key: cord-288807-sw6teq1n authors: cook, marion title: potential factors linked to high covid-19 death rates in british minority ethnic groups date: 2020-07-17 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30583-1 sha: doc_id: 288807 cord_uid: sw6teq1n nan black african 3·7 20·0 2·3 12·5 0 indicates absent or very low prevalence. na=not available, although sickle cell trait was found in babies of all ethnic groups in the nhs sickle cell and thalassaemia data report, 2013-14. 5 *predicted covid-19 fatalities based on geography and demographics and actual hospital deaths, relative to white british, by ethnic group. 6 †values for men. table: covid-19 fatalities and prevalence of haemolytic disorders and anaemia among uk ethnic groups mapping the presence of sickle cell and beta-thalassaemia in england: estimating and validating ethnic-specific rates european association for the study of the liver. easl clinical practice guidelines for hfe hemochromatosis the epidemiology of the haemoglobin level-a study of 1057 subjects in general practice nhs sickle cell and thalassaemia screening programme. data report 2013/14. trends and performance analysis are some ethnic groups more vulnerable to covid-19 than others? london: the institute for fiscal studies key: cord-305193-hbn69kmi authors: lang, min; som, avik; mendoza, dexter p; flores, efren j; reid, nicholas; carey, denston; li, matthew d; witkin, alison; rodriguez-lopez, josanna m; shepard, jo-anne o; little, brent p title: hypoxaemia related to covid-19: vascular and perfusion abnormalities on dual-energy ct date: 2020-04-30 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30367-4 sha: doc_id: 305193 cord_uid: hbn69kmi nan in any of the studies, and segmental increased perfusion to areas of infarction would be very atypical. furthermore, a peripheral halo of increased perfusion has not been described in pulmonary infarction, but has been described once pre viously in a case of bacterial pneu monia. 5 however, blood and sputum cultures were negative in the three patients with covid19 at our hospital and did not suggest bacterial coinfection. it might be possible that the inflammatory response to covid19 resembles more of a bacterial infection than a viral infection. overall, the combination of these imaging findings is novel for covid19 pneumonia. treatment for acute respiratory failure in patients with covid19 is challenging in part because of little understanding of the underlying pathophysiology. our findings are atypical for acute respiratory distress syndrome or thrombotic vascular disease and point to a possible central role for previously underappreciated pulmonary vascular shunting. more detailed assessments of vascular and perfusion changes in patients with covid19 are urgently needed. bpl reports royalties from elsevier as a textbook author and editor, outside the submitted work. aw reports personal fees from bayer pharmaceuticals, outside the submitted work. all other authors declare no competing interests. studies have shown that some patients with coronavirus disease 2019 (covid19) and acute hypoxaemic respiratory failure have preserved lung compliance, suggesting that processes other than alveolar damage might be involved in hypoxaemia related to covid19 pneumonia. 1 the typical imaging features of covid19 pneumonia, including peripheral groundglass opacities with or without consolidation, are also non specific and can be seen in many other diseases. 2 there has been increasing attention on microvascular thrombi as a possible explanation for the severe hypoxaemia related to covid19. 3, 4 dualenergy ct imaging can be used to characterise lung perfusion and is done as part of the standard protocol for imaging pulmonary embolism at our institution. three patients with covid19, as confirmed by nasopharyngeal rtpcr at our hospital, who did not have a history of smoking, asthma, chronic obstructive pulmonary disease, or other pulmonary conditions, underwent dualenergy ct imaging for elevated concentrations of ddimer (>1000 ng/ml) and clinical suspicion of pulmonary emboli. although no pulmonary emboli were observed in these individuals, we noted striking perfusion abnormalities that have not been previously described; in retrospect, at least nine other covid19 cases also shared these findings. in addition to the typical ct features of covid19 pneumonia, 2 we observed considerable proximal and distal pulmonary vessel dilatation and tortuosity, predominately within, or surrounding, areas of lung opacities. here, we present the first published images from dualenergy ct imaging of covid19 pneumonia that show profound vascular and perfusion abnormalities (figure; appendix). three major findings from dual energy ct were observed on the images of pulmonary blood volume perfusion: preferentially increased perfusion of the lungs proximal to areas of lung opacity, decreased areas of peripheral perfusion corresponding to peripheral lung opacities, and a halo of increased perfusion surrounding peripheral areas of consolidation. the observed pulmonary vascular dilation might be due to relative failure of normal, physiological hypoxic pulmonary vasoconstriction in the setting of overactivation of a regional vasodilatation cascade as part of a dysfunctional and diffuse inflammatory process. additionally, the mosaic perfusion pattern did not correspond to findings of bronchial wall thickening or secretions, making airway disease as the main underlying cause of hypoxaemia unlikely. therefore, these perfusion abnormalities, combined with the pulmonary vascular dilation we observed, are suggestive of intrapulmonary shunting toward areas where gas exchange is impaired, resulting in a worsening ventilation-perfusion mismatch and clinical hypoxia. although peripheral opacities with hypoperfusion can be seen in pulmonary infarction, no pulmonary emboli were observed clinical course and risk factors for mortality of adult inpatients with covid19 in wuhan, china: a retrospective cohort study dualenergy ct: spectrum of thoracic abnormalities chest ct findings in coronavirus disease19 (covid19): relationship to duration of infection incidence of thrombotic complications in critically ill icu patients with covid19 key: cord-275521-dlp055z8 authors: goldman, emanuel title: exaggerated risk of transmission of covid-19 by fomites date: 2020-07-03 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30561-2 sha: doc_id: 275521 cord_uid: dlp055z8 nan a clinically significant risk of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmission by fomites (inanimate surfaces or objects) has been assumed on the basis of studies that have little resemblance to real-life scenarios. the longest survival (6 days) of severe acute respiratory syndrome coronavirus (sars-cov) on surfaces was done by placing a very large initial virus titre sample (10⁷ infectious virus particles) on the surface being tested. 1 another study that claimed survival of 4 days used a similarly large sample (10⁶ infectious virus particles) on the surface. 2 a report by van doremalen and colleagues found survival of both sars-cov and sars-cov-2 of up to 2 days (on surfaces) and 3 days (in aerosols generated in the laboratory), but again with a large inoculum (10⁵-10⁷ infectious virus particles per ml in aerosols, 10⁴ infectious virus particles on surfaces). 3 yet another study found long survival (5 days) of human coronavirus 229e on surfaces with what i would still consider a substantially large viral load (10³ plaque-forming units) in a cell lysate. 4 however, using a cell lysate rather than purified or semipurified virus might enable initial viral proliferation or protection from the effects of the sample drying out. none of these studies present scenarios akin to reallife situations. although i did not find measurements of coronavirus quantities in aerosol droplets from patients, the amount of influenza virus rna in aerosols has been measured, with a concentration equivalent to 10-100 viral particles in a droplet, with even fewer infectious influenza virus particles capable of growth in a plaque assay. 5 by contrast, one study found human coronavirus 229e to survive for only 3 h, and human coronavirus oc43 to survive for 1 h, after drying on various surfaces including aluminum, sterile latex surgical gloves, and sterile sponges. 6 in a study in which the authors tried to mimic actual conditions in which a surface might be contaminated by a patient, no viable sars-cov was detected on surfaces. 7 a 2020 literature review 8 included most of the studies i have cited here (and others), but adds no new research, and in my view, does not critically evaluate previously published studies. i am not disputing the findings of these studies, only the applicability to real life. for example, in the studies that used a sample of 10⁷, 10⁶, and 10⁴ particles of infectious virus on a small surface area, 1-3 these concentrations are a lot higher than those in droplets in real-life situations, with the amount of virus actually deposited on surfaces likely to be several orders of magnitude smaller. 5 hence, a reallife situation is better represented in the work of dowell and colleagues 7 in which no viable virus was found on fomites. in my opinion, the chance of transmission through inanimate surfaces is very small, and only in instances where an infected person coughs or sneezes on the surface, and someone else touches that surface soon after the cough or sneeze (within 1-2 h). i do not disagree with erring on the side of caution, but this can go to extremes not justified by the data. although periodically disinfecting surfaces and use of gloves are reasonable precautions especially in hospitals, i believe that fomites that have not been in contact with an infected carrier for many hours do not pose a measurable risk of transmission in non-hospital settings. a more balanced perspective is needed to curb excesses that become counterproductive. i declare no competing interests. professor of microbiology, biochemistry and molecular genetics, new jersey medical school -rutgers university, newark, nj 07103, usa stability and inactivation of sars coronavirus stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 human coronavirus 229e remains infectious on common touch surface materials measurements of airborne influenza virus in aerosol particles from human coughs survival of human coronaviruses 229e and oc43 in suspension and after drying on surfaces: a possible source of hospital-acquired infections severe acute respiratory syndrome coronavirus on hospital surfaces persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents key: cord-277342-40d24mvm authors: chen, yu; li, lanjuan title: sars-cov-2: virus dynamics and host response date: 2020-03-23 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30235-8 sha: doc_id: 277342 cord_uid: 40d24mvm nan since december, 2019, coronavirus disease 2019 (covid-19) has affected more than 100 000 patients globally. 1 covid-19 is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and has a case-fatality rate of 2·3%, with higher rates among elderly patients and patients with comorbidities. 2 person-to-person transmission is efficient, with multiple clusters reported. clinically, patients with covid-19 present with respiratory symptoms, which is very similar to the presentation of other respiratory virus infections. radiologically, covid-19 is characterised by multifocal ground-glass opacities, even for patients with mild disease. 3 knowledge of virus dynamics and host response are essential for formulating strategies for antiviral treatment, vaccination, and epidemiological control of covid-19. however, a systematic study on these aspects has not been done. in the lancet infectious diseases, kelvin to and colleagues 4 report the viral load and antibody profiles of a cohort of 23 patients admitted to hospital with covid-19. in these patients, the viral load peaked during the first week of illness then gradually declined over the second week. viral load was also shown to correlate with age. furthermore, both igg and igm antibodies started to increase on around day 10 after symptom onset, and most patients had seroconversion within the first 3 weeks. finally, the igg and igm antibody level against the sars-cov-2 internal nucleoprotein and the surface spike receptor binding domain correlated with neutralising activity. these findings have several practical implications. first, the high viral load during the early phase of illness suggests that patients could be most infectious during this period, and it might account for the high transmissibility of sars-cov-2. furthermore, the high viral load on presentation suggests that sars-cov-2 could be susceptible to emergence of antiviral resistance. second, age was associated with viral load in this study, which could explain the high degree of severe disease in older patients with sars-cov-2. 5, 6 the high viral load in elderly patients is associated not only with low immunity but also with high expression of the ace2 receptor (the cellentry receptor for sars-cov-2) in older adults. 7 the timing of antibody seroconversion is crucial for determining the optimum timepoints for collecting serum specimens for antibody testing for diagnosis. furthermore, this information is important for immunologists to choose the best timepoints for obtaining peripheral blood b cells for development of therapeutic monoclonal antibodies. 8 the major strength of the study by to and colleagues is the systematic analysis of the serial viral load and antibody profile for up to 4 weeks, which provides insights into viral and host interactions during the acute and convalescent phases. another notable aspect is that self-collected posterior oro pharyngeal saliva samples were used, instead of nasopharyngeal specimens, for viral load monitoring. collection of nasopharyngeal specimens is an invasive procedure, and it is uncomfortable for the patient and poses an infection risk to health-care workers. self-collected saliva is much more acceptable to patients and is safer for health-care workers. this study clearly shows the feasibility of using saliva for viral load monitoring. the information provided by to and colleagues is solid scientific evidence on covid-19 for clinicians and scientists. nonetheless, many questions are still outstanding on the viral characteristics and host response during infection. sars-cov-2 has been detected in faeces, blood, and urine samples, 9, 10 and it is important to ascertain viral load dynamics in such samples, for prevention and control of the pandemic. furthermore, the relation between viral load and disease severity needs to be further clarified. studies with a larger sample size are needed to understand how different factors can affect viral load or antibody response. for example, immunocompromised patients might have higher viral load, prolonged viral shedding, and impaired antibody response. future studies in the paediatric population are vital, because children seem to have much milder disease than in adults. finally, a more detailed understanding of the innate and adaptive immune response against sars-cov-2 is important for understanding the pathogenesis and for designing vaccines. we declare no competing interests. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series asians do not exhibit elevated expression or unique genetic polymorphisms for ace2, the cell-entry receptor of sars-cov-2 timely development of vaccines against sars-cov-2 public health might be endangered by possible prolonged discharge of sars-cov-2 in stool epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore key: cord-288867-iicfe7il authors: bauch, chris t; anand, madhur title: covid-19: when should quarantine be enforced? date: 2020-05-20 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30428-x sha: doc_id: 288867 cord_uid: iicfe7il nan when children are little, they like to pose dilemmas as "would you rather" questions that involve difficult trade-offs. would you rather fight an elephant-sized duck or five human-sized rhinos? would you rather have a runny nose for a month or dry eyes? the options are not only both undesirable, but also incomparable. these questions are how we might think of some of the dilemmas created by the covid-19 pandemic, which presents us with difficult trade-offs in equity, economics, public health, and civil liberties. in the lancet infectious diseases, corey m peak and colleagues 1 explore one such dilemma. plainly put, they ask the question: should health authorities place potentially exposed individuals into a quarantine setting where their separation from others can be enforced, or should authorities simply let them go home, ask them to avoid contacts, and monitor them for covid-19 symptoms through phone calls or health-care visits? the authors name the two options individual quarantine and active monitoring, respectively. individual quarantine impinges more on civil liberties but is less risky from a public health perspective. aware of this dilemma, peak and colleagues 1 use a mathematical model of the early spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections to establish the conditions under which individual quarantine works better than active monitoring. under a broad range of model parameters the authors find that there is no difference in the effectiveness of the two strategies. they either both contain the outbreak, or they both do not contain it. however, an important exception occurs if the serial interval (time between symptom onset in an infector and their infectees) of the infection 2 is around 4·8 days, and if at least 75% of infected contacts are identified within 12 h, on average. under those circumstances, individual quarantine could contain an outbreak, whereas active monitoring could not. this prediction appears to be consistent with another recently published model of covid-19 containment. 3 crucially, this analysis assumes that testing is rapid and widely available, which is not true for many places. in view of evidence that the sars-cov-2 serial interval is probably about 5 days, 2 these observations raise the question: how hard is it to attain 75% success in tracing of infected contacts? this rate might be impractical with manual contact tracing, depending on the route of transmission. interviews with individuals are stymied by problems of cognitive bias, inability of individuals to remember their detailed movements, and difficulty in identifying contacts unknown to the patient. by contrast, digital technologies offer vast improvements in terms of location accuracy and contact identification; 3 these technologies put the 75% target within reach, as the experience of south korea with covid-19 has shown. 4 however, using digital surveillance to reach the target would then present two infringements on civil liberties: individuals must be tracked to capture enough infected contacts, and those contacts must be individually quarantined. and even if contact tracing fails to contain an outbreak, the combined effect of physical distancing and contact tracing is greater than the effect of either intervention on its own. 1 hence, the authors' analysis tells us that many decision makers will need to choose whether to use digital surveillance. mathematical models such as those used by peak and colleagues 1 can help decision makers to adopt an evidence-based approach to addressing the difficult dilemmas that we will continue to face during the covid-19 pandemic. however, we suggest that mathematical models should go beyond addressing strictly epidemiological questions. the pandemic has affected almost every aspect of our individual and collective lives, and our own reactions to the pandemic shape the outbreaks we experience. hence, we think researchers should broaden their focus to developing models that explicitly include relevant social processes, equity considerations, and economic impacts in the model structure. there is already a precedent for this approach in modelling endemic infectious diseases 5, 6 and in other fields of natural systems modelling. 7 on a final note, we speculate that the covid-19 quarantine and monitoring dilemma is in some ways not as difficult to address as the "would you rather" questions of children. contact tracing represents a race to trace. 8 to prevent exponential growth in the number of cases, public health must trace contacts of infected cases and reduce their chances of causing further spread faster than the virus propagates through the network of personal contacts. thus, intrusive action in the early stages of a pandemic might reduce how much longer those intrusive measures have to be applied, and to how many people. additionally, benefits for other fundamental rights are accrued as the pandemic unfolds, such as saving both lives and livelihoods. 9 we declare no competing interests. cbauch@uwaterloo.ca individual quarantine versus active monitoring of contacts for the mitigation of covid-19: a modelling study the serial interval of covid-19 from publicly reported confirmed cases quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing epidemiology & case management team, korea centers for disease control & prevention. contact transmission of covid-19 in south korea: novel investigation techniques for tracing contacts social factors in epidemiology poverty trap formed by the ecology of infectious diseases the impact of human-environment interactions on the stability of forest-grassland mosaic ecosystems emergency response to a smallpox attack: the case for mass vaccination the universal declaration of human rights key: cord-322290-zx3i8mpo authors: yelin, dana; wirtheim, eytan; vetter, pauline; kalil, andre c; bruchfeld, judith; runold, michael; guaraldi, giovanni; mussini, cristina; gudiol, carlota; pujol, miquel; bandera, alessandra; scudeller, luigia; paul, mical; kaiser, laurent; leibovici, leonard title: long-term consequences of covid-19: research needs date: 2020-09-01 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30701-5 sha: doc_id: 322290 cord_uid: zx3i8mpo nan weeks and months after the onset of acute covid-19, people continue to suffer. paul garner, a professor of epidemiology at liverpool school of tropical medicine, uk, wrote on the 95th day after the onset of symptoms that "i am unable to be out of bed for more than three hours at a stretch, my arms and legs are permanently fizzing as if injected with szechuan peppercorns, i have ringing in the ears, intermittent brain fog, palpitations, and dramatic mood swings." 1 other people also describe similar complaints. 2,3 78 of 100 patients in an observational cohort study who had recovered from covid-19 had abnormal findings on cardiovascular mri (median of 71 days after diagnosis) and 36 of those reported dyspnoea and unusual fatigue. 4 we are seeing patients in clinics dedicated to covid-19 convalescents, and for some of these patients the return to their former health trajectory is slow and painful. these patients are not only those recovering from the severe form of the acute disease (ie, post intensive care syndrome), but also those who had mild and moderate disease. a summary of the most common complaints, based on our clinical impressions, is shown in the appendix (p 1). rare long-term sequelae can result after other viral infections-eg, infectious mononucleosis, measles, and hepatitis b. long-term sequelae of covid-19 are unknown (as are many aspects of the acute disease). long-term consequences were observed in survivors of severe acute respiratory syndrome (sars) 5,6 but it is unknown whether lessons from sars are applicable to covid-19. other concerns are rising: does acute covid-19 cause diabetes? 7 or other metabolic disorders? will patients develop interstitial lung disease? we are still in the first months of the pandemic and we do not know what to tell our patients when they are asking about the course and prognosis of their ongoing complaints. the number of people affected by covid-19 is unprecedented. we owe good answers on the long-term consequences of the disease to our patients and healthcare providers. the obvious answer is in research. in the appendix (p 2) we have compiled a list of questions we think should be answered. this list is based on the authors' views and experience rather than on the literature, which is scant. for efficient research and for research that our patients (and we) can trust, some common problems in the description and research of acute covid-19 should be avoided. the main problem is fragmentation. for example, wynants and colleagues 8 described 47 models for predicting covid-19 infection and 16 prognostic models for covid-19 patients. most of these models had a high risk of bias and most of them did not have external validation. additionally, randomised controlled trials on interventions to treat the acute disease were stopped before enlisting the planned sample size. although much effort was invested in these studies, we have learned little. fragmentation also happens by discipline, 6, 7 and the follow-up (for clinical and research purposes) should be multinational, multidisciplinary, comprehensive, and homogenous. careful recording of symptoms and patient examination should allow understanding of which part of the sequelae is common to all severe infections, which symptoms might be explained by the anxiety caused by a new disease and by the isolation, 9 and which symptoms are secondary to a complicated form of covid-19 (eg, pulmonary involvement during the acute disease). if indeed covid-19 is causing long-term sequelae then are the mechanisms underlying the longterm consequences immunological? or caused by new or relapsing inflammation, ongoing infection, or sideeffects of immunomodulatory treatment? such data can serve to point at candidate management strategies to be tested in trials. support for research is needed on the trajectory of people recovering from covid-19. to avoid the problems we have witnessed in the research of the acute phase of the disease, a clear definition of patient inclusion criteria, a common protocol, and uniform definitions of outcomes and ways to measure them are required. additionally, data should be collected in real time and computational tools are needed to be able to do this (appendix p 3). the participation of an international and interdisciplinary group of researchers is essential. multisite and multinational projects are needed because a description from one group or one site cannot discern between universal features and features of the local health system or the local population. by comparing data from different sites and countries we can learn infectious diseases institute, rambam health care campus and the ruth and bruce rappaport faculty of medicine, technion israel institute of technology covid-19 at 14 weeks-phantom speed cameras, unknown limits, and harsh penalties covid-19: prolonged and relapsing course of illness has implications for returning workers persistent symptoms in patients after acute covid-19 outcomes of cardiovascular magnetic resonance imaging in patients recently recovered from coronavirus disease 2019 (covid-19) mental morbidities and chronic fatigue in severe acute respiratory syndrome survivors: long-term follow-up the long-term impact of severe acute respiratory syndrome on pulmonary function, exercise capacity and health status new-onset diabetes in covid-19 prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal covid-19 pandemic and mental health consequences: systematic review of the current evidence key: cord-332238-qw2ual51 authors: meyer, jaimie p; franco-paredes, carlos; parmar, parveen; yasin, faiza; gartland, matthew title: covid-19 and the coming epidemic in us immigration detention centres date: 2020-04-15 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30295-4 sha: doc_id: 332238 cord_uid: qw2ual51 nan individuals in us immigration and customs enforcement (ice) detention are at risk from serious consequences resulting from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection and inadequate access to appropriate medical care. this situation represents a moral and public health imperative for rapid action by the us department of homeland security (dhs) to mitigate the human toll of the pandemic. sars-cov-2 emerged in late 2019 in wuhan, china, causing coronavirus disease 2019 (covid-19), which has been rapidly spreading across geopolitical, social, and economic boundaries around the world. in the usa, a rapid increase in sars-cov-2 infections in every state of the country has resulted in a growing number of hospitalisations, admissions to intensive care units, and deaths in specific age groups and in many people with underlying medical conditions. 1 individuals who are incarcerated, including immigrants in ice detention, are among the most vulnerable to infection and complicated disease because of existing drivers of inequality. [2] [3] the incarceration of undocumented immigrants is a relatively new phenomenon in the usa. the annual average daily population in ice detention has risen more than seven times in the past 25 years to a peak of more than 50 000 individuals in 2019. 4 many individuals in ice detention have never been charged with a criminal offense. immigrants in ice detention around the country have expressed panic over conditions that put them at exceptionally high risk of an outbreak of covid-19 and proposed an immediate humanitarian response to mitigate the risk of infection. 5 rapid implementation of infection prevention and control measures in immigration detention is essential to the wider national public health response. although people who are incarcerated are confined, a high degree of interaction occurs between people in facilities and the community, including people being transported between facilities, releases and new intakes that generate a population turnover, and the comings and goings of staff, visitors, vendors, and contractors. inadequate implementation of infection prevention strategies will affect the spread of covid-19 in the community and burden an already stretched health-care system. because the transmission of sars-cov-2 is predominantly from person to person through droplets, a pillar of infection prevention is social distancing and disinfection, which is antithetical to closed detention settings. incarceration requires large groups of people to be held together in confined and often poorly ventilated spaces. many areas within the facility are communal, including housing, waiting rooms, eating areas, recreation spaces, and classrooms. disinfection and decontamination practices are also complicated by the ability of sars-cov-2 to survive for extended periods on materials that are highly prevalent in detention settings, such as metals and other non-porous surfaces. from a public health perspective, mitigation strategies in detention facilities should be complemented by routine screening and containment procedures. these entail screening all people who enter facilities, including individuals in detention, staff, visitors, and vendors, and quarantining those who screen positive for covid-19 exposure. people who screen positive for symptoms on intake or develop symptoms during detention must be medically isolated and receive appropriate medical care. ice facilities do not have the staffing capacity or facilities to screen, quarantine to monitor for symptoms, isolate infected individuals, or deliver medical management in a setting with high rate of infection. 6 clinical deterioration, often rapid with covid-19, will require the rapid transfer of individuals with covid-19 to local medical facilities for specialised care that might exceed the capacity of local health-care systems, particularly in the rural and semi-rural settings where many ice detention facilities are located. the combination of a captive population exposed to a highly infectious disease and substandard care has the potential to increase the incidence of infection and casefatality rates among detained individuals, put the public at greater risk, and consume substantial medical and financial resources. because of the existing barriers to adequate mitigation, containment, and provision of medical care in detention facilities, the policy response to this crisis must involve the release of individuals in ice detention and a halt of ice enforcement action in the community. these actions should include the immediate release on humanitarian parole of individuals at risk of severe disease and death due to covid-19 infection. an even more robust and effective response would be to release all individuals who do not represent a threat to public safety. this does not represent amnesty, but rather the use of existing structures within the dhs and the us department of justice to enforce immigration laws in the community setting. as physician advocates, we believe that prompt action in this brief and rapidly closing window represents not only the humanitarian and moral course, but also the best public health intervention to prevent unnecessary deaths. cdc covid-19 response team. severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states social inequalities and emerging infectious diseases prisoners co-infected with tuberculosis and hiv: a systematic review us immigration and customs enforcement fiscal year 2019 enforcement and removal operations report letter to us immigration and customs enforcement office of detention and removal office of inspector general. concerns about ice detainee treatment and care at four detention facilities we declare no competing interests. key: cord-011468-1ienwzy8 authors: durrheim, david n; gostin, laurence o; moodley, keymanthri title: when does a major outbreak become a public health emergency of international concern? date: 2020-05-19 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30401-1 sha: doc_id: 11468 cord_uid: 1ienwzy8 nan could the pandemic of the century have been averted? the process by which who decides whether to declare a public health emergency of international concern (pheic) under the international health regulations has drawn criticism. reports have condemned the 4-month delay by who after the international spread of ebola in west africa before declaring a pheic. 1 the democratic republic of the congo, now experiencing the second largest ebola outbreak in recorded history, notified who of the outbreak on aug 1, 2018, but who required four emergency committee meetings, including on oct 17, 2018 (216 confirmed cases, 139 deaths, and 64% case fatality ratio), and april 12 and june 14, 2019 (four confirmed cases in uganda). justifying their response, the emergency committee said that "the cluster of cases in uganda is not unexpected". 2 a pheic was finally declared at the fourth emergency committee meeting on july 17, 2019 (2501 cases and 1668 deaths), almost a year after initial notification. the international health regulations 3 do not require actual international spread, only a high potential for that spread, and thus the criteria for a pheic had already been met by the second emergency committee meeting. 4 notably, the pheic declaration coincided with increased resourcing and international focus, leading to a major reduction in ebola cases. global health scholars have criticised the emergency committee process as lacking transparency, using "irrelevant considerations, undue influence and political interference", 5 and delaying declaration when international health regulations criteria have been met. the coronavirus disease 2019 (covid-19) outbreak originating in china and reported to who on dec 31, 2019, suggests that little has changed. the pheic declaration for covid-19 occurred well after most public health experts had concluded that this outbreak posed a major international threat. at the first emergency committee meeting on jan 22, 2020 (309 cases and six deaths reported in mainland china; five confirmed cases in four countries or territories), the emergency committee said it did not have key facts from china. it extended the meeting to the next day, when cases had risen to 571, with 17 deaths and ten cases in seven other countries or territories. yet, the emergency committee could not achieve consensus, and the director-general concluded that the outbreak was "an emergency in china, but it had not yet become a global health emergency". 6 again, the process appeared "more political than technical", as a lancet editorial described ebola in the democratic republic of congo, adding that "the committee seems to have favoured local protectiveness over global galvanising". 7 by the time the emergency committee declared a pheic for covid-19 on jan 30, 2020, 7736 cases and 179 deaths had been confirmed in mainland china, with 107 cases confirmed in 21 other countries. delays in declaring a pheic could have serious detrimental consequences, lulling governments and donors into a false sense of security, because they could reason that if who does not consider the situation an international emergency, then it does not require a surge response. the legal definition of a pheic is clear, as "an extraordinary event that may constitute a public health risk to other countries through international spread of disease and may require an international coordinated response." 3 the purpose of the declaration is to focus international attention on acute public health risks that "require coordinated mobilisation of extraordinary resources by the inter national community" for prevention and response. 3 the pheic process requires urgent reform. first, the allor-nothing nature of the assessment generates confusion. we therefore propose a multilevel pheic process with each level defined by objective epidemiological criteria and paired with specific readiness actions. level 1 pheic alert should indicate a high risk outbreak in a single country, with the potential for international spread requiring concerted public health efforts to contain and manage it locally. level 2 pheic should imply that multiple countries have had importations and that limited spread has occurred in those countries. level 3 pheic would indicate large clusters in multiple countries, with evidence of ongoing local transmission. this tiering would provide less ambiguous risk signalling, while also encouraging second, who should convene an expert consensus meeting to establish objective, evidence-based epidemiological and containment criteria to transparently guide its decision making processes. the draft algorithm under annex 2 of the international health regulations 8 (appendix) already includes critical elements, but there are also subjective considerations, such as restraints on international travel and trade. the algorithm contains perverse relative weightings, treating the five categories as equivalent. the clear purpose of a pheic declaration is to catalyse timely evidence-based action, to spur increased international funding and support, and to limit the public health and societal impacts of emerging and reemerging disease risks. in the aftermath of the covid-19 pandemic, international health regulation reform must be an ethical imperative for more rapid and effective responses to novel infectious diseases. we declare no competing interests. usa (log); and centre for medical ethics and law toward a common secure future: four global commissions in the wake of ebola statement-on-the-meeting-of-theinternational-health-regulations-(2005)-emergency-committee-forebola-virus-disease-in-the-democratic-republic-of-the-congo ebola in north kivu, dr congo-is it an undeclared public health emergency of international concern (pheic)? transparency in ihr emergency committee decision making: the case for reform statement-on-the-meeting-of-theinternational-health-regulations-(2005)-emergency-committeeregarding-the-outbreak the lancet. the politics of pheic annex 2 of the international health regulations key: cord-337491-ztco6guw authors: kucharski, adam j; nilles, eric j title: using serological data to understand unobserved sars-cov-2 risk in health-care settings date: 2020-08-03 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30579-x sha: doc_id: 337491 cord_uid: ztco6guw nan during past outbreaks of severe acute respiratory syndrome and middle east respiratory syndrome, many infections occurred within health-care settings. 1 since the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), growing evidence of nosocomial transmission has been observed, but tracking such outbreaks is challenging because a substantial proportion of infected individuals might exhibit mild or no symptoms. 2 in the lancet infectious diseases, kasper iversen and colleagues 3 report results from a large seroprevalence survey of almost 30 000 hospital employees in denmark. 3 the authors found that 1163 (4·04%) of 28 792 staff were seropositive overall, which was slightly higher than the 3·04% (142 of 4672) prevalence observed among local blood donors (risk ratio [rr] 1·33 [95% ci 1·12-1·58]). seroprevalence was also higher among frontline health-care workers than among staff in other hospital roles (1·38 [1·22-1·56]; p<0·001). staff working in dedicated covid-19 wards showed substantially higher rates of seropositivity (1·65 [1·34-2·03]; p<0·001) than other frontline health-care workers working in hospitals, reflecting increased risk for this group, a pattern that has also been reported in neighbouring sweden. 4 although iversen and colleagues used a point-of-care lateral flow immunoassay, which is generally considered less conclusive than enzyme-linked immunosorbent assays or similar laboratory-based methods, 5 the authors did a comprehensive pre-study test assessment and estimated a sensitivity of 82·5-90·6% and specificity of 99·2-99·5%. high specificity is essential to minimise high rates of false positives when used in low-prevalence populations, such as the one studied. the results highlight the risk that sars-cov-2 can pose to health-care workers, particularly those in regular contact with patients with covid-19, and the importance of understanding possible routes of exposure in hospitals. given the potential for nosocomial transmission to amplify outbreaks, particularly when incidence is otherwise low in the community, 6 serological surveillance is a crucial tool. serological surveillance can help investigate the dynamics of infections that often go unobserved in the early stages of epidemics or when a large fraction of cases is asymptomatic or with mild symptoms. among the danish hospital staff who were seropositive, one in five reported no covid-19 compatible symptoms at all in the 6 weeks before sample collection. the study also shows the challenge of identifying a specific and sensitive clinical case definition for covid-19, with around half of seronegative participants reporting at least one covid-19-like symptom. this finding suggests that symptoms reported by seropositive individuals were not necessarily all linked to sars-cov-2 infection. the analysis found that loss of taste or smell-a symptom that was omitted from many early clinical definitions 7 -was strongly associated with seropositivity (rr 11·38 [95% ci 10·22-12·68]). however, the prevalence of asymptomatic sars-cov-2 infections and covid-19-like symptoms among seronegative staff illustrates the limitations of relying on symptom-based surveillance alone. this finding also shows the importance of developing screening tests that are easily done and sufficiently rapid to enable frequent and accurate detection of acute infection among at-risk staff. as well as indicating the degree of exposure to sars-cov-2, seroprevalence might provide an insight into the possible extent of antibody-mediated immunity. important questions remain about the precise role of humoral and cellular immunity following sars-cov-2 exposure, and whether seropositivity or antibody titres can be considered a proxy measure of protective immunity. 8 if the seroprevalence estimated in the danish hospital staff does indeed reflect the extent of immunity that would prevent infection, this would be substantially below the level required to generate localised herd immunity that could stop future nosocomial transmission. although seroprevalence studies provide a useful indication of existing antibody levels within a population, we still need to know more about the medium-term and long-term persistence of such responses, particularly among individuals who have had mild or asymptomatic infections. if antibody kinetics against sars-cov-2 reflect those against seasonal coronaviruses, as appears increasingly likely, 9 we would anticipate rapid antibody decay and seroreversion (from seropositive to seronegative) within several months to a year. 10 characterising antibody dynamics and how these vary within and between populations will be crucial for the interpretation of ongoing serological studies and might provide insight into population-level protection and prospects for future vaccine-induced immunity. faced with the possibility of second epidemic waves, large-scale studies of serological dynamics in at-risk populations, ideally capturing longitudinal trends, will be essential to inform our knowledge of future sars-cov-2 transmission dynamics and accompanying covid-19 risks, and how these risks can be reduced. we declare no competing interests. transmission characteristics of mers and sars in the healthcare setting: a comparative study screening of healthcare workers for sars-cov-2 highlights the role of asymptomatic carriage in covid-19 transmission risk of covid-19 in health-care workers in denmark: an observational cohort study sars-cov-2 exposure, symptoms and seroprevalence in health care workers antibody testing for covid-19: a report from the national covid scientific advisory panel report into a nosocomial outbreak of coronavirus disease 2019 (covid-19) at netcare st. augustine's hospital real-time tracking of self-reported symptoms to predict potential covid-19 targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals clinical and immunological assessment of asymptomatic sars-cov-2 infections coronavirus protective immunity is short-lasting key: cord-329200-o5hxpl8f authors: houlihan, catherine f; beale, rupert title: the complexities of sars-cov-2 serology date: 2020-09-23 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30699-x sha: doc_id: 329200 cord_uid: o5hxpl8f nan diagnosing previous infection with respiratory viruses is challenging. our understanding of individual and population-level immunity to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) remains incomplete and developing reliable serological assays to detect previous infection has been an intense focus of the global scientific effort. for public health planning we need scalable assays validated against large banks of samples from individuals who had proven seasonal (non-severe acute respiratory syndrome) coronaviruses and those who had well characterised symptomatic and asymptomatic confirmed sars-cov-2 infection. false-positive results, due to crossreactivity with seasonal coronaviruses, are important to avoid, particularly if seropositive-individuals consider themselves immune. in the lancet infectious diseases, the national sars-cov-2 serology assay evaluation group 1 provide the first large comparative investigation of the performance of four widely available commercial assays and a single in-house assay. antibody responses to sars-cov-2 are predominantly directed at the spike glycoprotein, which the virus requires for entry, and the nucleocapsid protein, which binds the viral rna genome. the sars-cov-2 igg assay (abbott, chicago, il, usa) and elecsys anti-sars-cov-2 assay (roche, basel, switzerland) assays detect antibody to the nucleoprotein, whereas the liaison sars-cov-2 s1/s2 igg assay (diasorin, saluggia, italy), and sars-cov-2 total assay (siemens, munich, germany) detect antibodies to the spike glycoprotein. the abbott and diasorin assays detect igg only, whereas roche and siemens detect total antibody. the diverse approaches taken by the four commercial assays highlight the challenge of choice posed to laboratories: all manufacturers report similarly high sensitivity and specificity. the authors compared these four assays and a novel 384-well elisa detecting total igg to a trimeric spike protein and used all five assays on 976 pre-pandemic samples presumed to be negative, collected between 2014 and 2016, and 536 serum samples from patients with laboratory-confirmed covid-19 from research studies in oxford, uk, or plasma donors. the authors report that all assays had a high sensitivity (92·7-99·1%) and specificity (98·7-99·9%). the most sensitive test assessed was the in-house elisa. the abbott, roche, and siemens assays were the most specific. the benefit of the huge sample bank available to these authors was the clearly documented time since pcr positivity, which allowed them to optimise the manufacturers' cut offs and improve sensitivity. only three cases did not give rise to any detectable antibody responses in all five of the assays, possibly because of a genuine lack of response in infected individuals, or a false-positive quantitative pcr result. a limitation of this work is the small number of pauci-symptomatic and asymptomatic cases analysed. antibody responses in these individuals are likely to be lower, and therefore the sensitivity of all assays might be somewhat less than that reported. also, data on sex, age, and immunocompromise status were incomplete, meaning that the results could be limited in their application to specific patient groups. this limitation could be especially important in children, who are more likely than adults are to have had a recent infection with a seasonal coronavirus. the expectation is that the best predictor of antibodymediated protection will come from neutralisation assays, in which the ability of patient serum to prevent live virus infecting cell cultures is measured. these assays are impractical to deploy at scale. the presence of antibodies against the spike protein of sars-cov-2 correlates well with neutralisation. 2,3 the diasorin, siemens, and in-house assays measured these potentially protective antibodies, with the inhouse elisa using trimerised spike protein, which shows a high correlation with neutralisation. 4, 5, 6 further work is required to investigate what titre of neutralising antibodies correlates with protection, how long neutralisation activity persists, and which assay best predicts that. identifying an appropriate assay will be crucial for assessing vaccine responses, and for assessing potential risk of reinfection, which has been shown with seasonal coronaviruses, 7 but not so far for sars-cov-2. consistent with this future possibility, the neutralisation potency of serum declines in the months post infection. 8, 9 as our understanding of immunity and the correlates of protection (both cellular and humoral) increases and the range of immunoassays multiplies, we will probably flickr -niaid use different assays to answer specific questions. for example, most vaccine candidates elicit responses to spike rather than nucleocapsid protein. measuring antibodies to spike will therefore indicate whether there has been a good response, whereas measuring antibodies to nucleocapsid would help identify whether the individual had nonetheless become infected. measuring the different antibodies might also have prognostic value; a report showed that a predominant humoral response to nucleoprotein is associated with poor outcome in patients admitted to hospital, compared with that of spike. 10 further investigation is required and the possibility of a one-size-fits-all immunological assay looks less and less likely. we declare no competing interests. the national sars-cov-2 serology assay evaluation group. performance characteristics of five immunoassays for sars-cov-2: a head-to-head benchmark comparison. lancet infect dis 2020; published online sept 23. https://doi.org/10.1016/s1473-3099(20)30634-4. severe acute respiratory syndrome coronavirus 2−specific antibody responses in coronavirus disease patients safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial 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 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 infection induces robust, neutralizing antibody responses that are stable for at least three months human coronavirus nl63 molecular epidemiology and evolutionary patterns in rural coastal kenya longitudinal analysis of clinical serology assay performance and neutralising antibody levels in covid19 convalescents clinical and immunological assessment of asymptomatic sars-cov-2 infections distinct early serological signatures track with sars-cov-2 survival key: cord-009476-4emc4o6n authors: madani, tariq a title: case definition and management of patients with mers coronavirus in saudi arabia date: 2014-09-22 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70918-1 sha: doc_id: 9476 cord_uid: 4emc4o6n nan the threat to global health security from emerging and re-merging respiratory tract infections will be ever present because of the genetic adaptability of microbes, and their ability to resist clinical interventions and public health measures aimed at their elimination. although much has been learned from previous outbreaks, present surveillance systems have their inherent weaknesses, and recent experiences with mers-cov 14 show that pandemic preparedness still faces major political and scientifi c challenges. 15 an important priority for control of infectious disease is to ensure that scientifi c and technological advances in molecular diagnostics and bioinformatics are well integrated into public health. more eff ective and wider partnerships based on equity and best ethical practice, across governments, health care, academia, industry, and with the public, are essential to eff ectively galvanise economic, political and scientifi c measures required to develop core capacities, including legislation, national focal points, and pandemic planning to reduce risk of global spread and reduce the burden of respiratory tract infectious diseases. an urgent need exists to establish trusting and eff ective meaningful collaborations between countries to tackle new emerging microbial threats. this will facilitate early and rapid detection of potential pandemic infectious diseases through public health actions within the framework of the international health regulations. 16 outbreak and prevent human-to-human and animalto-human transmission; an appropriate management algorithm, including best-practice guidelines for accurate diagnosis, infection control, intensive care, emergency medicine, and treatment; prioritise research related to the mers-cov outbreak such as case-control and cohort studies, seroprevalence studies, and clinical trials; and to eff ectively monitor outbreak control activities. a continously operating command and control centre was established in the minister's office. in addition to the advisory council, nine further platforms were established: interministerial to coordinate efforts between the ministry of health (moh) and other concerned ministries; capacitybuilding to recruit and mobilise qualified staff to work in hospitals treating patients with mers-cov, increase the number of beds in intensive care units, and provide state-of-the-art machines such as extracorporeal membrane oxygenation to treat patients with respiratory failure refractory to conventional ventilation; public relations to communicate relevant information to the public, health-care workers, and local and international media; clinical operation to coordinate management of patients and transfers between hospitals; public health to collect data related to patients and their contacts; data analysis to enter and analyse data; epidemiological to provide consultations on data analysis and interpretation; laboratory to ensure fast and reliable diagnostic testing; and, infection control to oversee infection control practice and staff training activities. a mers referral hospital run by well trained staff was designated in riyadh, jeddah, and dammam to receive and manage all patients infected with mers-cov. the moh enforced strict infection prevention as new clinical information became available, a revision of the mers-cov case defi nition seemed appropriate. 2 the new case defi nition (appendix) was developed based on reported health-care-associated mers-cov pneumonia (added as category 2 in the new case defi nition) and non-respiratory characteristics of patients with confi rmed infection who fi rst presented with acute febrile dengue-like illness with body aches, leucopenia, and thrombocytopenia (added as category 3). the new case defi nition added a fourth category for contacts of people with mers-cov who present with not only lower respiratory tract but also isolated upper respiratory tract features. this defi nition classifi ed the status of patients into three categories of suspect, probable, or confi rmed infection. the new mers-cov case defi nition was revised and approved by the advisory council after seeking external cdc expert opinion. an algorithm for mers-cov case management was developed (fi gure). according to this algorithm, patients with confi rmed mers-cov who have no evidence of pneumonia or who recover from pneu monia but remain positive for mers-cov, can be isolated at home after careful assessment of the home situation and suitability for isolation by the treating physician, highly trained social workers, or other health-care professionals by telephone or home visits. the cdc has released recommendations on how to assess the home situation and the advice to be given to patients on home isolation and his or her caregivers and household members, 3 and also released guidance for the public, clinicians, and public-health authorities in the usa on control of the mers-cov infection. 4 ministry of health, riyadh, saudi arabia; and department of medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia tmadani@kau.edu.sa tim uyeki, and ray arthur for their critical review of the case defi nition and the mers-cov case management algorithm, and fadwa mushtaq for her secretarial assistance world health organization. who experts probe middle-eastern respiratory syndrome coronavirus (mers-cov world health organization. who revised interim case defi nition for reporting to who-middle east respiratory syndrome coronavirus (mers-cov): as of us centers for disease control and prevention. interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus first confi rmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities health-care-associated bacterial infections are an important cause of morbidity and mortality in critically ill patients, especially patients needing mechanical ventilation. decolonisation with topical antibiotics, such as selective digestive tract decontamination (sdd) or selective oropharyngeal decontamination (sod), eradicates potentially pathogenic bacteria, preventing ventilator-associated pneumonia and bacteraemia. in two dutch studies, 1,2 sdd and sod reduced mortality, intensive-care unit (icu) length of stay, icu-acquired bacteraemia, and carriage with antibiotic-resistant bacteria. accordingly, both measures were deemed cost eff ective. 3 only studies that assessed the unit-wide implementation of sdd or sod provide evidence of a key: cord-272274-p3oulo34 authors: teboh-ewungkem, miranda i; ngwa, gideon a title: covid-19 in malaria-endemic regions: potential consequences for malaria intervention coverage, morbidity, and mortality date: 2020-09-21 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30763-5 sha: doc_id: 272274 cord_uid: p3oulo34 nan covid-19 in malaria-endemic regions: potential consequences for malaria intervention coverage, morbidity, and mortality has had a massive impact on the populations and economies of the world. as of sept 9, 2020, the virus has infected more than 27 million people in 216 countries and territories worldwide, and the number of deaths is approaching a million. 1 although the spread of covid-19 to africa has been slow and its direct impact in africa is below the level seen in other continents, the potential effects of covid-19 on strategies and methods to combat other diseases such as malaria-which pose significant burdens on substantial proportions of the world and the african population, and especially children-are a cause for great concern. thus, understanding how the covid-19 pandemic could indirectly affect malaria control intervention strategies is urgent in all malariaendemic regions, and especially those that are part of who's "high burden to high impact" initiative. 2 since 2010, active malaria intervention control strategies have had a positive effect on lowering malaria burden and morbidity in africa and worldwide. these strategies include the use of long-lasting insecticidetreated nets (itns), indoor residual spraying, 4 and timely access to antimalarial drugs, including the use of intermittent preventive treatment aimed at killing forms of the malaria parasite in infected individuals, [5] [6] [7] in addition to the other mechanisms aimed at disrupting the transmission of malaria by exploiting the feeding behaviour and gonotrophic and reproductive cycles of mosquitoes. [8] [9] [10] [11] however, despite the progress of the past decade, evidence suggests that the rate of reduction in malaria mortality in the who african region has slowed since 2016, although total deaths due to malaria decreased overall. 3 in particular, from 2017 to 2018, among the ten african countries with the highest malaria burden, ghana and nigeria reported absolute increases in the number of malaria cases, while case numbers did not change substantially in seven countries and only uganda reported a decrease. 3 given that this deceleration could be compounded by the covid-19 pandemic, there is an urgent need to quantify and analyse the potential impact of the pandemic on malaria control and intervention strategies. in the lancet infectious diseases, daniel weiss and colleagues 12 quantified the indirect effects of covid-19 on the distribution of itns and on access to effective antimalarial drugs-two key components of malaria control in africa. using a range of counterfactual scenarios based on different levels of reduction in itn and antimalarial drug coverage, the authors estimated the additional morbidity and mortality due to malaria that might be seen in the year 2020 across malaria-endemic africa. current data were used to generate geospatial estimates of malaria infection prevalence, clinical case incidence and mortality, plasmodium falciparum parasite rates, itn coverage, and effective treatment availability. the anticipated malaria burden in the absence of covid-19 disruptions served as a baseline for comparison. on the basis of their estimates, weiss and colleagues concluded that covid-19-related disruptions to malaria control efforts in africa could lead to significant reversals of the progress made over the past two decades in reducing malaria morbidity and mortality, with a possibility of a near doubling in mortality due to malaria under the worst case scenario (combined reductions of 75% in effective antimalarial treatment and 75% in routine itn distribution, with no mass itn distribution campaigns). 12 weiss and colleagues' work is very relevant and timely, and serves as a call to action for policy makers not to ignore the control measures needed to fight the threat of malaria when considering strategies to combat the covid-19 pandemic. the authors suggest that an integrated approach, in which equivalent efforts to ensure malaria control procedures are sustained amid the response to the covid-19 pandemic, is essential to the goal of continuing the downward trend in malaria mortality and morbidity. this work is important and urgent as it quantifies the potential increases in malaria-attributable morbidity and mortality in 2020 that might occur under plausible reductions in itn and antimalarial drug coverage, serving to highlight the huge potential impact of covid-19-related disruptions in malaria intervention and control strategies on malaria burden. additionally, weiss and colleagues' work prompts us to consider, in general, other indirect effects of the covid-19 pandemic on malaria transmission dynamics. for example, given that fever is a common symptom in both diseases, it is important to educate both health-care providers and the population as a whole on the potential for misdiagnosis of malaria or covid-19, as well as on the potential for co-occurence of the two diseases. guidance on the need for and importance of testing for malaria and other diseases during the pandemic should be communicated to health-care providers and resources made available to faciliate this. furthermore, communication of these messages to communities is important to ensure that people with malaria are not scared to visit hospitals and community clinics in fear of misdiagnosis, which could limit their timely access to safe and legitimate antimalarials. in addition, it is worth asking whether there could be an increased risk of mosquito bites for individuals or families observing isolation or quarantine (whether either individually or in groups) that warrants them to stay in the same locality for extended periods, especially if done so in the absence of itns. if so, then perhaps our concern with regard to malaria transmission should also extend to other mosquito-transmitted diseases. covid-19) pandemic world malaria report the effect of malaria control on plasmodium falciparum in africa between 2000 and 2015 the effect of intermittent preventive treatment on anti-malarial drug resistance spread in areas with population movement intermittent preventive treatment (ipt) and the spread of drug resistant malaria intermittent preventive treatment (ipt): its role in averting disease-induced mortalities in children and in promoting the spread of antimalarial drug resistance on a three-stage structured model for the dynamics of malaria transmission with human treatment, adult vector demographics and one aquatic stage ivermectin as a novel complementary malaria control tool to reduce incidence and prevalence: a modelling study fighting malaria with ivermectin: a novel malaria control tool mosquito feeding behavior and how it influences residual malaria transmission across africa key: cord-265769-96p07nyz authors: perlman, stanley; zumla, alimuddin title: mers-cov in africa—an enigma with relevance to covid-19 date: 2020-10-06 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30578-8 sha: doc_id: 265769 cord_uid: 96p07nyz nan three novel zoonotic coronaviruses that have jumped the species barrier and caused lethal disease in humans have focused global public health attention in the past two decades: severe acute respiratory syndrome coronavirus (sars-cov) in 2003, middle east respi ratory syndrome coronavirus (mers-cov) in 2012, 1 and sars-cov-2 in late december 2019 2 -the cause of the ongoing covid-19 pandemic. 2 all these coronaviruses are featured in the who blueprint list of priority pathogens for research and development, because they threaten global health security, they have epidemic potential, and there are no effective treatments or vaccines. 3 in africa, only sars-cov-2 has caused significant outbreaks and disruption to health services. while african governments implement national mitigation and containment strategies 4 to control the spread of covid-19, mers-cov continues to circulate in the middle east causing intermittent outbreaks with up to 34% mortality. 1 mers-cov is highly prevalent in dromedary camels on the arabian peninsula and, they are the main sources of primary human mers-cov infections. 5 however, there have not been any reported clinical cases from africa of confirmed human mers, despite 70% of the world's dromedaries residing in africa (with high mers-cov infection rates) and frequent occupational and domestic contact between dromedaries and humans. 6 little serological evidence of human infection exists. 7, 8 this anomalous disparity between human mers-cov infections in the arabian peninsula and africa has not been easily explained and there has been a longstanding need for further studies of mers-cov at the animal-human interface. in the lancet infectious diseases, chris ka pub mok and colleagues 7 present data that indicate that human mers-cov infection is an occupational hazard in dromedary abattoir workers in nigeria. they focus our attention on the potential for mers-cov transmission and human infection and consequent threat to people living in africa and to public health services. their observational cohort study of people working at a slaughterhouse in kano, nigeria, identified mers-cov-specific cd4+ or cd8+ t cells in pbmcs obtained from 61 workers with close contact to dromedaries, but not in 20 other workers from the same slaughterhouse with no dromedary contact. these results agree with others showing that mers-cov virus-specific t-cell responses are more sensitive than serological tests for detecting past infection. 8 thus, zoonotic mers-cov infections of dromedary-exposed individuals are probably taking place in nigeria, and, by extrapolation, the incidence of human mers infections in all regions of africa with dromedaries has probably been underestimated. further studies are required to determine the extent of human mers-cov infections across africa. genetic and phenotypic differences in west african viruses might also be relevant to zoonotic outbreak potential. the great diversity of mers-cov lineages, and the large number of mers-cov infected dromedaries in africa, 5 might, with time, lead to mers-cov adapting and transmitting more efficiently, with epidemic potential. the findings of ka pub mok and colleagues 7 also have implications for ongoing efforts on covid-19 diagnostics development, testing, and tracing in africa and for vaccine development in general. 4 mild cases of mers and covid-19 induce antibody responses that are transient or wane rapidly. 8 specific diagnostic tests for mers-cov and sars-cov-2 infected individuals based on relevant antigenspecific cd4+ or cd8+ t-cell responses could allow for more sensitive detection of past or present infection than serological tests and identify additional human mers and covid-19 cases. t-cell responses appear to be more stable and useful for detection of previous infection or vaccine response in settings where virusspecific antibody titres are absent. it would also be interesting to know if dromedary-exposed workers with mers-cov-specific t-cell responses are protected from developing severe mers on rechallenge, and by extrapolation, whether t-cell responses are protective against severe covid-19 disease, even if virus-specific antibody is not detectable. to confirm these results, these studies should be repeated at other sites in africa and with larger numbers of samples. furthermore, no mers-specific t-cell responses were detected in abattoir workers with no dromedary exposure. by contrast, low numbers of sars-cov-2-specific t cells have been detected in uninfected individuals. 9 these apparent differences in pre-existing immunity to the two coronaviruses are unexpected and need to be flickr -jean & nathalie reconciled. the results also suggest that covid-19 and mers vaccines should be formulated to induce t-cell responses to maximise the likelihood of long-term protection. covid-19 and mers provide unique opportunities for african and middle eastern countries with relevant stakeholders to align and synergise ongoing covid-19 and mers surveillance, diagnostics, vaccine development, and evaluation, with basic science and translational research activities. 1,10 african and middle eastern countries must invest more in surveillance and urgent priority research to fill major knowledge gaps in the epidemiology, transmission, pathogenesis, and evolution of mers-cov and sars-cov-2, especially because they are co-circulating. the need for an effective one human-environmental-animal health multidisciplinary consortium across africa, middle east, and asia to tackle the ever-present threat of lethal coronaviruses and other emerging infections remains a global priority. 10 middle east respiratory syndrome another decade, another coronavirus prioritizing diseases for research and development in emergency contexts. world health organization from easing lockdowns to scaling-up community-based covid-19 screening, testing, and contact tracing in africa-shared approaches, innovations, and challenges to minimize morbidity and mortality enzootic patterns of middle east respiratory syndrome coronavirus in imported african and local arabian dromedary camels: a prospective genomic study mers-cov in camels but not camel handlers t-cell responses to mers coronavirus infection in people with occupational exposure to dromedary camels in nigeria: an observational cohort study recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals confronting the persisting threat of the middle east respiratory syndrome to global health security we have a special interest in coronaviruses and infectious diseases with epidemic potential. we declare no competing interests. key: cord-279765-sb1ifyfx authors: isakova-sivak, irina; rudenko, larisa title: a promising inactivated whole-virion sars-cov-2 vaccine date: 2020-10-15 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30832-x sha: doc_id: 279765 cord_uid: sb1ifyfx nan the ongoing covid-19 pandemic is the only outbreak to date in which the time from identification of a pathogen to the presentation of the first clinical trial results for a specific vaccine against the pathogen was less than 9 months. by september, 2020, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) vaccine landscape included 39 candidates being tested in clinical trials and more than 200 candidates in preclinical development. 1 it is generally accepted that only vaccines can halt the spread of the pandemic virus; thus, several groups have already published interim results of phase 1/2 clinical trials of sars-cov-2 vaccines generated on various vaccine platforms. 2-6 it is critical to accumulate as many clinical data on the safety and immunogenicity of sars-cov-2 vaccines as possible, because this infection is new to the human population and all possible short-term or long-term rare adverse events are difficult to predict. in this regard, the study by shengli xia and colleagues 7 is timely because it provides valuable evidence for the safety and immunogenicity of a β-propiolactone inactivated aluminium hydroxideadjuvanted whole-virion sars-cov-2 vaccine candidate developed by china national biotec group and the beijing institute of biological products (bbibp-corv), which was tested in randomised, double-blind, placebocontrolled phase 1/2 clinical trials in healthy individuals aged 18 years and older. importantly, this was the first study of an inactivated sars-cov-2 vaccine to include participants older than 60 years-the most vulnerable age group for this infection. in the phase 1 dose-escalating trial, the vaccine was given at a two-dose schedule at three different concentrations (2 μg, 4 μg, and 8 μg per dose) and was well tolerated in both age groups (18-59 years and ≥60 years). the older age group had lower rates of solicited adverse events than the younger adults: the overall rates of adverse events within 28 days after vaccination were 34 (47%) of 72 participants in the group aged 18-59 years, compared with 14 (19%) of 72 participants in the group aged 60 years and older. at the same time, in both age groups the vaccine was similarly immunogenic: the geometric mean anti-sars-cov-2 neutralising antibody titres measured by a 50% virus neutralisation assay 14 days after the booster dose were 88, 211, and 229 in the group aged 18-59 years and 81, 132, and 171 in the group aged 60 years and older for 2 μg, 4 μg, and 8 μg vaccine doses, respectively. moreover, the authors tested crossreactivity of the neutralising antibodies against several drifted sars-cov-2 isolates and showed the potential of their vaccine to protect against evolutionary diverged viruses, should they appear in circulation. the early phase 2 trial of the bbibp-corv vaccine in healthy adults aged 18-59 years assessed the effect of shortening the interval between two doses from 28 days to 14 days or 21 days on the vaccine's immunogenicity. the 4 μg dose of the vaccine was the most immunogenic when given at the 21-day interval (neutralising antibody titre 283), but its immunogenicity significantly decreased when the interval was reduced to 14 days (neutralising antibody titre 170), suggesting that the interval cannot be shorter than 3 weeks. the current study is the second to report the interim results of safety and immunogenicity of inactivated sars-cov-2 vaccine, with the first being the another β-propiolactone inactivated aluminium-adjuvanted whole-virion sars-cov-2 vaccine developed by wuhan institute of biological products. 6 both studies showed very similar levels of adverse events and neutralising antibody titres post vaccination, which indicates the reproducibility of clinical results of similar vaccine modes produced by different manufacturers. although the use of whole-virion vaccines ensures the presence of all potential immunogenic epitopes, a critical consideration for the safety and efficacy of the vaccines is the structural stability of the major antigenic determinants. as has been shown for other inactivated vaccines, improper inactivation processes can alter the antigenic properties of epitopes, resulting in the induction of non-neutralising antibodies, which can lead to the disease enhancement rather than protection. 8 with this in mind, encouraging results have been obtained when testing bbibp-corv in various animal models, where no disease enhancement on sars-cov-2 challenge was found. 9 however, we need to acknowledge that for this new infection, all possible animal models have not yet been worked out for simulating antibody-dependent disease enhancement in humans. therefore, long-term careful monitoring of quantitative and qualitative characteristics of the flickr -niaid induced sars-cov-2 antibodies after vaccination with inactivated sars-cov-2 vaccines is critically important. in addition, more studies are needed to establish whether the inactivated sars-cov-2 vaccines are capable of inducing and maintaining virus-specific t-cell responses, because cd4-positive t-cell help is important for optimal antibody responses, as well as for cytotoxic cd8-positive t-cell activation, which, in turn, are crucial for viral clearance if neutralising antibody-mediated protection is incomplete. 10 finally, because the correlates of protection afforded by inactivated sars-cov-2 vaccines are yet to be identified, the results of a phase 3 trial of bbibp-corv vaccine (currently underway in abu dhabi, united arab emirates; chictr2000034780), will provide information on whether this vaccine is safe and efficacious against sars-cov-2 infection, and for how long the protective effect is maintained. we declare no competing interests. an mrna vaccine against sars-cov-2 -preliminary report immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial safety and immunogenicity of an rad26 and rad5 vector-based heterologous prime-boost covid-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from russia effect of an inactivated vaccine against sars-cov-2 on safety and immunogenicity outcomes: interim analysis of 2 randomized clinical trials safety and immunogenicity of an inactivated sars-cov-2 vaccine, bbibp-corv: a randomised, double-blind, placebo-controlled, phase 1/2 trial inactivated viral vaccines development of an inactivated vaccine candidate, bbibp-corv, with potent protection against sars-cov-2 immunological considerations for covid-19 vaccine strategies key: cord-293540-45awgabp authors: drancourt, michel; gaydos, charlotte a; summersgill, james t; raoult, didier title: point-of-care testing for community-acquired pneumonia date: 2013-07-23 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70165-8 sha: doc_id: 293540 cord_uid: 45awgabp nan disease, a fi nding that, although not previously noted in infl uenza, has been reported for some viral diseases. 15 during the infl uenza pandemic of 1957, which was caused by the h2n2 subtype, individuals who had previously been infected with the h1n1 virus were less likely to be infected with h2n2 infl uenza than were those who had not. 16 the low incidence of severe h5n1 infections in elderly compared with younger people might be related to the presence of cross-protective antibodies to neuraminidase that are induced by seasonal infl uenza a h1n1 viruses. 17 overall, we postulate that antibodies to pre-1957 infl uenza a h1n1 viruses protected elderly people against pandemic a h1n1 virus infection, but consequently aff ected the development of heterosubtypic immunity and the disease outcome of h7n9 virus infections. the rate and timing of pandemic a h1n1 virus infections might have revealed the diff erences in h7n9 disease outcome, by contrast with historical infections with seasonal infl uenza a h3n2 viruses. this counterproductive imprinting of immunity might increase susceptibility to h7n9 virus infection. for that reason, older individuals should be given priority for vaccination if sustained person-to-person transmission of h7n9 viruses emerges. to obtain a better understanding of the role of imprinting of the adaptive immune system in h7n9 disease severity, prospective cohort studies in which cross-reactive t-cell immunity and virus-specifi c serum antibodies are tested for are imperative before possible wider spread of the virus. community-acquired pneumonia is a life-threatening disease. an estimated 8% of patients are admitted to intensive care and overall estimated 30 day mortality is 4-11%. 1 many bacteria and viruses cause community-acquired pneumonia (and can co-infect), and the causative pathogen (or pathogens) cannot be predicatively identifi ed by any clinical, radiological, or biological methods. 2 accordingly, antibiotic treatment is empirical, and guidelines recommend a combination of a β lactam with a macrolide. 2,3 identifi cation of the causative pathogen is usually delayed because clinical specimens are processed in a core laboratory, which is often in a diff erent centre from the patient and the doctor. culturing, if done, can also take several days. to avoid this delay, we introduced point-of-care (poc) microbiology laboratories near emergency departments where patients with community-acquired pneumonia are seen fi rst. 4 poc laboratories have a rapid turnaround time (<1·5 h) and deliver results as text messages directly to doctors' mobile phones. 4 poc testing could be implemented in medical centres in large cities, where specimen transport delays diagnosis, and in remote areas without full microbiological facilities. however, it should be noted that not all pathogens that can cause community-acquired pneumonia can be detected by poc tests, and molecular tests for staphylococus aureus have not been approved by the us food and drug administration (fda) or the european conformity (ce). in the emergency department, communityacquired-pneumonia poc kits, comprising a plastic bag containing prelabelled tubes for clinical samples, prelabelled laboratory forms, and an informed consent form, can be used to take nasal or pharyngeal swabs and urine and serum samples. these samples can be used for the entire panel of poc tests. poc diagnosis of community-acquired pneumonia relies on immunochromatographic assays for the rapid antigen detection of pathogen-specifi c antigen and realtime pcr tests detecting pathogen-specifi c genomic sequences. three rt-pcr-based poc tests have been approved by the fda-genexpert flu a/b (cepheid, sunnyvale, ca, usa), simplexa flu a/b&rsv (focus diagnostics, cypress, ca, usa), and filmarray rvp (biofi re, salt lake city, ut, usa). the appendix lists fdaapproved and ce-approved tests. procalcitonin, a useful biomarker in bacterial community-acquired pneumonia, 5 can be rapidly semiquantifi ed (in 30 min) in serum via immunochromato graphic testing. urinary rapid anti gen detection of legionella pneumophila serotype 1 and streptococcus pneumoniae can be done in 20 min. a metaanalysis 6 showed that urinary detection of l pneumophila had a pooled sensitivity of 0·74 and a specifi city of 0·99. urinary detection of s pneumoniae had a specifi city of 0·96 and positive predictive value of 0·88-0·96, allowing clinicians to use a narrower spectrum of antibiotics. 7 rapid antigen detection of the infl uenza virus in nasal or pharyngeal swabs (done in 30 min) has a low sensitivity of less than 0·60; sensitivity correlates with viral load. 8 however, specifi city is around 1·00, resulting in a positive predictive value of more than 0·98-high enough to make a positive result reliable for medical decisions. a negative result does not rule out the presumptive diagnosis of the physician, which should be checked by a second-line molecular test in a core laboratory. poc tests have to be operator-independent, and thus we do not recommend implementation of microscope-based tests, such as direct fl uorescent antibody tests, for which skilled microscopists are needed. however, a liquid direct fl uorescent antibody format (fast-point; diagnostic hybrids, athens, oh, usa) is available for poc testing (appendix). it has not been approved by the fda or the ce, but detects infl uenza virus, respiratory syncytial virus, adenovirus, coronavirus, and parainfl uenza virus in 25 min. the latest generation real-time pcr kits can complete molecular testing of swabs for bacterial and viral pathogens in 60 min. 9 new generation real-time pcr kits are typically multiplexed assays testing as many as 22 potential pathogens in parallel. 9 this new capacity of poc tests increases the number of diagnoses 11 and underscores that community-acquired pneumonia can result from co-infection with several pathogens, 10 which will challenge common notions about causation and management. for example, we propose that infl uenza and s pneumoniae have to be tested for in parallel irrespective of which one is the presumed causative pathogen. furthermore, detection by poc testing of an abnormal increase in group a streptococci might suggest co-infection with infl uenza. 11 procalcitonin concentrations greater than 0·5 ng/ml suggest bacterial co-infection in infl uenza-a major risk factor for death. poc results could aff ect the major decisions that doctors have to make in emergency departmentseg, whether to admit the patient (the usual action for community-acquired pneumonia), which antibiotic (such as balancing the advantages of β-lactam and macrolides 12 ) or antiviral to prescribe, isolation of contagious patients. although barriers to the implementation of poc tests in developing countries have been identifi ed, poc tests have been successfully implemented in remote areas-eg, rural senegal, where patients and health-care providers are in close contact. 13 furthermore, assessment of the numbers of poc tests done on a weekly basis could help to predict epidemics even before the causative organism is known. see online for appendix in may, 2012, on the attorney general's order, the police publicised photos and identity details of 18 women working illegally as sex workers in athens, greece, who had been arrested and found to be hiv positive. the rationale for this decision was presumably protection of the public-people who had had sexual intercourse with these sex workers might recognise them and seek medical advice and hiv testing, whereas people who pay for sex could avoid contact with these particular workers. five of the sex workers were prosecuted for intentional grievous harm-ie, transmission of hiv. the case drew media attention and sparked controversy for a brief period, but was forgotten amid pressing problems related to the economic crisis. on march 11, 2013, the fi ve women were acquitted, but this news did not make headlines. some important ethical issues were raised, which need to be adequately addressed both in greece and internationally. sex work in greece is legal and regulated; sex workers must register at their local prefecture and carry a medical card that is updated every 2 weeks. however, fewer than 1000 women are estimated to be legally employed as sex workers, whereas roughly 20 000 women, mostly of foreign origin, are thought to be engaged in illegal sex work. 1 greece is not unique in this regard-by and large, the sex industry evades control worldwide. notwithstanding the diversity of sex work settings and its many problematic aspects, such as human traffi cking, sex workers' health has gained the attention of policy makers, which is shown by a growing body of published guidelines and strategies. 2 is the aim of these strategies to protect sex workers or to protect public health via protection of sex workers? if the outcome is the same either way, does the intent make a diff erence? the greek authorities' handling of the 18 hivpositive sex workers shows an obvious diff erence between the two aims. researchers from other countries have already pointed out that public health offi cials are concerned less about the health of sex workers than about that of the sex workers' clients or the larger community. 3 the public health response to sex workers is often one sided. 4 in breach of key this online publication has been corrected. the corrected version fi rst appeared at thelancet. com/infection on feb 24, 2014 systematic review and metaanalysis: urinary antigen tests for legionellosis current and potential usefulness of pneumococcal urinary antigen detection in hospitalized patients with community-acquired pneumonia to guide antimicrobial therapy evaluation of fi ve rapid diagnostic kits for infl uenza a/b virus respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria group a streptococcal infections during the seasonal infl uenza outbreak 2010/11 in south east england a clinical solution to antimicrobial resistance in communityacquired pneumonia: narrowing the spectrum of antimicrobial therapy: comment on "current and potential usefulness of pneumococcal urinary antigen detection in hospitalized patients with community-acquired pneumonia to guide antimicrobial therapy point-of-care laboratory pathogens diagnosis in rural senegal key: cord-315130-8g2ih8zl authors: bax, adriaan; bax, christina e; stadnytskyi, valentyn; anfinrud, philip title: sars-cov-2 transmission via speech-generated respiratory droplets date: 2020-09-11 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30726-x sha: doc_id: 315130 cord_uid: 8g2ih8zl nan the relative humidity (rh) of 27% in which our measurements were conducted is 3 percentage points below the cdc guidelines for healthcare facilities (30%-60%) 4 and within the rh reported for common office buildings (20%-40%). 5 we assume that the authors were more concerned with the effect of rh on the rate of evaporation from respiratory droplets. again, while the laws of physics dictate that higher rh increases the time needed for evaporation of water, and thereby decreases the time needed for a droplet's fall to ground, to first order this time scales with (1-rh) and has no significant impact for any of the small droplets observed in our work. for example, droplets of 20 micron diameter will fully dry out at 50% rh in ca 30 ms, 6 many orders of magnitude faster than the time needed to fall to ground if they were to remain fully hydrated. "the duration of recorded speech was 25 s, but the results were artificially extrapolated to 1 min." as clearly described in the results section and figure 1 of our pnas article, 7 light scattering observations resulted from 25 seconds of recorded speech. as explicitly stated in the discussion, these results were used to estimate the number of potential virions emitted in one minute of speaking. there is nothing artificial about normalizing measured results to standard units. for example, we could have stated "an average of 17 virions per second over a period of 25 seconds" but such a number would suggest a precision higher than warranted, considering the wide variation in viral load and the fractional uncertainty in the diameter of the fully hydrated particles. instead, our reported "1000 per minute" provides an order of magnitude estimate. "in the 2.33 min preceding the beginning of the speech, we counted at least 12 instances where flying particles were observed" indeed, even when using a high-efficiency particulate air filter, infiltration of particles from outside sources contributed to our low background particle count rate. as indicated in figure 1a of our pnas article, 7 the decay of observed particles returns to this low background of 0.4 particles per frame (for the green curve; smallest particles). this was explicitly stated in the figure legend and was used when fitting the decay curves. "the authors used fluorescent green light to illuminate particles" nowhere did we state or suggest that fluorescent light was involved in any of our measurements. the laser used in our study generated coherent green light with a wavelength of 532 nm. laser light is not fluorescent. our measurements recorded green light scattered from particles passing through the light sheet. "no report of the loudness, measured in decibels, was found in either manuscript, although in the videos it seems that in some cases the study participant was shouting, so the claim of normal speech is dubious." evidently, abbas and pittet failed to read the legend for figure 1 in our pnas paper 7 which reports that the speaker used "a loud (maximum 85 dbb at a distance of 30 cm; average 59 dbb)." notably, the average loudness (59 db) is consistent with the cdc's definition of conversational volume (60 db). 8 the "shouting" we assume the authors refer to is when the speaker had the mouth covered by a washcloth, 9 which was used to illustrate that even when shouting, the number of detectable speech-generated droplets remains close to background levels when wearing a mouth cover. "the authors were mistaken when stating that high viral loads were found in asymptomatic patients while referring to the study by wolfel and colleagues. only one patient reported being asymptomatic in the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) outbreak in bavaria, germany, and that patient was not included in wolfel and colleagues' study, which included only hospitalised patients." evidently, abbas and pittet misread or misunderstood the results reported by wölfel et al. 10 wölfel et al. report the high viral loads of nine individuals. this patient population was a subset of the 16 covid-19 cases reported at the end of january 2020 in bavaria, germany by boehmer et al. 11 we thank abbas and pittet for referring us to boehmer et al., 11 whose report provides further evidence that presymptomatic spread occurs, with at least one patient, but probably five additional patients, being infected by a presymptomatic carrier. moreover, it is important to note that wölfel's patients were hospitalized not for the severity of symptoms, but preemptively on the basis of a positive covid-19 test. 11 in fact, 4/9 patients (44%) never had a cough (figure 2 a,d,f,i) 10 and wölfel et al. state that "the clinical courses in the patients under study-all of whom were young-to middle-aged professionals without notable underlying disease-were mild". 10 indeed, patient no. 16 did not exhibit symptoms during his hospitalization ( fig.2 and table 2 ). 10 "the presence of a fan at the bottom of the black box during the speech and for 10 s after the end of speech does not represent real-life conditions" in "real-life" conditions, exhaled air emerges with high humidity at a temperature near 37 ºc and rapidly mixes with room air, as demonstrated by schlieren images. 12 speaking into an enclosure creates thermal and humidity gradients that are nominally eliminated by operating the internal fan for a short period of time during and after speaking. using the fan to achieve a more homogeneous distribution of droplets prior to the actual decay time measurement does not influence the rate at which droplet nuclei subsequently disappear from view. the dispersion of speech droplets achieved by operating the fan for a short period of time is not unlike that produced by air currents generated when a person walks past a speaker. to suggest that use of the fan does not represent real-life conditions is as perplexing as it is irrelevant. we stated that transmission by asymptomatic covid-19 carriers is plausible. there is now an abundance of evidence for this observation in the scientific literature, 13 as acknowledged by who on july 8, 2020. 14 while the nuances between presymptomatic and asymptomatic transmission, or even oligosymptomatic transmission, had not yet been extensively discussed within the context of transmission when our work 7 was submitted and published, the key point remains that disease carriers with no symptoms, that is, subjects who are by definition unlikely to be coughing or sneezing, may be transmitting the virus via speaking. the number of speech droplets observed in our studies, and in particular for the fraction that is sufficiently small to remain airborne for many minutes, is far higher than was previously considered by the medical community. 15 multiple studies have shown that the oropharyngeal viral load in asymptomatic or presymptomatic patients is similar to that of symptomatic patients, 16, 17 with infectivity appearing to peak prior to onset of symptoms. 10, [18] [19] [20] "second, the authors assumed an average viral load in saliva of 7 × 10⁶ copies per ml on the basis of a prospective study wherein viral load was measured in sputum. thus, they assume that viral load in sputum is the same as in saliva." wolfel et al. report throat viral loads as high as 7× 10 8 copies per throat swab. considering a throat swab to contain ca 100-150 µl of oral fluid, the viral load was as high as ~7× 10 9 copies per ml. wolfel et al. also explicitly state "there were no discernible differences in viral loads or detection rates when comparing naso-and oropharyngeal swabs (fig. 1b) ". sputum consists of "lower respiratory tract secretions along with nasopharyngeal and oropharyngeal secretions, cellular debris, and microorganisms". 21 as mentioned above, speech droplets originate from oral fluid, both at the vocal folds (mostly sputum) and at the front of the oral cavity (mostly saliva). 22 oropharyngeal swabs represent an intermediate location. vowel sounds have been associated with high levels of small speech droplets 23 and are minimally modulated by other narrow passages before entering the atmosphere. these droplets therefore are generated at the same physical location as cough droplets. it is important to note that the viral load of a disease carrier, that is, whether it is high or low, equally impacts the probability of disease transmission through the airborne, large-droplet, and fomite pathways. the relative probability of transmission through these pathways is primarily defined by the likelihood that secreted virions reach the respiratory tract of a bystander, not by the viral load of the droplets. only if abbas and pittet wish to argue that the fecal route dominates disease transmission does the absolute viral load of respiratory fluid secretions become relevant. "the group also assume that every rna copy detected is a potentially infectious virion" nowhere did we state or assume that every rna copy detected is a potentially infectious virion. viability of excreted virions will modulate all pathways equally. indeed, as highlighted in figure 1f and 1g of wolfel et al., 10 the ability to culture virus from respiratory secretions rapidly decreases after onset of symptoms whereas viral loads decrease substantially slower, indicating that a progressively smaller fraction of virions is viable in culture as the infection progresses. however, the viability of virions modulates all transmission pathways equally. surfing the covid-19 scientific wave the size and the duration of air-carriage of respiratory droplets and dropletnuclei on air-borne infection -study ii droplets and droplet nuclei guidelines for environmental infection control in health-care facilities indoor air humidity, air quality, and health -an overview mechanisms of airborne infection via evaporating and sedimenting droplets produced by speaking the airborne lifetime of small speech droplets and their potential importance in sars-cov-2 transmission what noises cause hearing loss visualizing speech-generated oral fluid droplets with laser light scattering virological assessment of hospitalized patients with covid-2019 investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series a schlieren optical study of the human cough with and without wearing masks for aerosol infection control substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) who confirms there's 'emerging evidence' of airborne transmission of the role of particle size in aerosolised pathogen transmission: a review sars-cov-2 viral load in upper respiratory specimens of infected patients viral rna load in mildly symptomatic and asymptomatic children with covid-19 temporal dynamics in viral shedding and transmissibility of 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 physiology of airway mucus clearance modality of human expired aerosol size distributions size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities key: cord-282530-55lhjfm8 authors: carsana, luca; sonzogni, aurelio; nasr, ahmed; rossi, roberta simona; pellegrinelli, alessandro; zerbi, pietro; rech, roberto; colombo, riccardo; antinori, spinello; corbellino, mario; galli, massimo; catena, emanuele; tosoni, antonella; gianatti, andrea; nebuloni, manuela title: pulmonary post-mortem findings in a series of covid-19 cases from northern italy: a two-centre descriptive study date: 2020-06-08 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30434-5 sha: doc_id: 282530 cord_uid: 55lhjfm8 background: covid-19 is characterised by respiratory symptoms, which deteriorate into respiratory failure in a substantial proportion of cases, requiring intensive care in up to a third of patients admitted to hospital. analysis of the pathological features in the lung tissues of patients who have died with covid-19 could help us to understand the disease pathogenesis and clinical outcomes. methods: we systematically analysed lung tissue samples from 38 patients who died from covid-19 in two hospitals in northern italy between feb 29 and march 24, 2020. the most representative areas identified at macroscopic examination were selected, and tissue blocks (median seven, range five to nine) were taken from each lung and fixed in 10% buffered formalin for at least 48 h. tissues were assessed with use of haematoxylin and eosin staining, immunohistochemical staining for inflammatory infiltrate and cellular components (including staining with antibodies against cd68, cd3, cd45, cd61, ttf1, p40, and ki-67), and electron microscopy to identify virion localisation. findings: all cases showed features of the exudative and proliferative phases of diffuse alveolar damage, which included capillary congestion (in all cases), necrosis of pneumocytes (in all cases), hyaline membranes (in 33 cases), interstitial and intra-alveolar oedema (in 37 cases), type 2 pneumocyte hyperplasia (in all cases), squamous metaplasia with atypia (in 21 cases), and platelet–fibrin thrombi (in 33 cases). the inflammatory infiltrate, observed in all cases, was largely composed of macrophages in the alveolar lumina (in 24 cases) and lymphocytes in the interstitium (in 31 cases). electron microscopy revealed that viral particles were predominantly located in the pneumocytes. interpretation: the predominant pattern of lung lesions in patients with covid-19 patients is diffuse alveolar damage, as described in patients infected with severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses. hyaline membrane formation and pneumocyte atypical hyperplasia are frequent. importantly, the presence of platelet–fibrin thrombi in small arterial vessels is consistent with coagulopathy, which appears to be common in patients with covid-19 and should be one of the main targets of therapy. funding: none. since the earliest reports of cases in china in december, 2019, countries around the world have faced outbreaks of covid-19, the disease caused by the novel coronavirus severe acute respiratory syndrome corona virus 2 (sars-cov-2). italy was the first country in europe to document a large number of cases of covid-19, and lombardy in particular was severely affected, with a total of 17 713 people testing positive for sars-cov-2 and 1593 admitted to intensive care units between feb 20 and march 18, 2020. 1 luigi sacco hospital in milan and papa giovanni xxiii hospital in bergamo were the first hospitals in lombardy faced with managing the epidemic crisis. sars-cov-2 is the seventh member of the coronavirus family identified to cause disease in humans. coronaviruses are enveloped, positive-sense, single-stranded rna viruses. 2 two other members of this family, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), cause acute diffuse alveolar damage, pneumocyte hyperplasia, and interstitial pneumonia. [2] [3] [4] at the mild end of the clinical spectrum, covid-19 can be asymptomatic or manifest as mild upper respiratory disease with fever and cough, while severe cases can result in pneumonia, leading to acute respiratory distress syndrome (ards) in around 15% of hospitalised patients. 5 to the date of our last literature review (may 3, 2020), the only published reports of the pathological features of covid-19 lung involvement were from small case series or isolated cases, both from china and other countries. in those reports, the main histological fea tures comprised exudative diffuse alveolar damage with massive capillary congestion, often accompanied by microthrombi. [6] [7] [8] [9] we describe the lung histopathological findings from a large series of patients who died from covid-19 in northern italy, with the aim of reporting the main micro scopic pulmonary lesions associated with sars-cov-2 infection and severe respiratory failure. we histologically analysed lung tissue samples from 38 consecutive patients who died from covid-19 between feb 29 and march 24, 2020, in two referral centres for the management of the covid-19 outbreak in northern italy: luigi sacco hospital in milan (20 autopsies) and papa giovanni xxiii hospital in bergamo (18 autopsies). all patients had sars-cov-2 infection confirmed by real-time pcr analysis of throat swab samples taken at the time of hospital admission, and all had undergone molecular tests for common respiratory viruses and bacteria by the microbiology laboratories at the respective hospitals, with negative results. the ethics committees of luigi sacco hospital and papa giovanni xxiii hospital approved the use of personal and sensitive patient data for scientific research related to the disease in this study. the study followed the italian general rules used for scientific research purposes (regulation n.72-26/03/2012). autopsies were done in airborne infection isolation autopsy rooms, with personnel using personal protective equipment in accordance with the italian recommendations. 10 a team of pathologists (lc, as, an, rsr, ap, pz, ag, and mn) with extensive experience in the field of infectious diseases was involved in the autopsy procedures in both hospitals. a median of seven tissue blocks (range five to nine) were taken from each lung, selecting the most repre sentative areas identified at macroscopic examin ation. tissues were fixed in 10% buffered formalin for at least 48 h. paraffin-embedded sections of 3-µm thickness were stained with haematoxylin and eosin. to better characterise the inflammatory infiltrate, immunohistochem ical staining was done on the most representative areas of randomly selected cases, and included staining with antibodies against cd45 (clones 2b11 and pd7/26) for the identification of leucocytes, cd3 (clone 2gv6) for t lymphocytes, cd68 (clone kp-1) for mono cytes, and cd61 (clone 212) for megakaryocytes. tissues were also stained with antibodies against ttf1 (clone 8g7g2/1) and p40 (clone bc28) to identify squamous metaplasia of pneu mocytes, and ki-67 (mib-1) to determine the proliferative index of epithelial cells. all antibodies were ready-to-use monoclonal antibodies (ventana, roche diagnostics, basel, switzerland) and staining was done with the benchmark ultra ihc/ish system (roche, basel, switzerland) in accordance with the standard protocols supplied by the manufacturer. masson trichome staining was applied to char acterise collagen and fibrin deposition, epithelial cells, and fibrosis. additionally, in cases with histological suspicion for bacterial or fungal infections, periodic acid-schiff staining and grocott methenamine silver staining were done to confirm morphological findings. additional samples from ten consecutive cases were assessed for the presence of viral particles with use of electron microscopy: two samples from each lobe were selected, fixed in glutaraldehyde, and examined with use of a zeiss em-109 transmission electron microscope (zeiss, cologne, germany) and an olympus megaview g2 transmission electron microscopy camera with an integrated imaging platform (item; olympus, munich, germany) evidence before this study we searched pubmed up to may 3, 2020, using the search terms "autopsy" and "covid-19", with no language restrictions. the search revealed ten reported cases. lung morphological features of covid-19 in deceased patients were described in a few isolated case reports and in small case series, including some reports in chinese. histological descriptions of lung pathology were extremely concise, without any analytical report of morphological details, and lacking any highlighting of distinctive lesions compared with other forms of interstitial pneumonia. furthermore, as only quantitative and aggregate data were available, the possible clinical implications of the findings could not be determined. we histologically examined the lung tissues of 38 patients who died from covid-19-to our knowledge the largest post-mortem series so far reported-in two main hospitals providing care to patients with progressive breathing failure in a peak epidemic area in italy. we focused on the detailed analysis of histological features in these patients to elucidate any distinctive lesions associated with covid-19. to our knowledge, these data represent the first relevant provisional information regarding tissue damage specifically induced by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), besides the previously described diffuse alveolar damage, a feature that characterises interstitial pneumonia regardless of infectious agent. although our observations are provisional, they were obtained in a large cohort of patients and revealed that histopathological lung damage was characterised by expected features of diffuse alveolar damage, as well as diffuse thrombotic vascular involvement. this latter finding could be relevant in the management and targeted treatment of patients infected with sars-cov-2, with the potential to modify outcomes. histological evaluation was done independently by two pathologists from each hospital, from the same team involved with the autopsy, who were masked to patient characteristics, symptoms, and diagnoses. each pathologist analysed all the slides from both hospitals, and any discrepant results were jointly reviewed. the histological features of cellular and interstitial damage were described and graded on a semiquantitative scale on the basis of the percentage of tissue involved: absent (0%), rare (<5%), focal (5-25%), multifocal (26-50%), plurifocal (51-75%), or diffuse (>75%). 11 to quantify pulmonary intracapillary megakaryocytes, each tissue sample was scanned at low magnification to identify the hotspot area in which megakaryocytes were most easily recognisable, and cd61-positive cells in these areas were counted. a high value was defined as the presence of more than four cd61-positive cells per 25 high-power fields, which is considered to be the average number of intracapillary megakaryocytes in the lungs of people without diffuse alveolar damage. 12 this study had no funding source. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. patients were 33 men and five women, with a mean age of 69 years (sd 12; range 32-86). time spent in the intensive care unit or intermediate medical ward (subintensive care) ranged from 1 day to 23 days (mean 7 days [sd 6]). regarding past comorbidities, data were available for 31 patients: nine (29%) had diabetes, 18 (58%) had hypertension, four (13%) had past malignancies, 11 (35%) had cardiovascular disorders, and three (10%) had mild chronic obstructive pulmonary disorders. at the time of hospitalisation, all patients had clinical and radiological features of interstitial pneumonia. of the 26 patients with available d-dimer results, all had high values (>10 × the upper reference limit). mean time from symptom onset to death was 16 days (sd 6; range 5-31). upon macroscopic examination, the lungs of all patients were heavy, congested, and oedematous, with patchy involvement. in all cases, histological examination revealed features corresponding to the exudative and early or intermediate proliferative phases of diffuse alveolar damage (figure 1). these features were also focally associated with patterns of interstitial pneumonia (presence of inflammatory lymphomonocytic infiltrate along the slightly thickened interalveolar septa), organising pneumonia (alveolar loose plugs of fibroblastic tissue), and acute fibrinous organising pneumonia (some alveolar spaces containing granulocytes and fibrin, with the formation of balloon structures). features indicative of the fibrotic phase of diffuse alveolar damage, such as mural fibrosis and microcystic honeycombing, were observed to be focal, suggesting that none of the patients had progressed to the fibrotic phase, possibly because of the short duration of the disease. histological examination of the main bronchi and bronchiolar branches revealed mild, non-specific alterations: focal squa mous metaplasia and mild transmural lymphocytic and monocytic infiltrates. lumina often contained residual dense mucoid material, and granulo cytes were present in ten cases. four (11%) patients also had bacterial abscesses (one or two per lung, <5 mm in diameter), and one (3%) had a single fungal abscess (<7 mm in diameter). the abscesses were presumed to have formed after hospital admission. morphological findings and their corresponding semiquantitative grades are reported in the table. the predominant histological pattern of the exudative phase of diffuse alveolar damage, which was observed in all cases, included capillary congestion, interstitial and intraalveolar oedema, dilated alveolar ducts and collapsed platelet-fibrin thrombi in small arterial vessels (<1 mm diameter) were found in 33 (87%) cases. moreover, type 2 pneumocyte hyperplasia, showing various aspects of cellular atypia, was present to some extent in all patients; interstitial myofibroblastic reaction was observed in 25 (66%) cases and alveolar granulation tissue in 22 cases (58%), whereas septal collagen deposition was found in 15 (39%) cases and alveolar loose plugs of fibroblastic tissue in 11 (29%). mural fibrosis was sometimes observed (24 [63%] cases), as was microcystic honeycomb ing (15 [39%] cases, most often with a focal pattern of distribution). although no clinical history of pre-existing fibrosing interstitial lung diseases was found in patients with mural fibrosis and microcystic honeycombing, the presence of mild fibrotic alterations not in continuum with other pre-fibrotic fea tures (myofibroblastic proliferation or organising pneu monia), as expected in a context of disease progression, might suggest that fibrosing interstitial lung diseases were pre-existing in some cases. the inflammatory component was represented by cd45-positive and cd3-positive lymphocytes infiltrating the interstitial space; a large number of cd68-positive macro phages were also found, mainly localised in the alveolar lumina. immunohistochemistry with anti-cd61 antibodies identi fied an increased number of megakaryocytes in the lung capillaries in 33 (87%) cases. ultrastructural examination (figure 2) revealed particles suggestive of viral infection in nine (90%) of the ten cases analysed. the particles had a mean diameter of about 82 nm and projection of about 13 nm in length. the particles, assumed to be virions, were mainly localised along plasmalemmal membranes and within cytoplasmic vacuoles, as described for other corona viruses. 13 infected cells were type 1 and type 2 pneumocytes; however, in two cases, particles were observed in alveolar macrophages, albeit scarcely. no particles resembling viruses were observed in multinucleated cells. ultra structural analyses of alveolar capillaries frequently showed platelet and fibrin plugs within the lumina, but no particles resembling virions were detected in endothelial cells. to our knowledge, we report the largest series of covid-19 autopsies focusing on pulmonary lesions. in all samples, a diffuse pattern of exudative and early proliferative phases of diffuse alveolar damage was alveolar multinucleated giant cells 19 (50%) 6 (16%) 9 (24%) 1 (3%) 1 (3%) 2 (5%) tissues were categorised on the basis of the percentage of tissue involved, as follows: absent (0%), rare (<5%), focal (5-25%), multifocal (26-50%), plurifocal (51-75%), or diffuse (>75%). *absent was defined as fewer than four cells per 25 high-power fields. found, while the fibrotic phase was rarely observed. the distinctive histopathological findings were atypical pneumocytes (reactive atypia) and diffuse thrombosis of the peripheral small vessels. sars-cov, mers-cov, and sars-cov-2 infections show many similarities in clinical presentation. 2 sars-cov and mers-cov particles have been observed and described in pneumocytes, macrophages, and lung interstitial cells by electron microscopy, immunohistochemistry, and in situ hybridisation. 3, 4, 11, 14 in two autopsy studies of patients who died from sars (eight cases from singapore 11 and 20 cases from toronto), 3 the predominant pattern of lung injury was diffuse alveolar damage, including the exudative and proliferative phases. inflammatory infiltrate, oedema, pneumocyte hyperplasia, fibrinous exudate, and organisation were found. the toronto case series 3 included a comparison group of matched control patients who presented with respiratory symptoms and signs, died in the same period as those with sars, and were negative for sars-cov. com pared with non-sars lung lesions, sars lesions were distinguishable by a prominence of vascular endothelial injury and extensive acute lung injury in varying stages of exudation and organisation. 3 autopsy studies of patients who died from mers are limited. in the only complete report available, 4 the authors described lung injury characterised by exu dative diffuse alveolar damage, pneumocyte hyperplasia, and septal inflammatory infiltrate. despite the relevance of lung involvement in patients with covid-19, few data regarding lung pathology are available. in a case report of a patient who died from covid-19 in china, the histological findings in the lungs included desquamation of pneumocytes, diffuse alveolar damage, and oedema. 7 in addition, tian and colleagues 9 described the pulmonary pathology of early-phase covid-19 in two patients with lung carcinoma; both patients showed signs of the exudative phase of diffuse alveolar damage. in our study, fibrin thrombi in the small arterial vessels (<1 mm diameter) were observed in 87% of cases, around half of which had involvement of more than 25% of the lung tissue as well as high levels of d-dimers in the blood. these findings might explain the severe hypoxaemia that characterises ards in patients with covid-19. 1 vascular micro thrombi are often identified in areas of diffuse alveolar damage, and are associated with diffuse endothelial damage. these features, although not pathognomonic, were frequent in our series, widespread in the lung samples of the patients examined, and the predominant distinctive vascular component. our data support the hypothesis proposed in clinical studies 15 that covid-19 is complicated by coagulopathy and thrombosis. furthermore, d-dimer values of greater than 1 µg/ml have been associated with fatal outcomes in patients with covid-19. 16 for these reasons, the use of anticoagulants has been suggested to be potentially beneficial in patients with severe covid-19, owing also to their anti-inflammatory properties, although their efficacy and safety are being closely monitored. [17] [18] [19] in this study, we looked for virions in a subset of patients, and found particles resembling virions to be present, albeit rarely, in the cytoplasm of pneumocytes and macrophages. the morphology of the observed particles (about 80 nm in diameter, enveloped, with spike-like projections, and an electron-lucent core with peripheral electron-dense granules of the sectioned nucleocapsid) and their intravacuolar cytoplasmic location are consistent with the reported ultrastructural features of coronaviruses, including sars-cov-2. 20 despite the low number of cases assessed, these findings might suggest that the virus remains in the lung tissue for many days, even if in small quantities, and might trigger the mechanism that leads to lung damage and causes it to progress. further histological and molecular analyses and extension of the case series are ongoing to better define the cellular and tissue distribution of the virus as well as inflammatory responses in different organs. although this report represents the largest european study of lung autopsy findings from cases of covid-19 to date and is based on the analysis of a large number of lung samples, it is limited by the absence of controls. future pathological studies should include an extensive analysis of cases of ards associated with other viral pneumonias. baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov pulmonary pathology of severe acute respiratory syndrome in toronto histopathology of middle east respiratory syndrome coronavirus (mers-cov) infection-clinicopathological and ultrastructural study clinical features of patients infected with 2019 novel coronavirus in wuhan, china covid-19 autopsies pathological findings of covid-19 associated with acute respiratory distress syndrome histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer management of the corpse with suspect, probable or confirmed covid-19 respiratory infection-italian interim recommendations for personnel potentially exposed to material from corpses, including body fluids, in morgue structures and during autopsy practice lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore megakaryocytes and platelet homeostasis in diffuse alveolar damage the intracellular sites of early replication and budding of sars-coronavirus immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan incidence of thrombotic complications in critically ill icu patients with covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy anticoagulant therapy in acute respiratory distress syndrome thromboembolic risk and anticoagulant therapy in covid-19 patients: emerging evidence and call for action sarscoronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology we declare no competing interests. key: cord-267476-j59tm40d authors: yong, sarah ee fang; anderson, danielle elizabeth; wei, wycliffe e; pang, junxiong; chia, wan ni; tan, chee wah; teoh, yee leong; rajendram, priyanka; toh, matthias paul han sim; poh, cuiqin; koh, valerie t j; lum, joshua; suhaimi, nur-afidah md; chia, po ying; chen, mark i-cheng; vasoo, shawn; ong, benjamin; leo, yee sin; wang, linfa; lee, vernon j m title: connecting clusters of covid-19: an epidemiological and serological investigation date: 2020-04-21 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30273-5 sha: doc_id: 267476 cord_uid: j59tm40d background: elucidation of the chain of disease transmission and identification of the source of coronavirus disease 2019 (covid-19) infections are crucial for effective disease containment. we describe an epidemiological investigation that, with use of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) serological assays, established links between three clusters of covid-19. methods: in singapore, active case-finding and contact tracing were undertaken for all covid-19 cases. diagnosis for acute disease was confirmed with rt-pcr testing. when epidemiological information suggested that people might have been nodes of disease transmission but had recovered from illness, sars-cov-2 igg serology testing was used to establish past infection. findings: three clusters of covid-19, comprising 28 locally transmitted cases, were identified in singapore; these clusters were from two churches (church a and church b) and a family gathering. the clusters in church a and church b were linked by an individual from church a (a2), who transmitted sars-cov-2 infection to the primary case from church b (f1) at a family gathering they both attended on jan 25, 2020. all cases were confirmed by rt-pcr testing because they had active disease, except for a2, who at the time of testing had recovered from their illness and tested negative. this individual was eventually diagnosed with past infection by serological testing. elisa assays showed an optical density of more than 1·4 for sars-cov-2 nucleoprotein and receptor binding domain antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320. interpretation: development and application of a serological assay has helped to establish connections between covid-19 clusters in singapore. serological testing can have a crucial role in identifying convalescent cases or people with milder disease who might have been missed by other surveillance methods. funding: national research foundation (singapore), national natural science foundation (china), and national medical research council (singapore). as of april 15, 2020, more than 1·9 million cases of coronavirus disease 2019 , and more than 120 000 deaths from the disease, have been recorded worldwide. 1 many initial cases reported outside of china were imported or were linked to travellers from china. 2, 3 however, as community transmission has become widespread, the source of cases of covid-19 in several countries has not been established. in singapore, a globally connected city-state in southeast asia, health officials have attempted to contain the spread of covid-19 through intensive epidemiological investigations coupled with isolation of cases and quarantine of close contacts. however, establishing the source of infection to ascertain the possible extent of spread can be difficult, because scant epidemiological data might be available. even when possible nodes of transmission are retrospectively identified through epidemiological investigations, nucleic acid-based tests would not be diagnostically useful if these infected individuals have recovered and no longer shed the virus. hence, serological tests are needed to identify convalescent cases and aid investigations and containment efforts. we present findings of investigations from jan 29 to feb 24, 2020, that linked two people with covid-19 from wuhan, china, to three clusters of covid-19 cases in singapore. serological testing had a crucial role in establishing a link between clusters, showing its use in identifying convalescent covid-19 cases and supporting epidemiological investigations. in singapore, several surveillance methods are used to identify people with covid-19. on jan 2, 2020, a suspect case-definition of covid-19 was circulated to all doctors in singapore; 4 doctors are legally required to notify the ministry of health of cases of covid-19. 5 from jan 31, 2020, singapore began testing all patients with pneumonia in hospital for severe acute respiratory syndrome coronavirus 2 (sars-cov-2); this testing was later expanded to include people with pneumonia in primary care. the diagnosis of covid-19 is confirmed either by a respiratory sample testing positive for sars-cov-2 using a laboratory-based rt-pcr 6 or by a serum sample testing positive for sars-cov-2 on serological analysis. 7 once individuals with covid-19 were identified, their activities from 14 days before symptom onset until they were isolated were mapped and their close contacts traced. contact tracing before symptom onset was done to identify the source of exposure that led to the case being infected, allowing for further active case-finding around the source. contact tracing after symptom onset until isolation was done to identify exposed individuals for quarantine to break the transmission chain. both these approaches were part of the containment strategy. a close contact was defined as anyone who had prolonged contact within 2 m of the case. all close contacts with active or recent symptoms were tested, whereas those who were asymptomatic and exposed while the case was symptomatic were quarantined. activity maps were reviewed and crosschecked to establish potential expo sures and identify possible epidemiological links between cases and clusters. all epidemiological investigations and outbreak contain ment measures were implemented under the infectious diseases act, 5 which allows use of data for analysis to control outbreaks. for laboratory confirmation of covid-19, we did rt-pcr testing for sars-cov-2, using previously published methods. 6 two serological platforms were developed for confirmation of specific antibody responses to sars-cov-2 in people with suspected infection or individuals with pcr-confirmed disease. a virus neutralisation test (vnt) was established at the duke-national university of singapore medical school absl3 facility using a sars-cov-2 virus isolate (betacov/singapore/2/2020; gisaid accession number epi_isl_407987) cultured from a patient in singapore; vnt was done using protocols previously published for severe acute respiratory syndrome coronavirus (sars-cov). 7 for elisa assays, we used recombinant nucleocapsid protein from sars-cov and sars-cov-2 expressed in mammalian cell culture using the pcdna3.1 vector (thermofisher scientific, carlsbad, ca, usa), according to previously published methods, 8 and a recombinant receptor binding domain (rbd) of the sars-cov-2 spike protein custom-produced by a commercial provider (genscript, piscataway, nj, usa). elisa wells were coated with 100 ng of the respective protein per well and serum samples were used at dilutions from 1/50 to 1/400, followed by horseradish peroxidaseconjugated goat anti-human igg (santa cruz, dallas, tx, usa) used at a dilution of 1/2000. the funders 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 data in the study and had final responsibility for the decision to submit for publication. as of april 6, 2020, singapore had recorded 1375 cases of covid-19, of which 554 were imported and 821 locally transmitted. three clusters were identified that involved two churches (church a and church b) and one family gathering. these clusters comprised 28 locally transmitted cases. the clusters were linked to two travellers (w1 and w2) from wuhan, china, who attended a church service at church a on jan 19, 2020 (figure 1). the clusters at evidence before this study we searched pubmed on march 3, 2020, for reports on serological testing in individuals with coronavirus disease 2019 (covid-19). we used the keywords ("covid-19", or "2019-ncov", or "sars-cov-2") and ("serology" or "serologic testing"). our search did not identify any reports of the epidemiological application of serological tests in covid-19. in one report, researchers described serological characteristics of covid-19, and in other publications, researchers have commented on the potential importance of covid-19 serological tests. in another study, epidemiological investigations were reported of the epidemic in singapore, but serological methods had not been used. in our epidemiological investigation, we used rt-pcr and serological testing to diagnose cases of covid-19 and establish links between clusters. rt-pcr testing alone is limited by its ability to detect convalescent cases of covid-19, because rt-pcr can only detect severe acute respiratory syndrome coronavirus 2 during the period of viral shedding, which is the acute phase of infection. serological testing can be useful in detecting previous infection in people with suspected infection who have recovered, assisting in epidemiological investigation and containment efforts. implications of all the available evidence covid-19 laboratory testing is focused on use of quantitative rt-pcr for diagnosis, and serological testing can be overlooked. we have highlighted the importance of serological testing for epidemiological investigation of covid-19 cases, and we urge further development of serological testing capabilities. church a and church b were linked by one individual from church a (a2), who probably transmitted the infection to the primary case of the church b cluster (f1) at a family gathering on jan 25, 2020. all cases were confirmed by rt-pcr testing, except for a2, who was diagnosed by serological testing. the clusters at church a and church b were detected in early february and mid-february, respectively. although w1 and w2 were diagnosed with covid-19 at the end of january, their possible link to church a was only discovered after the church a cluster was identified, through investigation and repeat interviews. 5 by that time, a2 had recovered from covid-19 and was not immediately linked to either cluster. family members who had been infected at the family gathering on jan 25, 2020, were first linked to f1 and were initially regarded as part of the cluster at church b. however, subsequent investigations into case histories indicated that the family cluster was a distinct cluster and that a2 was most probably the missing link between the two church clusters; this idea was substantiated when a2's serological results were confirmed to be positive. five locally transmitted cases of covid-19 (a1-a5) were linked to church a. these people attended a church service on jan 19, 2020, the same day w1 and w2 visited the church. although all five people had developed symptoms by feb 2, 2020 (figure 2), only a1, a4, and a5 were diagnosed (between feb 6 and feb 8, 2020), because they had been hospitalised for pneumonia and tested for sars-cov-2 as part of enhanced surveillance measures to test all patients admitted to hospital with pneumonia. a2 and a3 were not diagnosed when symptomatic in late january because their symptoms were mild and they did not meet the suspect case-definition at that time. a2 and a3 were tested only after mapping of activities and movements of other cases suggested that a2 could be the missing link between the clusters at church a and church b. an rt-pcr test of a nasopharyngeal specimen taken from a3 on feb 18, 2020, was positive, although this individual had clinically recovered from the illness, which persisted from jan 28 to feb 10, 2020. serological analysis of a serum sample obtained on the same day as the nasopharyngeal specimen was also positive. although a2 had two negative rt-pcr tests, the serological result was positive, indicating past infection. a2 and a3 attended a chinese new year family gathering on jan 25, 2020, at the home of f1. nine cases (a2, a3, and f1-f7) were linked to this family gathering. a2, whose symptoms started on jan 23, 2020, was unwell at this event and most probably was the primary date source of transmission. a3 developed symptoms later, on jan 28, 2020, and was, therefore, unlikely to be the source of infection at this gathering. 17 locally transmitted cases were linked to church b (b1-b16 and f1). thorough review of activity maps ascertained that f1, who had developed symptoms on jan 29, 2020, and continued to work at church b while ill, was the primary case of the church b cluster. in mid-february, epidemiological and clinical evidence strongly suggested that a2 was the missing link between the clusters at church a and church b through attendance at the family gathering on jan 25, 2020, while symptomatic. however, by the time this link was ascertained, more than 3 weeks had passed from symptom onset on jan 23, 2020, and symptoms had resolved completely a week previously, on feb 8-10, 2020. a2 had two negative rt-pcr test results from samples taken from the nasopharynx, with testing done 1 day apart on feb 18 and feb 19, 2020. the sample for serological testing was taken on feb 18, 2020, and a positive result was confirmed on feb 22, 2020. elisa results for a2 and a3 on feb 20, 2020 (figure 3a) showed a strong antibody response to the sars-cov-2 rbd protein, which was not seen in serum samples from sars-cov patients. the results were further confirmed by vnt on feb 22, 2020 ( figure 3b ). this investigation shows how sars-cov-2 serological analysis (elisa detecting igg and vnt detecting neutralising antibodies), in addition to use of traditional epidemiological methods, was important in establishing links among locally transmitted covid-19 cases and tracing the transmission chain to an imported source. detection of covid-19 can be difficult because of the nonspecific mild respiratory symptoms in many affected individuals and because some people might recover without being diagnosed. 9 although pcr tests offer a rapid diagnostic solution, they can only detect sars-cov-2 during the period of viral shedding, which is the acute phase of infection. the duration of viral shedding for covid-19 is not certain, 10 but sars-cov data indicate that 21 days after symptom onset, 53% of cases achieved viral clearance in nasopharyngeal aspirate samples. 11 as such, pcr testing alone is limited by its ability to detect convalescent cases. serological testing can be especially useful in detecting a previous suspected infection in people who have recovered. for seroconversion kinetics, past coronavirus studies indicate that all patients with middle east respiratory syndrome (mers) seroconverted 3 weeks after symptoms started, 12 and 93% of patients with severe acute respiratory syndrome (sars) seroconverted at an average of 20 days from symptom onset. 11 the first preliminary analysis of sars-cov-2 igm and igg indicated that the antibody response in covid-19 patients is similar to, if not earlier than, these times. 13 cross-reactivity of immunoglobulins to closely related viruses such as sars-cov is a potential issue, 14 but our rbd-based elisa showed sufficient differentiation power, a finding further supported by vnt results. igm serological testing might hold promise as a diagnostic method, although for sars and mers, limitations for its use have been noted. 12, 13 for mers, igm was not detected earlier than igg, and igm against prevalent human coronaviruses showed cross-reactivity. 12 preliminary data for sars-cov-2 igm are promising, 13 but more work is needed to assess the feasibility of igm serological analysis as a rapid diagnostic method to enhance covid-19 detection capabilities. for most people in the three clusters we report here, transmission of infection was accounted for by close contact with a symptomatic case. our findings suggest that covid-19 is largely transmitted by close contact, particularly when contact occurs over a prolonged period and in close congregation. two churches were the setting for covid-19 transmission in our report. a church cluster has also been reported in south korea. 15 churches host prolonged repeated activities, during which close contact occurs, thus providing the opportunity for disease spread through droplets or fomites. singing (a common practice in churches) can generate droplets in a similar quantity to coughing. 16, 17 repeated social interactions of church groups has also facilitated discovery of transmission, compared with other settings in which people might not know each other. the interactions are similar in nature to large family gatherings, which other cases in our report were linked to. other similar settings include school and workplaces, in which respiratory disease transmission is not uncommon and should be the focus for prepared ness, surveillance, and containment measures. risk of transmission could be reduced if symptomatic people do not attend events in which prolonged social interactions take place (eg, at the family gathering when a2 was unwell, and f1 who continued to work at church b while unwell). other risk reduction measures could include having smaller group activities and prevention of interactions across these groups. moreover, there may be common touchpoints within each setting that could result in contact transmission. to prevent transmission, people should practice increased personal hygiene and reduce physical contact to minimise indirect transmission risks. linking disease transmission to an imported source and contact tracing for each identified case has facilitated a high capture of cases in singapore. this successful linking of a large proportion of cases to imported sources provides encouraging evidence that the intense containment measures undertaken in singapore have been effective. the three clusters we report here occurred relatively early in the emergence of covid-19 in singapore, within 4 weeks of the first imported case. as the epidemic continues, it might be progressively difficult to establish linkages by relying on traditional epidemiological methods alone. challenges include difficulty in obtaining information from cases and contacts, which could be inaccurate because of recall and other biases. in our investigation, information for symptom onset of w1 and w2 was based on case history alone and could not be independently corroborated. 5 in such instances, determining the transmission chain would have to account for possible inconsistencies in case history and rely on triangulation with other methods. the development and adoption of additional laboratory techniques, such as serological tests and phylogenetic analysis by whole-genome sequencing, could help identify possible links between cases. serological testing is a key method in the response to the covid-19 epidemic. as shown in our study, it enabled detection of a convalescent case, which could be key in initial containment efforts to discover transmission links to support containment efforts. serological testing also detects people with mild or asymptomatic disease who have recovered, allowing for more accurate deter mination of the number of people probably infected in a cluster or the population. identifying people who were probably infected in household or school clusters could help ascertain attack rates by age, particularly among children who mostly manifest less severe disease. 18 calculating population-level attack rates is also important to estimate disease incidence and the case-fatality rate (cfr). thus far, the cfr for covid-19 has been based on pcrdiagnosed symptomatic cases. serological surveys would be important to estimate cfr more accurately and would better inform calibrated responses to covid-19. as the pandemic progresses, monitoring seroprevalence would enable countries to track transmission dynamics and population immunity levels and to inform disease control policies. such monitoring would necessitate the development of ready serological testing solutions that are cost-effective and the establishment of population-level surveillance prog rammes to obtain blood samples. development and application of serological assays has helped to unravel connections between three clusters of covid-19 in singapore, linking the disease to two travellers from china. serological assays should be considered to identify mild or subclinical infections in the community. vjml and ylt had the idea for the study and contributed to study design. cp, vtjk, jl, n-ams, and pyc contributed to the epidemiological investigation and data collection. vjml, ylt, sefy, pr, mphst, jp, and wew contributed to analysis and interpretation of epidemiological data. lw, dea, wnc, and cwt developed the serological methods and contributed to the analysis and interpretation. sefy, wew, and jp wrote the report, and vjml, mi-cc, sv, bo, ysl, lw, and dea contributed to critical revision. we declare no competing interests. (singapore) covid19 research fund (covid19rf-001). we thank our colleagues who contributed to the investigations, including the covid-19 epidemiology workgroup, contact tracing team, and hospital epidemiology teams; and viji vijayan, benson ng, and velraj sivalingam, (duke-national university of singapore absl3 facility) for logistics management and assistance. evidence of sars-cov-2 infection in returning travelers from wuhan, china transmission of 2019-ncov infection from an asymptomatic contact in germany circular no 04/2020: viral pneumonia of unknown cause in wuhan city, china. singapore: ministry of health infectious diseases act investigation of three clusters of covid-19 in singapore: implications for surveillance and response measures bats are natural reservoirs of sars-like coronaviruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china covid-19: a puzzle with many missing pieces clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes serological assays for emerging coronaviruses: challenges and pitfalls korea centers for disease control and prevention. the updates of covid-19 in korea singing and the dissemination of tuberculosis aerosol emission and superemission during human speech increase with voice loudness clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study the development of serological tests was funded in part by a joint grant under the national research foundation (singapore) and the national natural science foundation of china (nrf2016nrfnsfc002-013). the study was partly funded by the national medical research council key: cord-279557-hk77e3pp authors: drosten, christian; seilmaier, michael; corman, victor m; hartmann, wulf; scheible, gregor; sack, stefan; guggemos, wolfgang; kallies, rene; muth, doreen; junglen, sandra; müller, marcel a; haas, walter; guberina, hana; röhnisch, tim; schmid-wendtner, monika; aldabbagh, souhaib; dittmer, ulf; gold, hermann; graf, petra; bonin, frank; rambaut, andrew; wendtner, clemens-martin title: clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection date: 2013-06-17 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70154-3 sha: doc_id: 279557 cord_uid: hk77e3pp background: the middle east respiratory syndrome coronavirus (mers-cov) is an emerging virus involved in cases and case clusters of severe acute respiratory infection in the arabian peninsula, tunisia, morocco, france, italy, germany, and the uk. we provide a full description of a fatal case of mers-cov infection and associated phylogenetic analyses. methods: we report data for a patient who was admitted to the klinikum schwabing (munich, germany) for severe acute respiratory infection. we did diagnostic rt-pcr and indirect immunofluorescence. from time of diagnosis, respiratory, faecal, and urine samples were obtained for virus quantification. we constructed a maximum likelihood tree of the five available complete mers-cov genomes. findings: a 73-year-old man from abu dhabi, united arab emirates, was transferred to klinikum schwabing on march 19, 2013, on day 11 of illness. he had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment. the patient died on day 18, due to septic shock. mers-cov was detected in two samples of bronchoalveolar fluid. viral loads were highest in samples from the lower respiratory tract (up to 1·2 × 10(6) copies per ml). maximum virus concentration in urine samples was 2691 rna copies per ml on day 13; the virus was not present in the urine after renal failure on day 14. stool samples obtained on days 12 and 16 contained the virus, with up to 1031 rna copies per g (close to the lowest detection limit of the assay). one of two oronasal swabs obtained on day 16 were positive, but yielded little viral rna (5370 copies per ml). no virus was detected in blood. the full virus genome was combined with four other available full genome sequences in a maximum likelihood phylogeny, correlating branch lengths with dates of isolation. the time of the common ancestor was halfway through 2011. addition of novel genome data from an unlinked case treated 6 months previously in essen, germany, showed a clustering of viruses derived from qatar and the united arab emirates. interpretation: we have provided the first complete viral load profile in a case of mers-cov infection. mers-cov might have shedding patterns that are different from those of severe acute respiratory syndrome and so might need alternative diagnostic approaches. funding: european union; german centre for infection research; german research council; and german ministry for education and research. in june, 2012, a coronavirus belonging to a group of viruses that had previously only been detected in bats was cultured from respiratory secretions of a patient who had died from severe acute respiratory infection. 1 the same agent was retrospectively detected in clinical samples from a hospital outbreak of severe acute respiratory infection that occurred in jordan in april, 2012, marking the fi rst known occurrence of the virus in people. 2 the agent has been named middle east respiratory syndrome coronavirus (mers-cov). 3 as of june 10, 2013, 55 laboratory-confi rmed cases had been reported in jordan, saudi arabia, the uk, france, italy, germany, and tunisia. 3 31 individuals with laboratory-confi rmed infection had died. few virological data have become available for mers-cov cases, and there is no information about the viral genome sequence, which could identify important epidemiological characteristics. [4] [5] [6] here, we provide a full description of a fatal case of mers-cov infection imported to munich, germany, from abu dhabi, including a chronological profi le of virus concentrations in diverse body compartments. we fully sequenced the mers-cov genome, and therefore could do a chronologically calibrated phylogenetic analysis with all available mers-cov genome sequences. these data were complemented by novel sequence data from an unlinked case treated in germany in 2012. 7, 8 we report data for a patient who was admitted to the klinikum schwabing (munich, germany) in march, 2013. investigation was done as part of a public health intervention according to the german infection protection act. written consent for scientifi c assessment was obtained from the patient's spouse as part of the patient treatment contract. we did diagnostic rt-pcr and indirect immunofl uorescence, following who recom mendations. 7, 9 for serum neutralisation tests, we grew vero b4 cells to subconfl uence in 24-well plates. pre-incubation reactions contained 25 plaque-forming units of mers-cov (emc strain) in 100 μl of medium, mixed one-to-one with serum samples from the patient prediluted in medium. the starting dilution was a tenth. after 1 h incubation at 37°c, each well was infected for 1 h at 37°c with the total 200 μl pre-incubation reaction. supernatants were removed and overlaid with avicell resin as described by herzog and colleagues. 10 assays were terminated and stained after 3 days. we defi ned neutralisation titres as the serum dilution reducing the number of plaques in four parallel wells in summary by greater than 90%. antibodies were tested by immunofl uorescence assay. 7 all clinical materials stored in the ward and laboratories were gathered and submitted for virological diagnostic tests. from the time of laboratory diagnosis, respiratory, faecal, and urine samples were obtained. we designed two diff erent sets of primers generating overlapping amplicons (available on request). the fi rst set consisted of 70 amplicons, 386-800 bp in length, with all primers containing two strong watson-crick bps at their 3 ends, so as to bind the template with high affi nity. the second set consisted of 68 amplicons, 415-761 bp in length, with primers that had no more than two strong bps in their fi ve 3 terminal nucleotides and no strong pairings in the two 3 positions. this method of primer design can decrease sensitivity, but it prevents mispriming within the product, which can improve the success of amplifi cation. after rt-pcr, we sequenced all fragments on a roche 454 junior instrument (roche, penzberg, germany) and assembled in geneious (version 6.1.2). virus quantifi cation was done with standard calibration curves that were based on quantifi ed in-vitro transcribed rna for the upe target gene. 9 we constructed a maximum likelihood tree of the fi ve available complete mers-cov genomes with phyml 11 and the gtr+gamma model of molecular evolution; we assessed phylogenetic support with 1000 bootstrap replicates. we inferred a timescale by linear regression of genetic divergence from the root against time of collection of the samples. the root was placed such that the correlation coeffi cient was maximised. a phylogenetic tree based on all available mers-cov sequences was calculated with phyml 11 on a concatenated 4012 bp dataset with the hky substitution model. reduction of the dataset was determined by the small number of sequence fragments that could be retrieved from a stored clinical sample containing a small amount of the virus, derived from a patient treated in essen, germany. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and fi nal responsibility to submit for publication. on march 8 (day 0), the patient-a 73-year-old man from abu dhabi, united arab emirates-abruptly developed fl u-like symptoms, with fever and non-productive cough. he was admitted to mafraq hospital (abu dhabi) on day 2 (fi gure 1), and was diagnosed with pneumonia. he was intubated on day 9 because of progressive hypoxia and acute respiratory distress syndrome (fraction of inspired oxygen 60%; positive end-expiratory pressure 10 cm h 2 o). the patient had received intensive antimicrobial treatment with meropenem, levofl oxacin, vancomycin, caspofungin, aciclovir, and oseltamivir during his stay in an intensive care unit in abu dhabi, without major improvement in his pulmonary function. the patient was transferred to klinikum schwabing (munich, germany) on march 19, 2013 (fi gure 1). the patient had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment in the previous few years, such as high-dose chemotherapy with autologous stem-cell transplantation in 2009. at relapse of his multiple myeloma in november, 2012, he was given lenalidomide plus dexamethasone. relatives reported that the patient owned camels, and had taken care of a diseased animal shortly before onset of symptoms. no animal samples, or further details about potential sources or exposures could be retrieved. during his stay in munich, we recorded thrombocytopenia (table 1) . thrombocytopenia was also reported in the fi rst described case of mers-cov infection, 1 in two of four patients from a family cluster in saudi arabia, 12 and in the two cases reported from france. 5 the patient developed renal insuffi ciency on day 14, and required dialysis. despite continuous invasive ventilation and antibiotic treatment, the patient's health status deteriorated. death occurred on day 18 and was due to septic shock, with signs of haemolysis and acute coagulation disorder (fi gure 1, table 1). after hospital admission in munich, infection with mers-cov was suspected on the basis of treatment-refractory acute respiratory distress syndrome, combined with the geographical origin of the patient. bronchoalveolar fl uid was obtained on march 20 and 22 (days 12 and 14). mers-cov was detected in both samples by rt-pcr. we also detected herpes simplex virus type 1 dna 13 (6·4 × 10⁴ to 1·9 × 10⁷ copies per ml) and rhinovirus rna 14 (3·7 × 10⁵ to 2·1 × 10⁹ copies per ml) by (rt-)pcr in both samples. mers-cov rna concentrations in respiratory samples ranged from 933 to 1·2 × 10⁶ genome copies per ml. virus concentrations seemed to be higher in samples taken earlier in the course than in those obtained later (fi gure 2). concentrations were more variable in tracheobronchial samples than in bronchoalveolar lavage samples (fi gure 2), which was ascribed to variation in volumes of saline solution applied during removal of tracheobronchial samples. notably, suction catheters without opening at point of care and stored for as long as 5 days at 8°c in a refrigerator in the intensive care unit tested consistently positive but yielded up to roughly 3·5log 10 lower rna concentrations than did those in fresh tracheobronchial aspirates taken on the same days (fi gure 2). immunofl uorescence assays yielded endpