficient costimulatory molecules and/or their respective receptors and immaturity of dendritic cells and/or marginal zone b cells. cell surfaces in the body are replete with glycan molecules, and it is clear that self glycotopes can function as tolerogens and thus negatively shape the expressed antibody repertoire. the absence of antibodies to a and b blood-group substances in individuals expressing the respective blood groups represents a classical example. however, tolerance is imperfect, and antibodies against self-glycan structures can be present in healthy subjects. infectious agents or vaccines expressing glycotopes structurally related to self glycans can elicit antibodies reactive with self antigens. for example, neutralizing antibodies elicited by an inactivated severe acute respiratory syndrome coronavirus vaccine cross-react with the human serum glycoprotein asialoorosomucoid [15] . what is less clear is the role of self glycans in the positive selection of the b cell repertoire. a growing body of evidence indicates that positive selection occurs during murine-b cell development [16] and can involve selection against the self glycoprotein thy-1 [17] . the recent discovery of an endogenous cd1-restricted glycosphingolipid, isoglobotrihexosylceramide, suggests that self glycans may mediate the positive selection of t cells and/or nk cells [18] . despite our lack of understanding of the mechanism, it is well established that the refractoriness of infants to pss, a class of antigens designated as thymus-independent (ti) type 2, can be overcome when ps glycotopes are presented in the context of an immunogenic carrier protein. coupling of protein is thought to convert glycotopes into t cell-dependent immunogens. immunization with ps protein-conjugate vaccines likely involves activation of the follicular b cell population. follicular b cells take up ps protein conjugates via their ps-specific b-cell receptor, process the carrier protein moieties, and present the resulting peptides on cell surface major histocompatibility complex class ii molecules where they may engage peptide-specific t cells. ps-specific b cells thereby receive signals via cognate t cell-b cell interactions and cd40-cd40l engagements that are sufficient for activation, differentiation into antibody secreting cells, germinal-center formation, class switch recombination, and somatic hypermutation. memory b cells generated during this t cell-dependent process can be activated by subsequent stimulation with either ps-protein conjugates or by multi-epitope ps. different carrier proteins have been used in the formulation of ps-conjugate vaccines, and although they are thought to convert the glycotope into a t cell-dependent form, they possess disparate immunological properties. for example, hib psconjugate vaccines utilizing different carriers elicit distinct antibody populations, as has been discerned by idiotypic [19] and avidity analyses [20] . furthermore, studies of humans indicate that both th1 and th2 cytokine responses are elicited following immunization with pneumococcal conjugate vaccines [21] . the outer membrane protein complex derived from neisseria meningitidis is used as a carrier protein in some ps-conjugate vaccines. the hib ps-conjugate vaccine, having an outer membrane protein complex as the carrier, is uniquely capable of stimulating a serum anti-hib ps-antibody response in 2month-old infants after a single immunization. in contrast, conjugates utilizing bacterial toxoids as carriers usually do not elicit significant seroconversion until the second immunization [22] . these properties of the outer membrane-protein complex carrier may be related to its ability to engage toll-like receptor-2 [23] . thus, carrier proteins function not only as a source of peptide epitopes permitting the delivery of t cell help, but they also can possess mitogenic and adjuvant-like properties that stimulate the innate immune response. the immunological variability of different carriers and the increasing use of multivalent glycoconjugates utilizing the same carrier protein has generated concern about potential complications arising from high carrier doses, such as antigenic competition and carrier-induced epitope suppression. this concern has prompted the search for alternate carrier-protein strategies that include the synthesis of a peptide carrier that functions as a universal t helper-cell epitope by binding promiscuously to human leukocyte antigen-dr molecules [24] and the synthesis of recombinant carrier proteins containing strings of t cell epitopes derived from several pathogen-derived antigens [25] . pss have been classified as ti-2 antigens on the basis of their ability to stimulate antigen-specific b cells in the absence of t cells [26] . ps antigens, by nature of their multivalent, repeating glycotope structure, engage a sufficient number of b cell receptors that result in b cell activation and maturation to antibody secretion. the inability of ps immunization to lead to anamnestic responses suggests that memory b cells are not generated by this type of antigenic stimulation, presumably because of failure of the immunogen to associate with mhc-ii molecules and recruit t cell help. although b cell activation by ti-2 antigens can occur independently of t cells, it is becoming increasingly clear that accessory signals, in addition to those generated by b cell-receptor cross-linking, are required to generate antibody responses to ti-2 antigens. in vitro, highly purified b cells do not respond to ti-2 antigens. accessory cells, such as dendritic cells and macrophages, and costimu-latory signals are required for an effective response to ti-2 antigens. in addition, many ti-2 antigens are able to fix complement, and the formation of these complexes in vivo could facilitate b cell activation by providing costimulation through complement receptors. engagement of the complement receptor lowers the antigen signaling threshold for b cell activation [27] , and the coupling of complement c3d fragments enhances ps immunogenicity [28] . several molecules-such as the transmembrane activator and its ligand b lymphocyte stimulator, tyrosine kinase pyk-2, bruton tyrosine kinase, and phospholipase c-g2-have been identified as costimulatory signals and/ or receptors necessary for the generation of antibody responses to ti-2 antigens (reviewed in [29] ). understanding the mechanisms by which these molecules exert their effects will not only suggest new ways to enhance responses to carbohydratebased vaccines, but also new ways to suppress autoimmune responses directed against self glycans. the immune response to glycotopes presented in the context of the microbe is important for understanding carriage, the acquisition of naturally acquired immunity, and the response to infection [30] . the majority of older children and adults possess serum antibodies specific to a plethora of bacterial capsular glycotopes, and these antibodies mediate protective immunity. presumably, exposure to the homologous organism or to organisms expressing cross-reacting glycotopes drives the formation of these antibodies. these natural antibodies appear to derive from memory b cells, because molecular analyses of adult pneumococcal antibodies show that they have undergone both a class switch and hypermutation [31] [32] [33] . these findings suggest that exposure to glycotopes presented in the context of the bacterial surface generates both ps-specific b cell antibody secretion and memory development. in mice, cd4 + t cells and engagement of the toll-like receptor-2 and its adaptor protein, myd88, have been implicated in the response to pneumococcal glycotopes elicited by intact bacteria [34] . unlike conjugate vaccination, which usually occurs by the intramuscular route, natural exposure occurs predominantly across mucosal surfaces. thus, understanding the induction of natural immunity and the maintenance of the carrier state must take into account the migration patterns between secondary lymphoid tissues and the homeostatic mechanisms operating at mucosal surfaces [29] . it is likely that bacteria, purified ps, and glycoconjugate vaccines address overlapping as well as distinct subsets of b cells. as discussed above, follicular b cells are probably the major subset activated by ps-protein conjugates, whereas blood-borne bacteria and ps antigens are thought to stimulate marginalzone b cells (and b-1 b cells in the mouse) [35, 36] . marginal-zone b cells differ from follicular b cells in their rapid proliferative response and elaboration of igm antibodies early in the course of bacterial infection, and it has been suggested that these b cells represent an innate-like response that mediates immediate immunity before the slower adaptive response has developed [35] . human marginal-zone b cells express high levels of cd1c, which suggests that they might be well suited for presentation of bacterially derived lipid antigens. in humans, there is a subpopulation of circulating b cells that have the phenotype of marginal-zone b cells. these cells have been designated "igm memory b cells" because they express both cell-surface igm and the memory cell marker cd27. the v genes of peripheral blood igm memory b cells are mutated [37] , and a recent report indicates that these cells are mobilized in response to ps vaccination [38] . igm memory b cells with mutated v genes are present in patients with hyper igm syndrome who lack functional cd40-cd40 ligand interactions [39] . this finding has prompted the suggestion that 2 pathways of somatic hypermutation exist in humans: the traditional pathway dependent on t cells and cd40 receptor-cd40l receptor interactions, and another pathway independent of cd40 receptor-cd40l receptor interactions [39] . molecular modeling and x-ray crystallographic studies have verified the original proposal of kabat et al. [40] that antibodycombining sites recognizing glycotopes can exist as either shallow grooves or as deep clefts, with the former binding internal epitopes expressed along the length of the glycan polymer and the latter binding to epitopes on the nonreducing ends of the glycan chain. some glycotopes are expressed by short oligomers, whereas others require longer chain lengths for expression. studies of the type 14 pneumococcal ps have shown that the induction of osponically active antibodies is critically dependent on chain length and the expression of a conformational epitope [41] . similarly, the protective glycotope of type iii group b streptococcal ps is a conformationally dependent, helical structure requiring extended chain lengths for expression [42] . thus, antibodies prepared against short oligomeric glycotopes may show the requisite antigen-binding specificity, but they may not recognize native capsular glycotopes and mediate protection. this issue is particularly important in the design of vaccines using synthetic oligosaccharides. avidity has been shown to be a critical determinant of the efficacy of anticapsular antibody protection against hib [19, 20] , pneumococcus [43] and meningococcus [44] . variations in antibody avidity occur during the course of the immune response and can be influenced by vaccine type [20] and age [45] . the structural basis of avidity variation has been studied in detail in the human response to hib ps, in which a struc-turally conserved, canonical combining site dominates the repertoire. regions in the combining site that drastically impact hib ps-binding avidity have been localized to areas thought to represent antigen contact sites, and they include allelic polymorphisms in complementarity region (cdr)-2 of the heavy chain v region [46] and polymorphisms in the light chain cdr-3 that are generated by differential j-region use and utilization of different insertional residues at the v-j junction [14, 47] . recently, a new mechanism of antibody glycotope recognition was described elsewhere [48] . a monoclonal antibody that is specific for a mannose glycotope of hiv was found to have exceptionally high affinity and potent neutralizing activity against numerous viral isolates. x-ray crystallographic analysis demonstrated that the fragment antigen binding dimer possesses 4 binding sites for the mannose determinant. two combining sites constitute the traditional site created by the association of variable heavy and light chain domains, and 2 additional sites are created by domain exchange at the vh-vh interface. this capability for multivalent interaction provides a structural explanation for the high avidity of this antibody. thus, targeting glycotopes in addition to protein epitopes may be a worthwhile strategy in the development of an effective multivalent hiv vaccine that elicits broadly neutralizing antibody responses [49] . molecular mimicry has been a major area of investigation in carbohydrate immunology. numerous reports have shown that peptides binding to carbohydrate combining sites can be isolated from peptide libraries [reviewed in 50]. these peptides show apparent specificity in that their binding is peptide-sequence dependent but is usually limited to the combining site against which they were selected. a fundamental issue is whether these mimetics, when presented in an immunogenic form, are able to elicit carbohydrate-specific antibody responses. some studies suggest that this, indeed, is the case. peptide mimics have been shown to induce cross-reactive responses to carbohydrate antigens expressed on bacteria [51, 52] , fungi [53] , hiv envelope protein [54] , and tumor cells [55] , to name a few. in a mouse-tumor challenge model, immunization with a dna construct encoding a mimetic inhibited tumor growth [56] and antimimotope antibodies block cell adhesion of hiv gp120-expressing cells and human dendritic cells [57] . one could imagine a number of advantages of using peptidyl mimics rather than glycotopes. these include the ease of synthesis and purification, the ability to be formulated in a variety of ways not amenable to glycotopes, such as multiple antigen peptides and dna vaccines [58] , and the ability to engage cellular processing and activation pathways not normally within the capability of glycotopes. the dogma that the recognition of glycotopes is the exclusive provenance of antibody combining sites has been overturned by studies in the past decade that demonstrate that t cell receptors are able to recognize glycotopes if they are presented in the appropriate mhc-binding context [59] [60] [61] . functional and structural studies of glycopeptide antigens have demonstrated that mhc molecules tether the glycopeptide by binding the peptidyl moiety in the mhc-binding pocket, such that the glycotope can directly engage the t cell receptor. cytotoxic t cell responses to carbohydrate antigens can be induced by immunization with either naturally occurring or synthetic glycopeptides [62, 63] . glycolipid antigens can also be recognized by t cells and nk cells via a cd1-dependent mechanism [64] . cd1 molecules, which exist in multiple isoforms, are structurally related to mhc class i molecules. examples of cd1-restricted antigens include mycobacterial glycolipids and the self-derived ganglioside gm1. the t cells recognizing cd1-presented lipid antigens may utilize a variety of t cell receptor-a and t cell receptor-b chains. in addition, nk cells expressing an invariant t cell receptor-a chain paired with various b chains (in both mouse and man) also are cd1-restricted, and they recognize a glycosphingolipid known as a-galactosylceramide, a glycolipid originally identified in marine sponges. a-galactosylceramide binds with high affinity to cd1d and has potent immunoregulatory properties. administration of a-galactosylceramide induces production of ifn-g and il-4 and activates nk cells, t cells, and b cells. a synthetic analogue of a-galactosylceramide has potent protective activities in murine models of malaria and melanoma. cd-1 restricted t cells may produce either th1-type or th1/th2-type cytokine responses that could be important in antitumor and antimicrobial immune responses. although the precise role of cd1-restricted t cells in adaptive immune responses requires further study, some evidence suggests that cd1-restricted nkt cells are involved in immunity to mycobacterium tuberculosis, pseudomonas auruginosa, plasmodium yoelii, trypanosoma cruzi, and francisella tularensis (reviewed in [64, 65] ). a recent study of the capsular ps of bacteriodes fragilis [66] identified a new pathway of glycotope processing and presentation. this work originated from earlier studies demonstrating that ps-specific cd4 + t cells were involved in protection against sepsis caused by b. fragilis [67] . the capsular ps of b. fragilis (and some pneumococcal capsules) is zwitterionic and forms a helical structure. the ps is capable of inducing protective psspecific t cells in an antigen presenting cell-dependent fashion, and this effect depends on the zwitterionic character of the ps. the b. fragilis ps is processed by antigen presenting cells via an endosomal-lysozomal pathway that involves chemical cleav-age by a nitric oxide-mediated mechanism that generates lowmolecular weight carbohydrates capable of binding to mhc-ii and being presented to cd4 + t cells. given the diversity of glycan structures and their chemical contexts, it is likely that additional carbohydrate-processing pathways will be discovered. for example, the c-type lectin, known as specific intracellular adhesion molecule-grabbing nonintegrin-r1 (sign-r1), which is expressed on splenic marginal-zone macrophages, binds with high affinity to the pneumococcal type 14 capsular ps and mediates uptake of type 14 pneumococci [68] . the discoveries that foreign ps antigens and glycotopes can be processed by endosomal pathways, can be presented by mhc-i, mhc-ii, and cd-1 molecules, and can be recognized by t cells and endogenous lectins hold great promise for designing new glycan-based adjuvants and vaccines against tumors, bacteria, and parasites. research in carbohydrate immunology is being accelerated by the development of new technologies, such as carbohydrate microarrays, automated syntheses of oligosaccharides, and biophysical and computational methods for studying antigenantibody interactions. carbohydrate microarrays can be used to investigate lectin and antibody specificities, to identify new lectins, to screen blood for disease patterns, or to identify lead structures for vaccine design [69, 70] . a consortium for functional genomics has been established and has developed a novel glycan-array format that has been demonstrated to have applicability for profiling the specificity of a wide variety of carbohydrate-binding molecules [71] . these new technologies will contribute to the design of better-defined carbohydrate vaccines and will serve as essential tools in our understanding of the glycome and its interaction with the immune system. progress is being made in the development of chemical methods and instruments capable of efficient, high-capacity oligosaccharide synthesis [72] , and these advances are being applied to basic research and to the development of synthetic carbohydrate vaccines. experimental vaccines utilizing synthetic carbohydrates have been developed for a number of pathogens, including s. pneumoniae [73] , vibrio cholera [74] , shigella flexneri [75] , and plasmodium falciparum [76] . a hib vaccine containing a fully synthetic oligomer of the hib capsular ps has been tested in humans and has been demonstrated to elicit functionally active antibodies [77] . carbohydrate antigens represent a significant resource for the development of vaccines against infectious agents, particularly against pathogens for which traditional protein and/or whole organism-based approaches have not been successful. although we do not discuss it in this article, there is a large body of work exploring carbohydrate-based vaccines for the elicitation of both humoral and cellular immunity against tumors [78] . new carbohydrate-based vaccines are being developed for pathogens such as hiv and malaria, and we might expect to see clinical trials evaluating new adjuvants, such as a-galactosylceramide. aside from the need for research, the need for a variety of resources is essential. the availability of carbohydrate microarrays and appropriate animal models, including primate models and transgenic mouse platforms, should be enhanced. also, tetramers and analytical chemistry are needed to aid in the identification of glycolipid antigens recognized by cd-1. other infrastructural needs include good laboratory practice resources for the production of synthetic carbohydrate vaccines and for testing the safety of vaccine candidates in animals and more opportunities (i.e., computer capacity) to model carbohydrate antigen-antibody interactions. resources will be needed to ensure compliance with regulatory standards, such as a defined chemical composition and structure, the ability to manufacture with consistent physical and chemical characteristics, the lack of inherent toxicity, and the capability to induce protective immune responses. it is becoming increasingly apparent that the various structures and modes of glycotope presentation affect how they are perceived and processed by the body and dictate the outcome of the immune response directed against them. new glycotopeprocessing pathways and recognition capabilities are being identified, the signals and molecules involved in cell activation are being elucidated, and new synthetic and microarray technologies are being developed that will permit new experimental approaches to explore the interaction of the immune system with self and nonself glycans. these discoveries can be expected to translate into new 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description of a new l light chainassociated idiotype and the relation between idiotype expression, avidity, and vaccine formulation avidity and bactericidal activity of antibody elicited by different haemophilus influenzae type b conjugate vaccines. collaborative vaccine study group significant variation in serotype-specific immunogenicity of the sevenvalent streptococcus pneumoniae capsular polysaccharide-crm197 conjugate vaccine occurs despite vigorous t cell help induced by the carrier protein differences in the immunogenicity of three haemophilus influenzae type b conjugate vaccine in infants haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging toll-like receptor 2 (tlr2) and requires the presence of tlr2 for optimal immunogenicity development of experimental carbohydrate-conjugate vaccines composed of streptococcus pneumoniae capsular polysaccharides and the universal t-lymphocyte epitope (padre) rationally 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cluster recognition hiv vaccine design and the neutralizing antibody problem peptide mimotopes as surrogate antigens of carbohydrates in vaccine discovery a peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice peptide mimicry of the meningococcal group c capsular polysaccharide immunogenicity and efficacy of cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice peptide mimicry of carbohydrate epitopes on human immunodeficiency virus ) â�¢ vaccines plewski z. vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses a mimic of tumor rejection antigen-associated carbohydrates mediates an anti-tumor cellular response fucosylated lactosamines participate in adhesion of hiv-1 envelope glycoprotein to dendritic cells induction of a protective capsular polysaccharide antibody response to a multiepitope dna vaccine encoding a peptide mimic of meningococcal serogroup c capsular polysaccharide mhc interaction and t cell recognition of carbohydrates and glycopeptides glycopeptides bind mhc molecules and elicit specific t cell responses carbohydrates and antigen recognition by t cells immunization with glycosylated kb-binding peptides generates carbohydrate-specific, unrestricted cytotoxic t cells crystal structure of an mhc class i presented glycopeptide that generates carbohydratespecific ctl understanding the function of cd1-restricted t cells going both ways: immune regulation via cd1b-dependent nkt cells polysaccharide processing and presentation by the mhcii pathway structural features of polysaccharides that induce intrabdominal abscesses the c-type lectin sign-r1 mediates uptake of the capsular polysaccharide of streptococcus pneumoniae in the marginal zone of mouse spleen carbohydrate microarrays-a new set of technologies at the frontier of glycomics the use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens consortium for functional genomics automated carbohydrate synthesis to drive chemical glycomics synthetic 6b di-, tri-and tetrasaccharide-protein conjugates contain pneumococcal type 6a and 6b common and 6b-specific epitopes that elicit protective antibodies in mice synthetic fragments of vibrio cholera o1 inaba o-specific polysaccharide bound to a protein carrier are immunogenic in mice but do not induce protective antibodies preparation of synthetic glycoconjugates as potential vaccines against shigella flexneri serotype 2a disease synthetic gpi as a candidate anti-toxic vaccine in a model of malaria synthetic conjugate polysaccharide vaccine against haemophilus influenzae type b on the power of chemical synthesis: immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines this review is based largely on presentations made at the carbohydrate moieties as vaccine candidates workshop held at the national institutes of health, 6-7 october 2004. we acknowledge the valuable contributions of the workshop participants and particularly thank drs. kate rittenhouse-olson, liise-anne pirofski, and john schreiber for their critical review of the manuscript prior to submission. this workshop was sponsored by the national vaccine program office and the national institute of allergy and infectious diseases. potential conflicts of interest. all authors: no conflicts. key: cord-351348-lzo0dz7z authors: gu, silan; chen, yanfei; wu, zhengjie; chen, yunbo; gao, hainv; lv, longxian; guo, feifei; zhang, xuewu; luo, rui; huang, chenjie; lu, haifeng; zheng, beiwen; zhang, jiaying; yan, ren; zhang, hua; jiang, huiyong; xu, qiaomai; guo, jing; gong, yiwen; tang, lingling; li, lanjuan title: alterations of the gut microbiota in patients with covid-19 or h1n1 influenza date: 2020-06-04 journal: clin infect dis doi: 10.1093/cid/ciaa709 sha: doc_id: 351348 cord_uid: lzo0dz7z background: coronavirus disease 2019 (covid-19) is an emerging serious global health problem. gastrointestinal symptoms are common in covid-19 patients, and sars-cov-2 rna has been detected in stool specimens. however, the relationship between the gut microbiome and disease remains to be established. methods: we conducted a cross-sectional study of 30 covid-19 patients, 24 influenza a (h1n1) patients, and 30 matched healthy controls (hc) to identify differences in the gut microbiota by 16s ribosomal rna (rrna) gene v3-v4 region sequencing. results: compared with hc, covid-19 patients had significantly reduced bacterial diversity, a significantly higher relative abundance of opportunistic pathogens, such as streptococcus, rothia, veillonella and actinomyces, and a lower relative abundance of beneficial symbionts. five biomarkers showed high accuracy for distinguishing covid-19 patients from hc with an area under the curve (auc) up to 0.89. patients with h1n1 displayed lower diversity and different overall microbial composition compared with covid-19 patients. seven biomarkers were selected to distinguish the two cohorts with an auc of 0.94. conclusion: the gut microbial signature of patients with covid-19 was different from that of h1n1 patients and hc. our study suggests the potential value of the gut microbiota as a diagnostic biomarker and therapeutic target for covid-19, but further validation is needed. coronavirus disease 2019 (covid-19) is an emerging respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and rapidly spread throughout the world [1] . the clinical manifestations and transmission routes of seasonal influenza a (h1n1) are similar to those of covid-19 [2, 3] . who reported that the prevalence of influenza and influenza-like illness were high in china and the united states in the 2019-2020 season [4] . distinguishing covid-19 and h1n1 at the early stage of outbreaks is essential because occupational safety measures, treatment, and prognosis are different between these entities. sars-cov-2 binds to angiotensin-converting enzyme 2 (ace2) receptors to invade human cells, and these receptors are highly expressed in the intestinal epithelium [5] . ace2 might be associated with cardiopulmonary disease via alterations in the gut and/or lung microbiomes [6] . previous studies have found that 3.34-11.4% of covid-19 patients had gastrointestinal symptoms, such as vomiting and diarrhea, especially in critically ill patients [7, 8] . moreover, viral rna and live viruses were detected in fecal samples, suggesting that the digestive tract might be a site of viral replication and activity [9] . however, the interaction between covid-19 and intestinal microorganisms is not fully understood. the intestinal flora is involved in host nutrient absorption and metabolism and has a profound impact on human health and disease [10] [11] [12] . previous studies indicated that the intestinal flora was closely related to respiratory virus infection and could affect the occurrence and development of diseases through the gut-lung axis [13] . moreover, influenza infection can affect the composition of the intestinal microbiota [14] , and intestinal microflora disorders reduce host antiviral immune response, thereby aggravating lung damage caused by these infections [15] . this cross-sectional study analyzed the gut microbiome of covid-19 patients, h1n1 patients, and healthy controls (hc) by high-throughput sequencing of the 16s rrna gene to provide a theoretical basis for differential diagnosis and intestinal microbial intervention. the results showed that biomarkers could be used to identify changes in the structure, composition, and function of the microbiome between these a c c e p t e d m a n u s c r i p t patient groups. the study was approved by the research ethics committee of the first affiliated all subjects who received antibiotics, probiotics, or both within 4 weeks before enrollment were excluded. viral infections were confirmed by real-time reverse-transcription polymerase chain reaction. only laboratory-confirmed cases with clinical symptoms were included in the study. patient data, including laboratory test results, clinical manifestations, and disease course, were obtained from medical records and laboratory information systems. fecal samples from covid-19 and h1n1 patients were collected at admission, a c c e p t e d m a n u s c r i p t and fresh stools from healthy subjects were collected during physical examination. fecal samples were processed in the laboratory within 4 hours after collection and stored at -80°c until analysis. blood samples were taken from all study subjects for analyzing hematological variables, liver functions, kidney functions and serum cytokines, using routine clinical laboratory methods as described in the supplementary methods. given the potential presence of live virus in feces, all fecal samples were inactivated for biomarker identification, a two-step approach was adopted. first, a random forest model [20] was constructed for distinguishing between the two groups, and ten most predominant genera as candidate biomarkers were selected on the basis of a c c e p t e d m a n u s c r i p t importance values (using the r package "randomforest", ntree=500) (supplementary table s1 ). second, the differences in taxonomic composition taxa between two cohort groups were identified using the linear discriminant analysis (lda) effect size (lefse) analysis (http://huttenhower.sph.harvard.edu/galaxy/). candidate biomarkers with a lda>3.5 were selected as final biomarkers. the discriminatory ability of the biomarkers was evaluated by plotting receiver-operating characteristic (roc) curves and calculating the area under the roc curve (auc) using r software's proc package. continuous variables were reported as means ± standard deviations, and statistical comparisons were made using the independent t-test. non-normally distributed variables were expressed as interquartile range (iqr), and comparisons were conducted using the mann-whitney u test. for correlation analysis, spearman's rank test was performed. statistical analysis was performed using spss version 20.0 (spss inc., chicago, il). p values of less than 0.05 after multiple-comparison correction using the false discovery rate method were considered significant. the study population included 30 hospitalized patients with confirmed sars-cov-2 there were no significance differences in age, gender, and body mass index (bmi) between the groups. however, there were significant differences in platelet a c c e p t e d m a n u s c r i p t count, aspartate aminotransferase (ast), il-4, and tnf-α between h1n1 and covid-19 patients (p<0.05). the rate of hypertension did not differ significantly between these two groups (p=0.445). with regard to inflammatory markers, there was a significant difference in procalcitonin (p=0.016) but no significant difference in c-reactive protein (crp) (p=0.832) between h1n1 and covid-19 patients. there were significant differences in lymphocyte count, alanine aminotransferase (alt), il-2, il-4, il-6, il-10 and tnf-α between hc and covid-19 patients. according to clinical guidelines, covid-19 severity on admission was categorized as general in 15 patients and severe in 15 patients. the clinical data of these subjects were summarized and compared (supplementary table s2 ). there was a significant difference in white blood cell (wbc) count, neutrophil count, lymphocyte count, and lactate dehydrogenase (ldh) between these two groups (p<0.05). the characteristics of the gut microbiome in patients with respiratory virus infections were analyzed by 16s rdna gene sequencing of 84 fecal samples (one sample per patient). after merging and filtering, 4,105,869 high-quality sequence reads were generated, with an average of 48,879 sequences per sample for subsequent data analysis. the mean community richness and microbial diversity were significantly lower in covid-19 and h1n1 patients than in hc, according to shannon diversity index and chao diversity index ( figures 1a, b) . the number of otus in the covid-19, h1n1, and hc groups was 911, 960, and 922, respectively. more than 50% of 1242 otus were shared by the three groups, and 62.3% of otus overlapped between the covid-19 group and hc ( figure 1c ). pcoa of bray-curtis distances indicated differences in the fecal microbiota between covid-19 and h1n1 patients and between these groups and hc (anosim, r=0.36, p=0.001) ( figure 1d ). ternary plot showed that the relative abundance of streptococcus and escherichia-shigella was significantly higher in covid-19 and h1n1 patients, respectively ( figure 1e ). the analysis of group similarities indicated that differences in richness, diversity, a c c e p t e d m a n u s c r i p t and structure of the gut microbiota were not significantly different between general and severe covid-19 patients (anosim, p=0.426; supplementary figure s1 ), indicating that the experimental design was adequate. to investigate changes in the microbiota of covid-19 patients, we assessed relative abundance in the three groups at the phylum, class, family, and genus levels ( figure 2 lefse analysis was used to determine and distinguish the composition of the gut microbiome between the covid-19 group and hc. the gut microbiome of the figure s2) . figure s2) . the gut microbiota signature of covid-19 and h1n1 patients was analyzed to assess correlations between disease characteristics and the microbiome. the abundance of prevotella, ezakiella, murdochiella, and porphyromonas was higher in the h1n1 group than in covid-19 patients ( figure 4a ). in addition, seven final biomarkers (streptococcus, fusicatenibacter, collinsella, dorea, agathobacter, eubacterium hallii group, ruminococcus torques group) were selected reference the two-step schema in the method to distinguish the two cohorts, with an auc of 0.94 (95% ci, 0.87-1.00) ( figure 4b ). spearman analysis was conducted to evaluate the correlation between genera (abundance >0.1%) and clinical indexes, including wbc, crp, pct, d-dimer, il-2, il-4, il-6, and tnf-α in covid-19 and h1n1 patients, respectively. the significance thresholds were absolute correlation coefficients higher than 0.4 and p values lower than 0.05, as shown in figure 5 . intestinibacter, and prevotella) ( figure 5b ). increasing evidence indicates the intimate relationship between the gastrointestinal and respiratory tract, which is known as the gut-lung axis [13] . deriu stimulating the production of ifn-γ by these cells [14] . studies have identified sars-cov-2 rna in stool specimens of infected patients [9] , and rna analysis demonstrated that the duration of viral shedding from stool was longer than that from respiratory samples [32] . in addition, host ace2 receptors are highly expressed in the gastrointestinal epithelium [7] . sars-cov-2 may interfere with nutrient absorption by binding to ace2 receptors, causing gastroenteritis-like symptoms, and disrupting intestinal homeostasis. we declare no competing interests. m a n u s c r i p t a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical features of patients infected with 2019 novel coronavirus in wuhan, china estimates of global seasonal influenza-associated respiratory mortality: a modelling study a pneumonia outbreak associated with a new coronavirus of probable bat origin emerging targets for cardiopulmonary disease therapy epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms multi-omics evaluation of gastrointestinal and other clinical characteristics of sars-cov-2 and covid-19 covid-19: gastrointestinal manifestations and potential fecal-oral transmission structure, function and diversity of the healthy human microbiome the gut flora as a forgotten organ homeostatic immunity and the microbiota the gut-lung axis in respiratory disease respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated th17 cell-dependent inflammation microbiota regulates immune defense against respiratory tract influenza a virus infection abbreviations: na, not available; iqr, interquartile range; bmi, body mass index; il, interleukin; tnf-α, tumor necrosis factor-α. a p values indicate differences between h1n1 and covid-19 05 was considered to indicate significant difference between healthy controls and h1n1 and covid-19, respectively. lactate dehydrogenase, u/l na a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-354009-1ek4s8oe authors: wang, yun; liu, ying; struthers, james; lian, min title: spatiotemporal characteristics of covid-19 epidemic in the united states date: 2020-07-08 journal: clin infect dis doi: 10.1093/cid/ciaa934 sha: doc_id: 354009 cord_uid: 1ek4s8oe background: a range of near-real-time online/mobile mapping dashboards and applications have been used to track the covid-19 pandemic worldwide. it remains unknown about small area-based spatiotemporal patterns of covid-19 in the united states. methods: we obtained county-based counts of covid-19 cases confirmed in the united states from january 22 to may 13, 2020 (n=1,386,050). we characterized the dynamics of covid-19 epidemic through detecting weekly hotspots of newly confirmed cases using spatial and space-time scan statistics and quantifying the trends of incidence of covid-19 by county characteristics using the joinpoint analysis. results: along with the national plateau reached in early april, covid-19 incidence significantly decreased in the northeast (estimated weekly percentage changes [ewpc]: -16.6%), but remained increasing in the midwest, south and west regions (ewpcs: 13.2%, 5.6%, and 5.7%, respectively). higher risks of clustering and incidence of covid-19 were consistently observed in metropolitan vs rural counties, counties closest to core airports, most populous counties, and counties with highest proportion of racial/ethnic minority counties. however, geographic differences in the incidence have shrunk since early april, driven by a significant decrease in the incidence in these counties (ewpc range: -2.0% – -4.2%) and a consistent increase in other areas (ewpc range: 1.5% – 20.3%). conclusions: to substantially decrease the nationwide incidence of covid-19, strict social distancing measures should be continuously implemented, especially in geographic areas with increasing risks, including rural areas. spatiotemporal characteristics and trends of covid-19 should be considered in decision-making on the timeline of re-opening for states and localities. since the first cluster of the coronavirus disease 2019 (covid-19) was reported, 1,2 the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has triggered massive outbreaks and then evolved to a worldwide pandemic of covid-19. as of may 13, 2020, 4,347,018 confirmed cases and 297,197 covid-19 related deaths have been reported worldwide. 3 in the united states, the first covid-19 case was reported on january 21, 2020, 4 and national outbreak of covid-19 beginning in early march of 2020 had caused 1,386,050 confirmed cases and 83,167 deaths from covid-19 as of may 13. 5 it is urgent to "flatten the epidemic curve" for covid-19 in the united states. remarkable efforts have been made to map the coronavirus spread using near-real-time interactive online/mobile gis dashboards, websites, and applications in and out the united states. 3, [5] [6] [7] these maps provide timely information on descriptive statistics of the outbreak situation. however, no studies have comprehensively assessed small area-based characteristics of covid-19 spreading in the united states. using government record-based surveillance data, we examined the spatiotemporal variations in covid-19 as well as its associated geographic characteristics across the country. the results would enhance our understanding of small area-based spatiotemporal dynamics of covid-19 outbreak, thus help inform multilevel strategies to control the spread of coronavirus and appropriate allocations of public health and healthcare resources in the united states. a c c e p t e d m a n u s c r i p t 5 we obtained the counts of covid-19 cases diagnosed from january 22 to may 13, 2020 in the united states from the usafacts, a not-for-profit initiative standardizing and providing the government record-based data publicly available. 5 the daily-updated numbers were cumulated to form a time-series database of confirmed covid-19 cases across all the us counties. the study is exempted from the ethics review due to the use of publicly accessible data source. to identify the characteristics of counties with a high burden of covid-19, we examined county-level geographic and sociodemographic factors, including rural-urban context, distance to the nearest core airport, population density, percentage of non-white minority population, percentage of population 65 years or older, and percentage of population below the federal poverty line. using the rural-urban continuum codes of u.s. department of agriculture, 8 rural-urban context was defined as metropolitan (codes 1-3), urban (codes 4-7), and rural areas (codes [8] [9] . there are 30 core airports with the highest volume of traffic across the country. 9 the euclidean distance from the populationweighted centroid of a given county to its nearest core airport was calculated to measure spatial relationship of that county with core airports. population density was computed as the number of population per square miles of lands. county-level information on land areas, population sizes, and other three socioeconomic variables were retrieved from combined 2014-2018 american community surveys to reduce potential marginal error of survey. a c c e p t e d m a n u s c r i p t 6 statistical analysis: we first created an epidemic curve to visualize the progression of newly confirmed covid-19 cases by four us government-defined regions (northeast, midwest, south, and west) over 11 distinct time periods from january 22 through may 13, 2020, including the first six epi-weeks in combination (january 22 nd -march 4 th ) and individual epi-weeks from march 5 th to may 13 th . using a spatial and space-time scan statistics (satscan), 10, 11 we examined spatiotemporal clustering of confirmed covid-19 cases through detecting the higher-than-expected geographic hotspots across the country. the satscan applies a pre-defined circular window with varied sizes and time periods to scan the study area and identify the most likely clusters of the event of interest using a space-time permutation statistical model, and uses a monte carlo simulation approach to generate 999 random datasets in computing the statistic for the statistical inference of a cluster. in this study, we defined the parameters of the scanning window as 150 miles of maximum geographic radius and the day as the minimum temporal scanning unit. geographic clustering was detected in each of 11 time periods to characterize the dynamics of geographic clustering of newly confirmed covid-19 cases. the most likely high-risk clusters/hotspots were captured based on the monte carlo rank with p<0.05. we further examined the associations of county characteristics with covid-19 clustering using logistic regressions. the outcome was whether a given county was identified as part of a hotspot or not. the analysis was performed separately for each of the 7 th -16 th epi-weeks. considering the colinearity between county characteristics, county-level variables were not mutually adjusted for. statistical significance was tested as two-sided with p<0.05. as of may 13, 2020, a total of 1,386,050 covid-19 cases were confirmed in the united states over 16 epi-weeks. figure 1a shows the overall temporal trend of weekly counts of newly confirmed covid-19 cases by four us regions. covid-19 had occurred sporadically until early march (first six epi-weeks); 103 confirmed cases were reported mainly in the west region. the number of weekly confirmed covid-19 cases subexponentially increased across the country from the 7 th to 11 th epi-week, followed by a slowly decrease over recent five weeks. during the entire observation period, the largest proportion of cases was from the northeast (48.6%), followed by the in the first six epi-weeks, covid-19 cases were reported in 27 counties from the west coast and northeast states with the highest county-level incidence of 3.4/100,000 persons. starting in the 7 th through the 16 th epi-weeks, sars-cov-2 spread to broad geographic areas. as of may 13, 92.0% of us counties had the confirmed covid-19 cases, and the median county-level cumulative incidence rate was 88.0/100,000 persons (interquartile range: 36.1-219.3/100,000 persons) with the highest reaching 14,426/100,000 persons (supplemental figure 1) . the incidence of covid-19 reached the peak in the northeast in the 12 th epi-week (214.2/100,000 persons), followed by a significant reduction of 16.6% weekly until the 16 th epi-week. however, the incidence consistently increased in the midwest, south and west regions from the 10 th to 16 th epi-weeks with a significant ewpc of 13.2%, 5.6% and 5.7%, respectively. overall, covid-19 incidence reached the national plateau in the epi-week 11 (66.6/10,000 persons), followed by a slight and insignificant decrease in recent five weeks ( figure 1b and table 2 ). figure 3 illustrates the trends in the incidence of covid-19 by county characteristics. over 16 epi-weeks, the incidence was significantly higher in metropolitan vs urban/rural areas, areas closest vs farthest to core airports, most vs least populous areas, and areas with highest vs lowest percentage of minorities, and lowest vs highest percentage of population aged 65 years and older ( figure 3a-e) . the incidence dramatically increased from the 7 th epi-week and reached the peak in the 11 th epi-week in metropolitan areas (75.7/100,000 persons), counties closest to core airports (91.7/100,000 persons), most populous counties (79.6/100,000 persons), and counties with highest percentage of minorities (100.9/100,000 persons), followed by a significant decrease thereafter (ewpc= 2.0%, 2.8%, 2.6%, a c c e p t e d m a n u s c r i p t 10 and 4.2%, respectively) ( table 2) . notably, the incidence consistently increased from epi-week 7-16 in rural (0.04 to 35.0/100,000 persons) and urban areas (0.1 to 37.5/100,000 persons) ( figure 3a ). unlike metropolitan areas, the incidence remained increasing in rural and urban areas after the 11 th epi-week with a significant ewpc of 17.8% and 18.1%, respectively (table 2) . similarly, a consistent increase in the incidence of covid-19 from epi-week 7-16 was also observed in counties farther from core airports and in less populous and lower minority counties ( figure 3b -d and table 2 ). the incidence in counties with lowest or highest percentage of elderly people increased from epi-week 7-11 and remained steady thereafter. overall, geographic disparities in the incidence of covid-19 by county characteristics had decreased since the 11 th epi-week. there was no significant difference in the incidence of covid-19 for highest vs lowest percentage of population below the federal poverty line ( figure 3f ). using a national time-series database of confirmed covid-19 cases, we examined the spatiotemporal patterns of covid-19 in the united states during the starting 16 epi-weeks. covid-19 cases sporadically occurred in the west coast and northeast states in the first six epi-weeks and increased rapidly across the country thereafter until the 11 th epi-week, and then slightly decreased since the 12 th epi-week. despite a remarkable reduction in newly confirmed cases from the northeast in recent four weeks, the risk of covid-19 infection remained consistently increasing in the midwest, south and west regions. geographic clustering of covid-19 was first identified in southern and northern california, and then rapidly expanded nationwide. higher risks of covid-19 clustering and incidence were observed in metropolitan vs rural counties, counties closest to core airports, most populous counties, and counties with highest proportion of racial/ethnic minority. however, the differences have shrunk since the 11 th epi-week, which was driven by a significant decrease in the incidence in these counties and a consistent increase in other areas in recent five weeks. it might be a result of social distancing measures well implemented recently in high-risk areas in early stage of the a c c e p t e d m a n u s c r i p t 11 outbreak, and also suggests that recent region-to-region spreading and community transmission occurred in other areas. further studies are needed to assess the effectiveness of public health and behavioral interventions on covid-19 infection and implemental barriers, which is essential for promoting the strict adherence to social distancing guidelines and enhancing personal protections (including appropriately wearing face masks as needed and timely washing hands) to prevent the sars-cov-2 spreading and thus substantially decrease the incidence of covid-19 locally and nationwide. a significant association between short distance to core airports and covid-19 clustering suggests a critical role of air transportation in sars-cov-2 spreading across the country. air transportation was believed to accelerate and amplify the spread of influenza, sars-cov or mers-cov. 13 a recent study showed that the rail transport is related to the transmission and regional outbreak of covid-19 in china. 14 in the united states, the airports may contribute substantially to the early travel-related region-to-region transmission. from march 18 to april 22, at least 42 states, 3 counties, 10 cities, the district of columbia and puerto rico joined illinois, new york, and california in the lockdown orders. 15 however, airlines, as one of essential transportation services, are generally exempted from the orders and still operating. we flagged the importance of airports in spreading covid-19 even after the lockdown of most regions in mid-march. airlines have put in place stringent safeguards for those still flying, including supercharged cleaning, reduced in-flight services, and the spacing out of passengers on flights. it is crucial to maintain strict management and monitoring of major airports to maximize the reduction of region-to-region transmission. while covid-19 incidence in metropolitan areas has decreased since the 11 th epi-week, we identified a consistent increase in the incidence of covid-19 in rural areas over the 16 epi-weeks. this was probably a sign of that the local spread of covid-19 extended beyond metro/urban enclaves and the secondary community transmissions took place around geographic hotspots and spread to rural areas. rural areas with a lower population density are not safe for this pandemic because rural residents tend to be older and have limited access to health care. 16, 17 therefore, the a c c e p t e d m a n u s c r i p t 12 restrictive social-distancing measures are necessary in rural areas, and adherence to social distancing should be enhanced for rural residents. the pandemic of covid-19 poses different challenges for the us states currently designing its coping strategies. the population density is a key driver for transmission of infectious disease. we observed that predominately minority counties were at higher risk of covid-19. this was consistent with the reports of african americans accounting for about 70% of covid-19 related deaths but just about 30% of the population in chicago, milwaukee county and louisiana. 18 the disproportionate burden of covid-19 in minority populations may largely result from inequities in adherence to social distancing measures. our analysis indicates that the incidence of covid-19 was lower in areas with higher percentage of elderly people. it could partly result from the lower mobility of older vs younger people. however, we found a comparable risk of covid-19 clustering in counties with highest vs lowest percentage of elderly people in recent weeks. it might be relevant to the outbreaks of covid-19 in nursing homes in some geographic areas. 19, 20 therefore, senior-heavy areas should not be ignored in the allocation of prevention efforts on covid-19 because senior people typically have multiple chronic health conditions and higher risk of developing more serious complications from covid-19. 21 lack of a stable pattern in the association between poverty and the risk of covid-19 indicates that socioeconomic factors might not play a critical role in the risk of coronavirus infection. the study has some limitations. the confirmed cases of covid-19 might not reflect the actual number of persons infected with sars-cov-2 due to unknown/untested asymptomatic cases. 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regression with applications to cancer rates the roles of transportation and transportation hubs in the propagation of influenza and coronaviruses: a systematic review the association between domestic train transportation and novel coronavirus (2019-ncov) outbreak in china from 2019 to 2020: a datadriven correlational report. travel medicine and infectious disease the new york times. see which states and cities have told residents to stay at home access and issues of equity in remote/rural areas. the journal of rural health : official journal of the american rural health association and the national rural health care association rural-urban differences in usual source of care and ambulatory service use: analyses of national data using urban influence codes covid-19 and african americans nursing home care in crisis in the wake of covid-19 long-term care policy after covid-19 -solving the nursing home crisis. the new england journal of medicine clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study transmission of 2019-ncov infection from an asymptomatic contact in germany. the new england journal of medicine asymptomatic cases in a family cluster with sars-cov-2 infection. the lancet infectious diseases centers for disease control and prevention. social distancing, quarantine, and isolation: keep your distance to slow the spread how will countrybased mitigation measures influence the course of the covid-19 epidemic? hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states can china's covid-19 strategy work elsewhere? governmental public health powers during the covid-19 pandemic: stay-at-home orders, business closures, and travel restrictions. jama. 2020. 30. pan a public health interventions for covid-19: emerging evidence and implications for an evolving public health crisis reopening society and the need for real-time assessment of covid-19 at the community level population density (quartile) we thank the gis and spatial statistics supporting from the health behavior, and communication & outreach core, which is affiliated with washington university institute of clinical translational sciences funded by the national center for advancing translational sciences, national institutes of health (ul1 tr002345) and washington university alvin j. siteman cancer center funded by the national cancer institute, national institutes of health (p30 ca091842). a c c e p t e d m a n u s c r i p t 15 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t 20 key: cord-351231-aoz5jbf1 authors: bartlett, john g. title: why infectious diseases date: 2014-09-15 journal: clin infect dis doi: 10.1093/cid/ciu441 sha: doc_id: 351231 cord_uid: aoz5jbf1 infectious diseases is a broad discipline that is almost unique in contemporary medicine with its ability to cure and prevent disease, to identify specific disease causes (microbes), and to deal with diverse, sometimes massive outbreaks. the value of the infectious disease practitioner is now magnified by the crisis of antibiotic resistance, the expanding consequences of international travel, the introduction of completely new pathogen diagnostics, and healthcare reform with emphasis on infection prevention and cost in dollars and lives. infectious disease careers have great personal rewards to the practitioner based on these observations. it is unfortunate that we have been so effective in our work, but relatively ineffective in convincing the healthcare system of this value. students of medicine have multiple career options with various attractions and concerns. so it is with the discipline of infectious diseases. as with all medical specialties, infectious diseases has unique features that are important to highlight: among medical specialties, this one is consistently changing, often unpredictable, usually exciting, and incredibly rewarding for health impact. it is also often challenging and seemingly underappreciated, at least until needed. these facts appear to be relatively idiosyncratic to this discipline with a menu of priority pathogens that is in constant flux and weaponry that will change in unpredictable ways. the extraordinary kinetics and ability to intervene successfully using public health, preventive vaccines, and disease-limiting antimicrobials are its great strengths. the following are some of the highlights and unique features of a career in the science and practice of infectious diseases. the menu includes the litany of epidemics, heroic efforts to conquer disease, our expectations with antimicrobials, vaccines and public health, and challenges that may lead to transformative interventions. the field of infectious diseases is kinetic, unpredictable, and layered with surprises that sometime require heroic efforts from a diverse field of scientists and practitioners. • on 2 october 2001, a patient with fever and confusion was seen at a florida medical center by dr larry bush, an infectious disease physician. he examined the cerebrospinal fluid, saw boxcar gram-positive rods, diagnosed anthrax, and predicted bioterrorism [1] . this was strong stuff at a time no one had thought much of bioterrorism anywhere in decades and especially in an obscure, small town in florida. the ensuing epidemiologic investigation showed anthrax spores in this patient's workplace, the local postal service, and a letter received by the patient. this was the index case of the anthrax bioterrorism epidemic that shook the country in 2001.the result was a major national preparedness response to not only bioterrorism, but also preparedness for natural disasters, epidemics, and other major public health threats. • in 1977, dr alan steere, a rheumatologist from yale school of medicine, led an investigation of an outbreak of arthritis involving 39 children and 12 adults in connecticut. most of the patients had asymmetric swelling and pain of large joints, especially knees, and some also had an erythematous, annular rash [2] . it was initially called "lyme arthritis," but most physicians thought it was simply juvenile rheumatoid arthritis. dr steere was convinced it was an infection and moreover, that it was arthopod-born based on epidemiology and clinical features. his relentless pursuit of the pathogen was finally rewarded with the discovery of the newly recognized spirochete in the blood and in typical skin lesions [3] . the specific agent was subsequently defined in a another extraordinary effort, this time by dr willie bergdorfer, who had spent much of his career studying the microbiology of the hindgut of ticks; he successfully isolated the pathogen that he considered his last and most important scientific project [4, 5] . that agent is named in his honor: borrelia burgdorferi [6] . • dr robin warren, a pathologist in australia, made the historic discovery that gastric biopsies from patients with gastritis showed a large burden of curved bacteria. no one paid attention until a young gastroenterologist, dr barry marshall, agreed to study the association. this pairing was considered an "odd couple"; dr warren was described as quiet, thoughtful, and persistent whereas dr marshall was self-described as brash and determined [7] . subsequent studies consistently showed the association between this curved microbe with gastritis and peptic ulcer disease, but there was almost uniform opposition from both gastroenterologists and infectious disease physicians. larry altman, noted medical editor for the new york times, wrote that never in his experience had he witnessed such fierce opposition from the medical community to the possibility that peptic ulcer disease was caused by a microbe (l. altman, personal communication, 16 may 2012) [8] . this prompted 2 proponents, drs barry marshall and alan morris, to perform the ultimate experiment-they swallowed a flask of helicobacter pylori (and suffered from the experience; l. altman, personal communication, 16 may 2012) [9] . the long-term result of this unrelenting battle is now well known: h. pylori is accepted as the cause of peptic ulcer disease and its sequela, guidelines for diagnostic testing and treatment are based on h. pylori as the pathogen, this agent is listed as a class 1 carcinogen, and nobel prizes were awarded to drs warren and marshall [10] . • in early september 2012, dr april pettit, an infectious disease physician in tennessee, saw a patient with aspergillus meningitis following an epidural steroid injection [11] . this prompted her to notify dr marion kainer at the tennessee health department, who then set up shop with a sleeping cot in the health department to facilitate a nonstop investigation [12] . this was the beginning of the infamous national epidemic of exserohilum rostratum meningitis associated with the contaminated steroids that led to 741 cases and 55 deaths in 18 states. credit here is to dr pettit for recognition and prompt notification, to dr kainer for her aggressive response on behalf of the victims, and to the centers for disease control and prevention (cdc) for the hasty intervention. (somewhat disappointing is the fact that compounding pharmacies are still unregulated.) • in 1981, dr anthony fauci read the 3 july edition of morbidity and mortality weekly report [13] describing gay men with kaposi sarcoma or pneumocystis carinii pneumonia in california. for the first time in his life, dr fauci had what he called "chill pimples" ("chill bumps"), and this led to a career change to find the cause, treatment, and prevention of aids. this must be now viewed as possibly the most remarkably successful attack on an important infectious disease since fleming discovered penicillin. • in february 2003, severe acute respiratory syndrome (sars) was a newly described, severe disease in humans that was often fatal and appeared to travel by air routes, but had no established pathogen or treatment. dr klaus stohr at the world health organization (who) identified the finest virology laboratories in the world and asked them to collaborate to define the pathogen with the condition that all information would be shared on the internet and there was no ownership of the data. the 11 participating labs with varying skills were spread throughout the world, so the daily conference calls started with "good morning, good afternoon, and good evening." the etiologic agent was described in an unauthored "global alert" on 16 april 2003 and in the lancet with "multicentre collaborative network" as the authors (see [14] ). this unselfish collaboration under strong leadership is credited with the rapid solving of a global crisis that eventually showed 8000 cases with 774 deaths in 25 countries. • these anecdotal experiences (bioterrorism, lyme disease, peptic ulcer disease, iatrogenic fungal meningitis, human immunodeficiency virus [hiv]/aids, and sars) illustrate the unpredictable challenges and some of the unique responses that have left a major imprint on medicine. it is noteworthy that all started with strong leadership and came to closure with either elimination or successful management. epidemics of infections are predictable to occur, but largely unpredictable in time, place, microbe, and consequences. the following highlights some of the recent epidemic records and surprises in this category: • west nile virus was first reported in new york city in 1999 and reached a 10-year zenith for reported cases in 2012 with 5674 cases, including 51% with the dreaded neuroinvasive form of the disease [15] . • coccidioidomycosis: the total number of reported cases increased 10-fold in 13 years, from 2265 in 1998 to 22 401 in 2011 [16] . • malaria reported in us travelers reached a record high of 1925 cases in 2011 [17] . • chikungunya virus reached a record number of cases in the caribbean with >1000 reported cases, including 10 in us travelers to st martin [18] . this pathogen is highlighted because global warming is expected to make it endemic in the southern united states and because of its substantial morbidity with possible long-standing arthritic complications [19] . • measles: the largest number of annual reported cases in the united states in 17 years was noted during 2011-2013. it now appears that 2014 will be worse [20] . measles was declared eradicated in 2000 but has now become a problem, primarily in those refusing vaccination, but also in some with documented vaccination [21] . measles continues to be an important infectious disease challenge due, in part, to the extraordinary public health issues that cost one health system $800 000 to deal with the potential epidemiologic consequences of a single case [22] . • pertussis: this infection is resurgent in the united states and europe, with increased cases including epidemics in children and adults and involving both vaccinated and unvaccinated individuals [23] . this is thought to reflect waning immunity to the acellular vaccine and the need for a new vaccine [24] . • meningitis: 2013-2014 reporting showed 4 outbreaks of neisseria meningitidis meningitis, 2 on college campuses and 2 among gay men in new york city and los angeles [25] . • influenza: this is a continual concern based on the everpresent threat of pandemics with devastating consequences (1918-1919, 1957-1958, 1968-1969, 1977-1978, 2009-2010) that seem difficult to predict or control [26, 27] . limitations of current expertise were illustrated with influenza a(h1n1) swine flu, as the standard concept based on historic precedent is that new influenza epidemics come from asia in the wintertime, but this one came in the eastern hemisphere in the summertime [27] . the more recent threats that could pose serious consequences are influenza a(h7n9) and influenza a(h5n1) [28] [29] [30] . both show high mortality rates, but little evidence so far of that single critical mutation permitting attachment to the hemagglutinin antigen to permit sustained person-to-person transmission [26] . • middle east respiratory syndrome (mers) coronavirus: this coronavirus is a major global concern with analogies to the sars coronavirus in terms of its perceived potential to become a global epidemic with high mortality and no apparent treatment [31, 32] . of immediate importance in the united states is recognition of risk with appropriate diagnostic testing, isolation, and management of persons with severe, unexplained pneumonia associated with recent travel to the arabian peninsula (mers) [32]. • foodborne disease: widespread foodborne epidemics are now a common consequence of the massive food distribution system that permits contaminated beef or lettuce from mexico to reach stomachs in distant multistate areas, with medical consequences involving hundreds or thousands of people. this includes the more recent emergence of the gii.4 sydney strain of norovirus. these outbreaks seem likely to continue, with unpredictable pathogens in unpredictable places [33] [34] [35] . • heartland virus: a recently encountered tick-borne disease in tennessee and missouri with 10 cases and 2 deaths [36] . • polio-like virus infection with extremity paralysis has been recently reported in 5 and possibly 25 children in california [37] . • ebola virus: who has reported an outbreak in guinea involving a new clade of this usually fatal infection [38] . this listing could continue almost indefinitely. the point is that epidemics are the domain of infectious diseases and public health, with the expectation for management or prevention of outbreaks with requirements for detection, reporting, isolation, and case management. the listing here includes diverse pathogens, some life-threatening diseases, infections with important public health implications, an upsurge of pediatric infections in adults, many travel-related infections, multiple public health threats, and the continuous concerns for influenza and foodborne disease. the major weaponry of the infectious disease catalog includes antibiotics, vaccines, and public health. these categories are remedial reading, but some facets are worthy of emphasis. the value of antibiotics seems obvious. the first patient to receive penicillin was a young woman with β-hemolytic streptococcal bacteremia with fever of 39.4°c-41.2°c daily for 4 weeks. she received penicillin intravenously starting 14 march 1942, promptly recovered, and survived to age 90 years [39] . this would appear to be "evidence-based medicine" with an n = 1. the following statement from dr walsh mcdermott in 1982 summarizes this breakthrough especially well: "penicillin gave more curative power to a barefoot, itinerant care provider in the deepest reaches of africa than the collective powers of all physicians in new york city" [40] . the more recent experience with bacterial resistance and sparse pipeline threatens this miracle, but antiviral development is quite different, primarily for hiv and hepatitis c virus (hcv). it now appears that patients with hiv can achieve near-normal longevity [41] . hcv infection is even more impressive in terms of speed of progress and ability to cure. the hcv treatment story reflects the efficiency of basic science to define targets, pharmaceutical skills of industry, well-organized trial networks, and a regulatory agency (us food and drug administration [fda]) that facilitated product development [42] . the impact of vaccines is also impressive. a comparison of annual incidence of vaccine-preventable diseases in the united states reported for the period prior to availability of the designated vaccine compared with its incidence in 2013 shows the decrease in polio as 100%; diphtheria, 100%; rubella, 99.9%; mumps, 99.6%; invasive type b haemophilus influenzae, 99.2%; and pertussis, a disappointing 88%. a recent report concluded that the global total for lives saved by vaccines exceeds 100 million [43] . the impact could be substantially greater with more global access, fewer refusals, and a better pertussis vaccine. a recent cdc analysis of annual costs associated with 5 major nosocomial infections totaled $9.2 billion per year in the united states with the following rank order by median cost/case: central line bacteremia, $45 814; ventilator-associated pneumonia, $40 144; surgical site infection, $20 785; clostridium difficile infection, $11 285; and catheter-associated urinary tract infection, $896. this illustrates the challenge and the priorities [44] . another challenge is epidemics involving nosocomial pathogens, as shown with the klebsiella pneumoniae carbapenemaseproducing bacteria (kpc) in the national institutes of health (nih) clinical center. this began with a patient transferred from a new york city hospital with a kpc infection and became the source of an institutional outbreak that required extraordinary efforts to control, including a wall constructed to isolate cases, removal of plumbing (as a possible source), use of matrix-assisted laser desorption/ionization time-of-flight (maldi-tof) molecular diagnostics to detect cases and carriers, hydrogen peroxide room aerosols, and "whole house" surveillance cultures. the epidemic was finally halted, but the toll was 18 cases and 6 fatalities over 6 months [45, 46] . another kpc epidemiologic investigation showed widespread distribution of this microbe from a long-term acute-care facility in the chicago area [47] , and others have demonstrated distribution of kpc by air travel from india to europe [48] . these epidemics require extensive resources and specialized skills; they will be expected to increase substantially in the era of "bad bugs." there is no specialty field in medicine that demonstrates shifting priorities like infectious diseases. to illustrate this point, i have summarized the "hot topics" discussed in the "what's hot in infectious diseases" presentation to the annual meeting of the american college of physicians in 2004, compared with the presentation in 2014, to illustrate the nearly complete change of priorities in a relatively short time. avian influenza, rabies (first survival without vaccine), west nile virus, bioterrorism, transfusion-associated jacob-creutzfeldt disease, usa300 strain of methicillin-resistant staphylococcus aureus (mrsa), sars, and chlamydia pneumoniae and its role in coronary artery disease and influenza. carbapenemase-producing gram-negative bacilli, colistin, constant infusion of β-lactam antibiotics, molecular diagnostics, a litany of epidemics, new pathogens (mimivirus, borrelia miyamotoi, emmonsia species, and bradyrhizobium enterica), c. difficile gene sequencing, the microbiome, and hcv. note that the 10-year interval resulted in a completely new agenda for what was considered timely and important in the field based on rapid changes in topical microbes, new epidemics, and new diagnostics, (but not new antimicrobials). it is impossible to predict the menu for 2024. it is now known that genes for resistance to antimicrobial agents were well established in bacteria at least 3 million years before evidence of human life [49] . the use of antibiotics has selected for these genes by mendelian laws, making it increasingly difficult to control previously treatable infections. this problem was predicted by the father of antibiotics, alexander fleming, who, in 1945, wrote that ". . . the public will demand the drug and . . . then will begin an era . . . of abuses. the microbes are educated to resist penicillin and a host of penicillin-fast organisms is bred out which can be passed to another individual and perhaps from there to others until they reach someone who gets a septicemia or a pneumonia which penicillin cannot save. in such a case, the thoughtless person playing with penicillin treatment is morally responsible for the death of the man who finally succumbs to an infection with penicillin-resistant organisms. i hope this evil can be averted" [50] . also of note is the prediction by nobel laureate joshua lederberg: "the future of humanity and microbes will likely evolve as . . . episodes of our wits vs their genes" [51] . it now appears that fleming's prediction is a harsh reality and evolutionary microbial resistance genes are gaining the upper hand, reflecting the combination of massive antibiotic use and lack of new pharmacologic agents. the result is the alarming escalation of antibiotic resistance that is global and applies to nearly all categories of treatable pathogens, leading some to predict "the postantibiotic era." this resistance has been declared a "crisis" by the infectious diseases society of america, the cdc, who, the us congress, and the us president. a disturbing observation in the united states is the conspicuous absence of a national plan to deal with resistance, including the lack of a living record of antibiotic consumption and resistance correlated by location and trajectory. this is in sharp contrast to the european union, which includes 28 countries with 24 official languages and diverse cultures, but has systematically collected data on antibiotic consumption and microbial resistance patterns for 15 years [52, 53] . this has resulted in multiple publications with data reviews, studies of interventions, messages to consumers such as an ebug internet program for students, a european antibiotic awareness day, standardized methods to collect data [54] and a recent 12-point plan with budget to address the issue [55] . their data are striking in showing the dramatic association between per capita antibiotic use and national resistance patterns. for example, antibiotic consumption in greece is nearly 4 times that of the netherlands, so we expect more resistance problems in greece, but the magnitude of this difference is alarming: bacteremic carbapenemase strains among all bacteremic k. pneumoniae isolates appear to be about 150 times more common in greece, and mrsa as a percentage of all s aureus isolates is about 40 times higher [51] . the european union appears to have a mature and substantive model to learn from, with the important caveat that it functions well because there is no claimed ownership, as there are 28 equal partners. there are also some good national programs that have successfully addressed specific problems to learn from: • eu data for 2002 showed that france had embarrassingly high antibiotic use rates, accompanied by increasing resistance by s. pneumoniae. this prompted a national campaign targeting prescribers and consumers on antibiotic abuse and its consequences. the goal was a 25% reduction in antibiotic prescriptions for the entire country; they achieved a 26% reduced resistance [56] and also achieved the largest decrease in per capita antibiotic consumption for any nation in the history of the global antibiotic fund [57] . • a recent report from israel showed a national campaign to reduce the incidence of kpc. analysis of their results with a prevention bundle showed a reduction from 55 per 100 000 patient-days to 4.6 per 100 000 patient-days [58] . • the united kingdom addressed the issue of the epidemic nap-1 strain of c. difficile through gene sequencing and aggressive antibiotic control. the result was a national 61% reduction in c. difficile infection rates [59] . the 3 examples given are based on national data addressing major challenges with impressive results. in the united states, this remains a unanswered challenge, but is also an opportunity for the skills of the infectious disease discipline in terms of data collection, evaluation, interventional trials, and policy implementation into practice, primarily in the form of antibiotic stewardship. recent reports using gene sequencing suggest that conventional methods of infection control could substantially improve this effort. examples: (1) results from the united kingdom have largely disproven conventional teaching regarding the epidemiology of c. difficile infection [59] ; (2) this technology also appears to contradict some contemporary concepts about transmission patterns of s. aureus [60] ; and (3) it has proven to be a valuable tool in outbreak investigation of kpc infections in a hospital [45] and in a large community outbreak of kpc involving multiple facilities [47] . it seems clear that as this technology gets faster and cheaper, it will be embraced as an infection control standard [61] , although there needs to be caution and skill in interpreting results [62] . this work in developing countries is another attractive career option based on need, probability of impact, and unique special programs such as the president's emergency plan for aids relief, the bill & melinda gates foundation, and others. some of this is direct patient care, but possibly very attractive targets for impact are the development and implementation of innovative programs that deal with the vast need combined with minimal resources [63] . the new healthcare system should value infectious disease expertise based on its important role in addressing resistance and costs associated with nosocomial infections. nevertheless, it is feared that the current structure and payment system are not constructed as a good fit to prioritize infectious disease skills. specifically, there is no code for preventing infections, conserving antibiotic use, or preventing resistant pathogens. this might be an erroneous conclusion, or the situation may change as the system matures and becomes serious about addressing the crisis. "bundles" to deal with healthcare efficiencies are in vogue and could be a strength of infectious diseases. an example is the 5-step central line bacteremia prevention bundle that proved effective in trials [64] . generalized adoption of this bundle was predicted to save 18 000 lives and $1.8 billion per year in us hospitals [65] , and subsequent actions by clinicians, regulatory agencies, and stakeholders have resulted in an estimated 63% decline in central line bacteremia rates [66] . • stewardship: solving or reducing the problem of antibiotic resistance largely depends on antibiotic development and reducing antibiotic abuse. the major on-site forces for improving smart antibiotic use at the point of care are antibiotic stewards-preferably infectious disease or pharmacy personnel trained in this skill to improve the speed of detecting resistant or epidemic pathogens. the tools are obvious to infectious diseases-trained clinicians, but often require methods that are not well inculcated into hospital or clinic practice. methodology with proven value for antibiotic conservation include shortcourse regimens (virtually always wins or ties in trials), use of procalcitonin to facilitate decisions on when to start or stop antibiotics, use of molecular diagnostics to improve pathogentargeted antibiotic decisions, outpatient infusion therapy to reduce inpatient risk (and cost), optimal use of the agents we have, waiting room with notices that the doctor will prescribe antibiotics only according to guidelines, acknowledgement of possible microbiome harm, and possible use of social network media [52, 53] . nevertheless, there must be caution: the new us healthcare system represents socialized medicine largely managed by capitalists, which invites both quality and chicanery. for example, the centers for medicare and medicaid services' "6-hour rule" for treating community-acquired pneumonia had improved outcome advantages, but also led to overprescribing, declined use of diagnostics, perceived antibiotic abuse, and increases in c. difficile infections. given the priority of cost containment and its relevance to infectious diseases, infectious disease training should probably include attention to the business of medicine. • molecular microbial diagnostics: these are rapidly being developed and introduced into clinical use for detection of epidemic pathogens or resistance genes with advantages of speed, precision, and sensitivity. most polymerase chain reaction (pcr)-based tests define a specific pathogen with extraordinary sensitivity within 60 minutes. the fda has approved these pcr tests to detect at least 14 viruses and 10 bacteria [67] . these tests may also be useful for early detection of epidemic pathogen or resistance genes [68] . it seems clear that the introduction of molecular tests for general use may be difficult to interpret in the context of clinical care, so these new tests will require a substantial stewardship from the infectious disease community. this was illustrated in a trial to guide antibiotic decisions based on results of a pcr-based diagnostic to detect mrsa in purulent soft tissue infections that had no significant impact on antibiotic selection [69] . gene sequencing will be a new and important role for the infectious diseases-trained clinician as it becomes more readily available for defining transmission patterns to inform infection control practice. • microbiome: study of the microbiome at various anatomical sites represents a major nih-sponsored initiative that could possibly translate into important opportunities to treat or prevent multiple conditions [70, 71] . this work is at the dawn of development, but the early reads suggest a potential role in obesity, allergies, autoimmune disease, cancer, diabetes, heart disease, and other conditions [71, 72] . it is also apparent that antibiotics have a profound and long-lasting impact on the microbiome [71] . this field requires a transformation in our conventional understanding of infectious diseases, as the "pathogens" are communities of microbes that communicate in contrast to the koch postulate of "one microbe, one infection." an example is a recent report showing that volunteers fed steak and eggs (lecithin) have conversion by gut flora to trimethylamine-n-oxide, which is a marker of atherosclerosis [72] . this microbial interaction could be altered with antibiotics. the long-term goal is to define associations and intervene possibly with antibiotics and probiotics; this work may also illustrate potential harm to redefine risk-benefit ratios for antibiotics. • bundles: another potentially important role for infectious diseases-trained clinicians is the development of bundles that prevent infectious disease complications. an example is central line bacteremia, as described above [64] [65] [66] . that experience can now be applied to multiple iatrogenic infection risks associated with specific patients or procedures, possibly prioritizing those with the greatest healthcare consequences as described above. the role of infectious diseases is to define the bundle, design the study, and then implement them when results are convincing or even mandated. specialized skills in the management and study of infectious diseases are an increasingly important specialty in contemporary medicine. the roles of practitioners in the discipline are diverse, usually important, and sometimes critical, but commonly undervalued by contemporary priorities in healthcare systems and healthcare reform. it would be difficult to find another discipline in medicine that has such extraordinary diversity, surprises, value in patient care, and clinical relevance for both domestic and international applications. for many trained in medicine, joining the field of infectious diseases is simply the right thing to do. supplement sponsorship. this article was published as part of a supplement titled "the john bartlett festschrift: celebrating a career in medicine," sponsored solely by the department of medicine of the johns hopkins school of medicine in recognition of john bartlett's contributions to medicine. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. index case of fatal inhalation anthrax due to bioterrorism in the united states lyme arthritis: and epidemic of oligoarticular articular arthritis in children and adults in three connecticut communities the spirochetal etiology of lyme disease interview with willie bergdorfer, ph.d. interview by vicki glaser lyme disease-a tick-borne spirochetosis? how the discovery of borrelia bergdorferi came about nobel prize winners robin warren and barry marshall two win nobel prize for discovering bacterium tied to stomach ailments long-term follow-up of 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ecological perspective the microbiome explored: recent insights and future challenges intestinal microbial metabolism of phosphaditylcholine and cardiovascular risk key: cord-295525-emrwcx0m authors: to, kelvin kai-wang; tsang, owen tak-yin; yip, cyril chik-yan; chan, kwok-hung; wu, tak-chiu; chan, jacky man-chun; leung, wai-shing; chik, thomas shiu-hong; choi, chris yau-chung; kandamby, darshana h; lung, david christopher; tam, anthony raymond; poon, rosana wing-shan; fung, agnes yim-fong; hung, ivan fan-ngai; cheng, vincent chi-chung; chan, jasper fuk-woo; yuen, kwok-yung title: consistent detection of 2019 novel coronavirus in saliva date: 2020-02-12 journal: clin infect dis doi: 10.1093/cid/ciaa149 sha: doc_id: 295525 cord_uid: emrwcx0m the 2019 novel coronavirus (2019-ncov) was detected in the self-collected saliva of 91.7% (11/12) of patients. serial saliva viral load monitoring generally showed a declining trend. live virus was detected in saliva by viral culture. saliva is a promising noninvasive specimen for diagnosis, monitoring, and infection control in patients with 2019-ncov infection. the 2019 novel coronavirus (2019-ncov) was detected in the self-collected saliva of 91.7% (11/12) of patients. serial saliva viral load monitoring generally showed a declining trend. live virus was detected in saliva by viral culture. saliva is a promising noninvasive specimen for diagnosis, monitoring, and infection control in patients with 2019-ncov infection. keywords. 2019 novel coronavirus; saliva; diagnostics; viral load; transmission; covid-19. in 2003, the severe acute respiratory syndrome coronavirus (sars-cov) caused a devastating global outbreak with a casefatality rate of 10% [1] . in december 2019, a sars-cov-like coronavirus, the 2019 novel coronavirus (2019-ncov), has emerged in hubei province of china and has spread rapidly in mainland china and to other parts of the world [2, 3] . the 2019-ncov belongs to betacoronavirus genus lineage b, and is phylogenetically closely related to bat sars-like coronaviruses [2] . however, the spike, orf8, and orf3b proteins differ significantly from other known sars-like coronaviruses, which may confer differences in pathogenicity and transmissibility from sars-cov [4] . similar to sars-cov, the 2019-ncov can be efficiently transmitted between humans. cases of familial clustering have been reported [2] . rapid and accurate detection of 2019-ncov is crucial in controlling the outbreak in the community and in hospitals. nasopharyngeal and oropharyngeal swabs are the recommended upper respiratory tract specimen types for 2019-ncov diagnostic testing. however, the collection of these specimen types requires close contact between healthcare workers and patients, which poses a risk of transmission of the virus to the healthcare workers. furthermore, the collection of nasopharyngeal or oropharyngeal specimens causes discomfort and may cause bleeding, especially in patients with thrombocytopenia [2] . hence, nasopharyngeal or oropharyngeal swabs are not desirable for serial monitoring of viral load. sputum is a noninvasive lower respiratory tract specimen, but only 28% of patients with 2019-ncov in 1 case series could produce sputum for diagnostic evaluation [3] . saliva specimens can be provided easily by asking patients to spit into a sterile bottle. since no invasive procedures are required, the collection of saliva can greatly minimize the chance of exposing healthcare workers to 2019-ncov. we have previously demonstrated that saliva has a high concordance rate of greater than 90% with nasopharyngeal specimens in the detection of respiratory viruses, including coronaviruses [5, 6] . in some patients, coronavirus was detected only in saliva but not in nasopharyngeal aspirate [5] . saliva has also been used in screening respiratory viruses among hospitalized patients without fever or respiratory symptoms [7] . sars-cov can be detected in saliva at high titers [8] . given the benefits of saliva testing, we have tested 2019-ncov in saliva from patients with suspected 2019-ncov infection based on clinical and epidemiological criteria as outlined by the centre for health protection of hong kong. here, we report the results of the saliva testing. in hong kong, 2019-ncov testing was performed by public health laboratory services branch in hong kong for patients who fulfilled the reporting criteria or enhanced surveillance criteria [9] . a patient is considered to have laboratory-confirmed infection if 2019-ncov was detected in their nasopharyngeal or sputum specimens. saliva was collected by asking the patient to cough out saliva from their throat into a sterile container, and 2 ml of viral transport medium was added as we described previously [5, 6] . this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw 13-372). saliva specimens were subjected to total nucleic acid extraction by nuclisens easymag (biomerieux) as we described previously [5] . each specimen was mixed with lysis buffer. after extraction, the total nucleic acid was recovered using 55 μl of elution buffer. in-house 1-step real-time reverse transcription-quantitative polymerase chain reaction (rt-qpcr) assay targeting the s gene of 2019-ncov was performed using quantinova sybr green rt-pcr kit (qiagen) in a lightcycler 480 real-time pcr system (roche), as we described previously [2] . viral culture of 2019-ncov was conducted in a biosafety level-3 facility. vero e6 cells were seeded with 1 ml of minimum essential medium (mem) at 2 × 10 5 cells/ml in culture tubes and incubated at 37°c in a carbon dioxide incubator for 1-2 days until confluence for inoculation. each saliva specimen was inoculated in duplicate; 1 tube contained tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin (0.5 μg/ml) in serum-free mem and the other tube contained mem with 1% fetal calf serum. each tube was inoculated with 0.2 ml of saliva and was incubated in a slanted position so that the inoculum covered the monolayer for 60 minutes at 37°c. then 1 ml of either mem or trypsin mem was added and incubated in a roller apparatus at a speed 12 to 15 revolutions per hour. virus-induced cytopathic effect was examined daily for up to 7 days. a total of 12 patients with laboratory-confirmed 2019-ncov infection in hong kong were included. the median age was 62.5 years, ranging from 37 to 75 years. there were 5 female and 7 male patients. at the time of writing, all patients were still hospitalized. saliva specimens were collected at a median of 2 days after hospitalization (range, 0-7 days) ( figure 1 ). the 2019-ncov was detected in the initial saliva specimens of 11 patients (91.7%). for patient k, the first saliva specimen collected on the day of hospital admission tested negative. the median viral load of the first available saliva specimens was 3.3 × 10 6 copies/ml (range, 9.9 × 10 2 to 1.2 × 10 8 copies/ml). serial saliva specimens were available for 6 patients. the viral load was highest in the earliest available specimens for 5 patients (83.3%). for patient h, the viral load was slightly higher on day 1 after hospitalization (6.8 × 10 7 copies/ml) than on the day of hospital admission (5.7 × 10 7 copies/ml). for patient b, viral shedding in saliva was still detected on day 11 after hospitalization. in 33 patients whose nasopharyngeal specimens tested negative for 2019-ncov, all saliva specimens also tested negative. at the time of writing, viral cultures were positive for 3 patients and negative for 2 patients. in this study, we have demonstrated that 2019-ncov could be detected in the saliva specimens of 11 of the 12 patients studied. serial saliva specimens showed declines in salivary 2019-ncov rna levels after hospitalization. viral culture demonstrated that live viruses were present in the saliva of 3 patients. there are several advantages in using saliva specimens for the diagnosis of 2019-ncov. first, saliva specimens can be provided by the patient easily without any invasive procedures. therefore, the use of saliva specimens could reduce the risk of nosocomial 2019-ncov transmission. cases of 2019-ncov infection among healthcare workers have been found, with at least 1 reported death [10] . second, the use of saliva will allow specimen collection outside the hospitals where airborne-infection isolation rooms are not available, such as in outpatient clinics or in the community. in the setting where a large number of individuals require screening, saliva would represent a practical noninvasive specimen type. third, since healthcare workers are not required to collect saliva specimens, the use of saliva specimens will eliminate the waiting time for specimen collection, and hence the results would be available much sooner. this is especially important in busy clinical settings where the number of available staff is limited. among patients with serial saliva specimens available, there was a general decline in viral load for most patients, but 1 patient had viral shedding in the saliva for at least 11 days after hospitalization. the use of saliva is preferred over nasopharyngeal or oropharyngeal specimens for serial viral load monitoring because this would reduce the discomfort to the patient and reduce the health hazards to healthcare workers during repeated sampling. our experience with sars in 2003 showed that viral load often peaked at day 10 after symptom onset. thus, early detection and isolation of cases was strategic for infection control and provides the window of opportunity for antiviral therapy to decrease the peak viral load. the positive viral culture indicates that saliva contains live viruses that may allow transmission. respiratory viruses are considered to be transmitted from person to person through direct or indirect contact, or via coarse or fine droplets. saliva can be emitted through cough, and respiratory droplets containing influenza virus can be found even during normal breathing [11] . therefore, 2019-ncov may be transmitted via saliva directly or indirectly even among patients without coughing or other respiratory symptoms. our findings reinforce the use of surgical masks as a control measure. sars-cov has been shown to infect epithelial cells in salivary gland ducts in rhesus macaques [12] . the presence of 2019-ncov in patients' saliva suggests the possibility of salivary gland infection. however, it should be noted that saliva specimens not only contain saliva secreted from major or minor salivary glands but also contain secretions coming down from the nasopharynx or coming up from the lung via the action of cilia lining the airway. further studies are required to delineate the sources of 2019-ncov in saliva. our results have demonstrated the potential for saliva to be a noninvasive specimen type for the diagnosis and viral load monitoring of 2019-ncov. because saliva can be provided by patients without any invasive procedures, the use of saliva specimens will reduce the risk of nosocomial transmission of 2019-ncov and is ideal for situations in which nasopharyngeal specimen collection may be contraindicated. coronavirus as a possible cause of severe acute respiratory syndrome a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical features of patients infected with 2019 novel coronavirus in wuhan genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan additional molecular testing of saliva specimens improves the detection of respiratory viruses saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study respiratory virus infection among hospitalized adult patients with or without clinically apparent respiratory infection: a prospective cohort study detection of sars-associated coronavirus in throat wash and saliva in early diagnosis severe respiratory disease associated with a novel infectious agent-letters to doctors wuhan reports first doctor death from coronavirus emit consortium. infectious virus in exhaled breath of symptomatic seasonal influenza cases from a college community epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques key: cord-355734-pz64534w authors: antonio-villa, neftali eduardo; bello-chavolla, omar yaxmehen; vargas-vázquez, arsenio; fermín-martínez, carlos a; márquez-salinas, alejandro; bahena-lópez, jessica paola title: health-care workers with covid-19 living in mexico city: clinical characterization and related outcomes date: 2020-09-28 journal: clin infect dis doi: 10.1093/cid/ciaa1487 sha: doc_id: 355734 cord_uid: pz64534w background: health-care workers (hcws) could be at increased occupational risk for sars-cov-2 infection. information regarding prevalence and risk factors for adverse outcomes in hcws is scarce in mexico. here, we aimed to explore prevalence of sars-cov-2, symptoms, and risk factors associated with adverse outcomes in hcws in mexico city. methods: we explored data collected by the national epidemiological surveillance system in mexico city. all cases underwent real-time rt-pcr test. we explored outcomes related to severe covid-19 in hcws and the diagnostic performance of symptoms to detect sars-cov-2 infection in hcws. results: as of july 5 (th), 2020, 35,095 hcws were tested for sars-cov-2 and 11,226 were confirmed (31.9%). overall, 4,322 were nurses (38.5%), 3,324 physicians (29.6%), 131 dentists (1.16%) and 3,449 laboratory personnel and other hcws (30.8%). after follow-up, 1,009 hcws required hospitalization (9.00%), 203 developed severe outcomes (1.81%), and 93 required mechanical-ventilatory support (0.82%). lethality was recorded in 226 (2.01%) cases. symptoms associated with sars-cov-2 positivity were fever, cough, malaise, shivering, myalgias at evaluation but neither had significant predictive value. we also identified 341 asymptomatic sars-cov-2 infections (3.04%). older hcws with chronic non-communicable diseases, pregnancy, and severe respiratory symptoms were associated with higher risk for adverse outcomes. physicians had higher risk for hospitalization and for severe outcomes compared with nurses and other hcws. conclusions: we report a high prevalence of sars-cov-2 infection in hcws in mexico city. no symptomatology can accurately discern hcws with sars-cov-2 infection. particular attention should focus on hcws with risk factors to prevent adverse outcomes and reduce infection risk. the pandemic caused by the sars-cov-2 has created new challenges in health-care systems worldwide (1) . recently, the pandemic has had significant increases in the number of cases in the americas, where it has caused considerable pressure on health-care facilities and led to a substantial number of deaths (2, 3) . health-care workers (hcws) have a fundamental role in caring and managing patients with covid-19, being the primary workers involved in the daily management of the pandemic at an individual level. notably, this population could be at increased occupational risk of acquiring sars-cov-2 infection, which ultimately could lead to an increased risk of associated covid-19 complications. although it has been emphasized that hcws with respiratory symptoms should be isolated as soon as possible and protective equipment has been provided in several facilities, there is no consensus on the essential symptoms to promptly identify positive cases to mitigate transmission risks (4) . this was previously reported in the influenza a(h1n1) pandemic in 2009 with increased risk for influenza in early periods of the pandemic for hcws, which alongside social disparities could represent a challenge to hcws, particularly in developing countries (5, 6) . the situation in mexico is complex, given that sars-cov-2 infections coexist with a high prevalence of comorbidities associated with covid-19 complications in a large proportion of patients, including hcws. furthermore, healthcare systems within mexico are highly fragmented and quality of care and the ability to protect hcws within each institution is highly heterogeneous due to structural inequalities, which overall could increase the disparities in risk among hcws within marginalized communities (7) . given these fundamental differences, hcws living in mexico are at a substantial risk for sars-cov-2 infection and adverse covid-19 outcomes. there is a need to understand these trends and outcomes related to covid-19 in hcws to generate evidence which could inform public policy and promote development of recommendations to improve work environments amongst hcws by reducing transmission risk and, ultimately improve quality of care. here, we sought investigate the epidemiology of sars-cov-2 infection within hcws and its a c c e p t e d m a n u s c r i p t 4 related outcomes in mexico city. we also assessed the predictive ability of individual symptoms and comorbidities to identify hcws with sars-cov-2 infection for prompt isolation of affected cases. we analyzed data collected within the national epidemiological surveillance system database in mexico city, which is an open-source dataset comprising daily updated suspected covid-19 cases that have been tested using real-time rt-pcr to confirm sars-cov-2 according to the berlin protocol (8), and were certified by the national institute for diagnosis and epidemiological referral (9, 10) . health-care related professions included subjects whose occupations were reported as physicians, nurses, dentists, laboratory personnel and other involved hcws. demographic and health data were collected and uploaded to the epidemiologic surveillance database by personnel from each corresponding health-care facility. available variables include age, sex, nationality, state and municipality where the case was detected, immigration status as well as identification of individuals who self-identify as indigenous. health information includes the status of diabetes, obesity, chronic obstructive pulmonary disease (copd), immunosuppression, pregnancy, arterial hypertension, cardiovascular disease, chronic kidney disease (ckd), and asthma. evaluated symptoms included fever, cough, odynophagia, dyspnea, irritability, diarrhea, chest pain, shivering, headache, myalgia, arthralgia, malaise, rhinorrhea, polypnea, vomiting, abdominal pain, conjunctivitis, cyanosis, and sudden onset of symptoms. date of symptom onset, hospital admission, and death are available for all cases as are outpatient or hospitalized status, information regarding the diagnosis of clinical pneumonia, icu admission, and whether the patient required mechanical ventilation support (mvs). severe outcome was defined as a composite definition comprising death, requirement for mvs or icu admission (11) . we aimed to investigate comorbidities and symptoms associated with sars-cov-2 positivity using a mixed effects logistic regression model, including facility of treatment as a random effect to account for the variability in case distribution and treatment across healthcare facilities. we excluded hcws who were suspected cases at the time of inclusion without a definitive result for sars-cov-2. two separate models were designed to explore separately comorbidities and symptoms associated with sars-vov-2 positivity. we further explored the diagnostic test capacity, area under the curve, sensitivity, specificity, positive and negative predictive values (vpp, vpn, respectively) of each symptom to predict sars-cov-2 positivity. we fitted cox proportional risk regression models to explore risk factors associated to covid-19 related 30-day lethality, hospitalization, or severe outcome estimating time from symptom onset up to death, clinically reported outcome, or censoring, whichever occurred first. factors associated with using mechanical ventilation support (mvs) were evaluated using a mixed effects logistic regression model. we also performed a kaplan-meier analysis to assess differences in covid-19 outcomes comparing physicians and other hcws using the breslow-cox test. we evaluated cox proportional risk regression model performance using harrel's c-statistic and model assumptions were verified using schöenfeld residuals. logistic regression model performance was evaluated using the nagelkerke r 2 and the a c c e p t e d m a n u s c r i p t 6 hosmer-lemeshow test. a p-value <0·05 was considered as statistical significance threshold. all analyses were performed using r software version 3.6.2. as of the writing of this report (july 5 th , 2020) we identified 35,095 hcws assessed for sars-cov-2. amongst them, 11,226 (31.9%) had confirmed sars-cov-2 infection. overall, positive cases have been increasing since late february, with a notable peak of confirmed cases in late may and early june (supplementary figure 1) . in the group of confirmed cases 4,322 (38.5%) were nurses, 3,324 (29.6%) physicians, 131 (1.16%) dentists, 299 (2.66%) laboratory personnel and 3,150 (28.1%) referred as supportive hcws. covid-19 outcomes included 1,009 (9.00%) hcws who required hospitalization, 203 with a severe outcome (1.81%), and 93 who required mvs (0.82%). lethality attributable to covid-19 was recorded in 226 (2.01%) hcws. overall, 341 (3.04%) hcws referred no associated symptomatology but tested positive for sars-cov-2. compared with negative cases, those with confirmed sars-cov-2 test were older, predominantly female, and with a higher prevalence of diabetes and obesity. as expected, confirmed cases had a higher rate of adverse clinical outcomes ( table 1) . symptoms associated with increased probability of sars-cov-2 positivity in hcws included fever, cough, malaise, shivering, myalgias, arthralgias, rhinorrhea, chest pain, and polypnea at the time of clinical assessment. diarrhea, sudden onset of symptoms, irritability, headache were all associated with decreased likelihood of having a positive sars-cov-2 test. next, we sought to investigate the predictive performance of each symptom to predict sars-cov-2 positivity; as expected, all symptoms were highly unspecific. nevertheless, cases reporting headache, cough, and fever had a higher sensitivity for a positive sars-cov-2 test; cyanosis, polypnea, and dyspnea had greatest specificity but with poor sensitivity (supplementary table 1 ). we found that pregnancy, male sex, prolonged time for clinical assessment (≥7 days since beginning of symptoms), obesity, and diabetes were conditions a c c e p t e d m a n u s c r i p t 7 which increased likelihood of sars-cov-2 positivity; whilst active smoking and puerperium had a decreased likelihood of being positive cases (figure 1) . in confirmed sars-cov-2 cases, we found that symptoms at clinical assessment which increased risk for hospitalization were dyspnea, fever, and polypnea; while hcws with diarrhea, odynophagia, and conjunctivitis had a decreased risk for this outcome. exploring for conditions related to hospitalization, we found that cases with age ≥65 years, hiv/aids, diabetes, obesity, and arterial hypertension had increased risk of being hospitalized. hcws with age ≥65 years, dyspnea, fever, or polypnea at the moment of clinical assessment had increased risk for severe outcome. moreover, age ≥65 years, diabetes, and dyspnea at evaluation were conditions associated with mvs. predictors of lethality in hcws were clinical pneumonia at evaluation, age ≥65 years, diabetes, and obesity (figure 2) . overall, compared with non-hcws with positive sars-cov-2 test living in mexico city, (figure 3) . finally, a particular group of interest was those hcws who belonged to an indigenous group, which had up to 11-fold increased risk for covid-19 lethality (hr 11.44 95%ci 3.57-36.) after adjusting for covariates, although no differences were found in the risk for hospitalization and severe outcome. in this work, we report the prevalence of sars-cov-2 infection, related symptomatology, and covid-19 clinical outcomes using a city-wide based surveillance reports of hcws living in mexico city. amongst health-care workers, the group of physicians tend to have an increased risk of severe covid-19 outcomes, which is a remarkable occupational risk. moreover, certain factors, such as associated comorbidities and symptoms at the time of evaluation may predispose an increased risk of adverse outcomes. these findings should be considered by authorities in relation to relevant occupational hazards in hcws, particularly in physicians; given the vital role of hcws in managing the impact of the pandemic on individual patients, promoting the application of stricter regimes to reduce the probability of infection and adverse outcomes attributable to covid-19 amongst hcws is of paramount importance. hcws have increased occupational hazard to acquire sars-cov-2 infection compared with general population, attributable to direct contact during care of hospitalized patients. prevalence and lethality rates of sars-cov-2 infections reported in our population are slightly higher compared with previous reports in china, europe and the united states (4, (12) (13) (14) (15) (16) (17) . similarly, in mexico the number of positive cases reported in hcws has increased since the first confirmed cases were reported in late february. to reduce transmissibility and improve outcomes, it has been proposed that hcws who develop respiratory symptoms should be isolated to mitigate the spread between peers. however, as a c c e p t e d m a n u s c r i p t 9 our results confirm and based on previous reports, there is no sustainable evidence which allows using specific symptoms to identify sars-cov-2 positive cases in a clinical context (4) . hence, global and local authorities should instead steadily encourage the rational use of personal protective equipment in hcws, regardless of contact and care for patients with sars-cov-2 and systematic testing amongst hcws to identify asymptomatic cases. although previous reports have shown the effectiveness of personal protective equipment, there is still insufficient evidence if this strategy could mitigate the infection amongst all hcws at long-term, which sets an area of opportunity for further studies (18, 19) . our results also show that comorbidities in hcws, particularly those related to chronic noncommunicable diseases (e.g., diabetes, obesity and arterial hypertension), and the presentation of severe respiratory symptoms at the time of clinical assessment, increases the risk of adverse covid-19 outcomes. certain groups, such as pregnant women, older workers, and those referred to belong to an indigenous group, are at higher risk for severe related outcomes. previous reports by our group had shown the relationship between the presence of cardiometabolic diseases and risk of complications associated with sars-cov-2 infection in mexico (20) (21) (22) (23) . although not completely understood, this relationship could be explained by immunological over responsiveness observed in confirmed cases with diabetes and obesity (20, 23, 24) , particularly given recent evidence relating changes associated with an enhanced immune response to sars-cov-2 with risk of respiratory and multi-organ failure (23, 25) . conversely, in patients with prior immunosuppression, an increased risk for associated co-infections has been reported, which could explain risk for adverse outcomes in hcws with hiv/aids or pregnancy (26, 27) . finally, we found that physicians are a group of risk for developing adverse events compared to other hcws. this is consistent with a previous report, as it has been shown that physicians tend to spend more time in areas where patients with sars-cov-2 are assisted (28) . furthermore, prolonged shift times, work overload, phycological distress and exposure to probable cases amongst peers could lead physicians to be considered a group with significant occupational risk for developing covid-19 related outcomes (29) (30) (31) . a c c e p t e d m a n u s c r i p t 10 nevertheless, hcws have overall a lower risk of having a positive test and adverse outcome, compared with other professions. this could be explained by potential factors such as prompt and appropriate medical attention and social assurance which could be provided to hcws. interestingly, we also found that groups of physicians who self-reported as belonging to an indigenous community were at increased risk of death attributable to covid-19. although preliminary, these results may denote an inequality of access to timely care given the significant social discrepancies reported in mexico (7, 32, 33) . more studies should focus on the risk of adverse outcomes attributable to social conditions in medical personnel. our study had some strengths and limitations. first, we analyzed a large dataset which included information on confirmed positive and negative sars-cov-2 cases in mexico city, providing a unique opportunity to investigate covid-19 specific risk factors in hcws. a potential limitation of this study is the use of data collected from a sentinel surveillance system model, which is skewed towards investigating high-risk cases or only those with specific risk factors which on the one hand increases power to detect the effect of comorbidities and on the other hand might not be representative of milder cases of the disease. this is particularly true for asymptomatic cases amongst hcws, which were heavily underrepresented in our study and its prevalence must be assessed with widespread testing amongst hcws to reduce in-hospital transmission amongst hcws. furthermore, the reduced odds of sars-cov-2 positivity could be related to a higher number of tests being carried out amongst medical personnel compared to the general population, which is consistent with the positivity rate (). another potential limitation of our study is that our assessment of hcws is limited to those living in mexico city, which may not capture the whole picture in the country or the large socio-economic inequalities which might lead to higher rates of infection amongst hcws in disadvantaged areas. in summary, we present the first report of a city-wide based surveillance system which assessed clinical symptomatology and related outcomes attributable to covid-19 in hcws living in mexico city. we found that no specific symptoms can accurately discern among m a n u s c r i p t m a n u s c r i p t m a n u s c r i p t m a n u s c r i p t global, regional, and national estimates of the population at increased risk of severe covid-19 due to underlying health conditions in 2020: a modelling study covid-19-looking beyond tomorrow for 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e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-353342-2n6kqyeo authors: corman, victor m.; albarrak, ali m.; omrani, ali senosi; albarrak, mohammed m.; farah, mohamed elamin; almasri, malak; muth, doreen; sieberg, andrea; meyer, benjamin; assiri, abdullah m.; binger, tabea; steinhagen, katja; lattwein, erik; al-tawfiq, jaffar; müller, marcel a.; drosten, christian; memish, ziad a. title: viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection date: 2016-02-15 journal: clin infect dis doi: 10.1093/cid/civ951 sha: doc_id: 353342 cord_uid: 2n6kqyeo background. the middle east respiratory syndrome (mers) coronavirus causes isolated cases and outbreaks of severe respiratory disease. essential features of the natural history of disease are poorly understood. methods. we studied 37 adult patients infected with mers coronavirus for viral load in the lower and upper respiratory tracts (lrt and urt, respectively), blood, stool, and urine. antibodies and serum neutralizing activities were determined over the course of disease. results. one hundred ninety-nine lrt samples collected during the 3 weeks following diagnosis yielded virus rna in 93% of tests. average (maximum) viral loads were 5 × 10(6) (6 × 10(10)) copies/ml. viral loads (positive detection frequencies) in 84 urt samples were 1.9 × 10(4) copies/ml (47.6%). thirty-three percent of all 108 serum samples tested yielded viral rna. only 14.6% of stool and 2.4% of urine samples yielded viral rna. all seroconversions occurred during the first 2 weeks after diagnosis, which corresponds to the second and third week after symptom onset. immunoglobulin m detection provided no advantage in sensitivity over immunoglobulin g (igg) detection. all surviving patients, but only slightly more than half of all fatal cases, produced igg and neutralizing antibodies. the levels of igg and neutralizing antibodies were weakly and inversely correlated with lrt viral loads. presence of antibodies did not lead to the elimination of virus from lrt. conclusions. the timing and intensity of respiratory viral shedding in patients with mers closely matches that of those with severe acute respiratory syndrome. blood viral rna does not seem to be infectious. extrapulmonary loci of virus replication seem possible. neutralizing antibodies do not suffice to clear the infection. the middle east respiratory syndrome coronavirus (mers-cov) was first isolated in 2012 in saudi arabia [1] . since 2012, at least 1595 laboratory-confirmed cases of mers-cov infection, mostly with respiratory tract illness, have been reported; 571 of these were fatal [2] . known cases and outbreaks have been linked to countries in the arabian peninsula [3] . large nosocomial outbreaks, such as in jeddah, kingdom of saudi arabia in 2014 and the republic of korea in 2015, have demonstrated the potential of the virus to spread in healthcare settings [4] [5] [6] . due to the sporadic nature of the disease, with cases and small outbreaks distributed over a wide geographic area, investigation of the natural history of infection has been limited. except for individual case descriptions, chronological data summarizing the main viral diagnostic parameters, such as viral load or antibodies, are lacking. better knowledge of the kinetics of viral shedding from different body regions could help prevent nosocomial transmission and inform clinical management. knowledge of serological features, such as the kinetics of antibody production, could guide decisions regarding diagnostic protocols and provide essential information regarding immunity and virus elimination. quantitative data, such as viral loads and antibody titers, could enable comparisons with related diseases, in particular, severe acute respiratory syndrome (sars), for which studies of natural history were conducted in the aftermath of the 2002-2003 epidemic [7] . patients were seen during a hospital-associated outbreak between 5 march and 1 may 2014. there was no prospective planning of statistical power. patients were selected for mers-cov testing by reverse transcription polymerase chain reaction (rt-pcr) based on general clinical condition, oxygen saturation, and their need for invasive or noninvasive ventilation. samples of patients who tested positive were taken at least daily, starting from 0-7 days after initial submission of samples for mers-cov diagnosis. the day of the first sample testing positive by rt-pcr was defined as the day of diagnosis. the mean delay between first positive sampling and return of laboratory results was 3 days. stored samples, for retrospective analysis, were not available. specimens were taken from tracheal secretions via suction catheters, from the throat and the eyes using sterile swabs, and from urine and stool via sterile containers. baseline information on enrolled patients is provided in supplementary table 1 . institutional review board approval was obtained from the research ethics committee at prince sultan military medical city. real-time rt-pcr was performed on rna extracts using the upe and orf1a target genes as described in [8, 9] . raw rna concentrations were transformed to absolute viral loads by conversion factors, according to sample type (supplementary table 2 ). virus isolation, with increased sensitivity via the use of caco2 human colon carcinoma cells, was performed as described in [10] . a recombinant enzyme-linked immunosorbent assay (elisa; anti-mers-cov elisa igg, euroimmun, lübeck, germany) was based on soluble mers-cov spike protein s1 domain expressed in hek-293t cells [11] . the test was conducted as described previously [12, 13] . sera were tested twice and the arithmetic mean of the 2 measurements was used. detection of immunoglobulin m (igm) antibodies was done using immunofluorescence slides carrying vero cells infected with full mers-cov, as described in corman et al [9] . these were converted into a homogenous reagent format by an in vitro diagnostics manufacturer (anti-mers-cov-iift; euroimmun). all sera were depleted of immunoglobulin g (igg) antibodies using eurosorb (euroimmun) reagent according to manufacturer instructions. a mers-cov microneutralization test (nt) was performed as described in [13] [14] [15] . predilution before setting up log 2 -dilution series was 1:10, defining 1:20 as the lowest possible significant titer for categorizing a sample as positive. statistical analyses were done using spss software (version 22). in all cases, correlation analyses and preliminary multiple regression analyses were conducted to exclude confounding due to patient age or disease duration. to determine kinetic virological parameters in mers-cov infection, we followed 37 hospitalized patients. mean age was 63 years (range, 24-90 years), and 73% of patients were male. mers-cov infection had been established in all cases by rt-pcr. sixty-five percent of all patients died during the course of study. sequencing of full or partial genomes from 35 of the study patients revealed the existence of at least 6 closely related virus lineages (supplementary figure 1 and table 1 ). some sequences had already been seen in an earlier study [5] . patients belonged to at least 3 nosocomial transmission clusters. three cases could not be associated with clusters. at time of positive diagnosis, patients had spent 11 days in hospital on average, with a maximum of 108 days. only 20 of the 37 patients had been hospitalized for less than a week. because of the unresolved timing of transmission events in nosocomial clusters and the existence of comorbidities in most patients, it was impossible to determine the day of onset of symptoms in the majority of patients. unambiguous knowledge of the day of onset of symptoms was available for only 9 patients. mean and median duration between symptom onset and admission was 3 days (range, 0-8 days). in these 9 cases, mean and median duration between onset and diagnosis was 8 days (range, 1-16 days). the mean age of the 9 cases was not significantly different from the mean age of all patients under study. to provide a common point of reference in the clinical course of all patients, the day of diagnosis (day of first rt-pcr-positive sample) was defined as day 0 in the subsequent analyses. eight hundred twenty-three specimens from the 37 patients were tested, including 661 tests for viral load in 6 different sample categories (supplementary table 2 ). because of the variable latency between diagnosis and enrollment, clinical samples were not evenly distributed over patients' courses of disease (supplementary figure 2 ). absolute viral rna concentrations and positive proportion of samples were determined in 661 samples. data are illustrated in figure 1 and supplementary table 2 . lower respiratory tract (lrt) samples had the highest viral loads, up to 6.3 × 10 10 copies/ml (mean, 5.01 × 10 6 copies/ml). average viral loads in all other sample types were significantly lower (2-tailed t test, p < .0001 for all comparisons). virus isolation trials using the 6 stool samples with the highest rna concentration had negative outcomes. almost half of all sera showed detectable viral loads during the first week after diagnosis (25 of 51 sera tested). virus isolation from 20 viremic serum samples (10 with and 10 without neutralizing antibodies) failed, despite a highly optimized protocol [10] . there was an inverse correlation between in vitro serum neutralization activity and viremia in 45 sera (pearson r = −0.31, p < .03). however, viral rna and neutralizing antibodies were codetected in several cases, suggesting that the detected viral rna may only in part represent infectious virions ( figure 2a ). concentrations of rna in serum did not correlate significantly with those in lrt samples collected on the same day (n = 31 pairs of serum and lrt samples; spearman correlation, p = .08; figure 2b ). distributions of average lrt viral load per patient were summarized in 3 subsequent time windows (figure 3 ). in particular, during the first 5 days after diagnosis, average viral loads were not normally distributed but had a skewed distribution with a preponderance of patients with high viral load. of note, the 17 patients in the 2 highest viral load categories (right-most columns in the top panel in figure 3 ) did not show a significantly increased proportion of fatal outcomes (χ 2 test, p = .12). the average viral load during the first week after diagnosis was 5 × 10 7 copies/ml in fatal cases and 3.9 × 10 6 copies/ml in survivors (2-tailed t test, p < .007). divergence of viral loads between survivors and fatal cases was more pronounced in the second week (1.6 × 10 5 and 7.8 × 10 6 copies/ml, respectively; p < .0006). serological courses could be followed for 35 patients. almost half of these (n = 17) were already reactive (via elisa) on the day of diagnosis. among 27 patients with complete serological follow-up during the first week after diagnosis, 89% (n = 24) had antibodies by end of the week in both elisa and neutralization tests. eighteen of these patients tested positive for igm by immunofluorescence assay (ifa) (titers >1:10) by end of the week. only 1 of the igm-positive patients did not have a concomitant positive elisa result by end of the week. all of the 12 patients with 2 weeks of serological follow-up seroconverted (elisa and neutralization tests). eleven of these 12 patients developed igm detectable by ifa. antibody kinetics averaged over all samples and tests are summarized in figure 4 . information on outcome was available for 34 patients with serological follow-up. all 12 patients who survived their infection showed seroconversion (elisa and nt) during the first week. all developed igm antibodies concomitantly. among 22 fatal cases, 14 showed seroconversion by elisa prior to death, the latest seroconversion occurring by day 11 postdiagnosis. twelve of 22 fatal cases developed neutralizing antibodies, and 11 developed detectable igm. antibody levels (elisa optical density [od] and log 2 nt titers) during the first week after diagnosis were not significantly different between surviving and fatal cases (2-tailed t test, p = .8). during the second week after diagnosis, the average elisa od values in survivors were significantly higher than in fatal cases (2.9 vs. 2.1, 2-tailed t test, p < .02). also, average nt titers were higher in survivors during week 2, but with less significant discrimination (2 7.5 vs 2 5.4 in survivors and fatal cases, respectively; 2-tailed t test, p < .06). elisa od values and log 2 nt titers were compared against log 10 viral loads in lrt samples. from 30 patients, elisa and viral load data were available based on matched serum and respiratory tract samples taken on the same days (198 matched data pairs, covering days 0-17 postdiagnosis). because of workload and biosafety, the number of nt assays had to be restricted. however, combined elisa, nt, and viral load data were available from 26 patients, with 91 matched datasets that covered days 1-17 after diagnosis. supplementary figure 3 summarizes the distribution of matched samples over time. pearson test identified significant linear correlation between antibodies and log 10 viral loads (elisa, r = −0.6; nt, r = −0.51; p < .001 in both analyses). however, plots of elisa and nt antibody results in matched sample pairs yielded no evidence of mutually exclusive occurrence of virus and antibodies ( figure 5a and 5b) . discussion we studied quantitative viral excretion and serum antibody kinetics of a substantial group of hospitalized patients infected with mers-cov. the time of diagnosis chosen as a chronological reference point represents the time when an infection is suspected in hospitalized patients, or when outpatients report to hospitals due to worsening symptoms. even though the day of onset was unknown in many of the studied cases, the presented virological courses represent the typical situation encountered during mers treatment in hospital settings. by providing absolute quantitative measures of virus excretion, we can for the first time compare results between mers and sars, a disease that is now thought to have involved higher pandemic potential than mers [16] . in our patients and elsewhere, the lrt was found to be the main source of mers-cov excretion [17] . unfortunately, there are few data on lrt virus excretion for sars-cov, because endotracheal sampling was widely discouraged during sars outbreaks to avoid nosocomial risks. we have shown, in one of the few available studies, that sars-cov was excreted from the lrt at mean concentrations of 1.2-2.8 × 10 6 copies/ml, reaching a maximum of 10 10 copies/ml [18] . that study was conducted in a similar clinical context (a treatment center in singapore) with similar timing of samples and clinical courses. determination of viral load was performed by the same laboratory, using equivalent calibrators and conversion factors. from this comparison, we can conclude that average and peak lrt viral loads in mers are equal to those in sars [18] . more data are available for upper respiratory tract (urt) viral loads in sars patients. peak urt rna concentrations can reach up to 5 × 10 5 copies/sample between days 7 and 10 after onset [18, 19] . the corresponding number for mers ( peaking at 4.1 × 10 6 copies/sample), is equivalent or higher. also, the 47.6% proportion positive for mers in urt exceeded the 38% proportion positive in 98 urt samples for sars patients in hong kong [19] . the timing of excretion is more difficult to compare, as many patients in studies on sars were outpatients who entered hospital because of their sars infection, whereas our mers patients were mostly inpatients [20] . in sars cases that occurred before hong kong authorities started active community contact tracing, the average time from symptom onset to admission was 4-7 days [20] . because case definitions and diagnostics were well-established during sars in hong kong, diagnostic samples would have been taken immediately upon admission. this can be aligned with the timing in our study based on a subcohort of patients for whom an onset of symptoms could be reliably determined. in these patients, the time from onset of symptoms to the initiation of laboratory diagnostics (8 days) was similar to that of early hong kong sars cases. the shedding peak in sars patients occurred after approximately 10-12 days from symptom onset, which is very similar to the shedding maximum observed in our study, under the assumption that the day of first diagnosis in our mers cases plausibly falls around the eighth day of symptoms (ie, directly after admission) [21, 22] . in summary, neither the virus concentration nor the timing of respiratory shedding provides an explanation for differences in transmissibility between mers-cov and sars-cov. experimental data suggest a higher sensitivity of mers-cov to respiratory epithelium-associated type i interferon, which might provide a plausible explanation for its lower transmissibility in comparison with sars-cov [23] . many other explanations are possible, however. the detection of mers-cov in serum is another similarity with sars. up to 79% of serum samples were found to contain sars-cov rna during the first week of illness, and around 50% during the second week [24] [25] [26] . these numbers match our observations for mers. free viremia seems unlikely as a cause of nosocomial transmission of mers, as no infectious virus was isolated from serum. there is evidence of sars-cov replicating in peripheral blood mononuclear cells, macrophages, and dendritic cells, albeit at low levels [27] [28] [29] [30] . in the present study, the absence of correlation of serum viral load with lrt viral load points to potential extrapulmonary replication. viremia despite the presence of neutralizing antibodies indicates a body region that is not accessible to neutralizing antibodies but which releases virus into the blood. sars-cov has been shown to replicate in several extrapulmonary organs without evidence of tissue damage [27] . one organ implicated in mers is the kidney. kidney failure has been reported in many cases, and mers-cov was originally isolated in kidney cells that express dpp4, the mers-cov entry receptor [11] . however, earlier healthcare-associated outbreaks have been centered near dialysis centers and nephrology departments, and have affected metabolically compromised patients who are predisposed to kidney failure when suffering from systemic disease affecting blood pressure and circulation. although patients with sars-cov showed viral rna detection rates up to 50% in urine [7, 22] , we rarely found urine-associated mers-cov rna in this study. as in sars, kidney failure in mers patients might well be explained by severe inflammatory reaction combined with the administration of potentially nephrotoxic drugs during intensive care [31] . nevertheless, it will be highly important to conduct postmortem examinations, particularly of the kidney, of patients who die during acute mers-cov infection. a clear difference from sars was the detection of viral rna in stool. in sars, the rna prevalence in stool samples was so high that testing of stool was proposed as a reliable and sensitive way to routinely diagnose the disease [7, 22] . active replication in the gut with live virus isolation has been demonstrated [32] . for mers, we found stool-associated rna in only 14.6% of samples, with rather low rna concentration, and had no success in isolating infectious virus. based on these data, fecal excretion may not have played a relevant role for nosocomial spread of mers-cov among the patients under study. as in sars, mers-cov nosocomial transmission was repeatedly ascribed to the potential of some patients to act as super-shedders or super-spreaders [6, 20] . our analysis of viral loads, particularly in the early acute phase of disease, supports the existence of a limited number of patients with extraordinarily high viral loads. as these patients were not more likely to die of the infection, they might not have had more severe symptoms, and thus might have been able to engage in social contact despite their disease. the course of mers antibody development resembles that of sars. patients infected with sars seroconverted during weeks 2 and 3 after onset [7] . most of the patients studied here had already seroconverted during the first week after diagnosis, which putatively represents the second week after onset. as in sars, igm was not detected earlier than igg, which limits its diagnostic utility, in particular when considering that igm against more prevalent human coronaviruses may cross-react with mers-cov [12, 33] . with current methodology, igm testing should be restricted to cases that require proof of recent and overcome mers-cov infection. information on the prognostic value of antibody response in sars is less clear. in the present study on mers, 36% (elisa) and 45% (nt) of fatal cases failed to mount an antibody response prior to death. however, differences became apparent only in the second week after diagnosis, pointing to only a weak protective effect against lung disease. the development of antibodies in serum was not followed by a rapid elimination of viral rna from the lung. neutralizing antibodies normally include immunoglobulin a (iga) secreted in respiratory fluids and saliva. we have recently shown that anti-mers-cov iga is indeed secreted in respiratory fluids [10] , suggesting that the development of iga comes too late to confer timely reduction of viral replication in infected mucosa. based on these data, vaccines against mers-cov should be designed so as to include and enhance cellular immune responses. supplementary materials are available at http://cid.oxfordjournals.org. consisting of data provided by the author to 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monocytes/macrophages acute renal impairment in coronavirusassociated severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital acknowledgments. we thank artem siemens, heike kirberg, monika eschbach-bludau, sebastian brünink, stephan kallies, and tobias bleicker (institute of virology, bonn, germany) for excellent technical assistance. we also thank dr terry c. jones, department of zoology, university of cambridge, united kingdom, for editing the final manuscript.financial support. this work was supported by european commission grant prepare (contract number 602525), as well as the deutsche forschungsgemeinschaft (dfg dr772/7-1).potential conflicts of interest. all authors: no potential conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed.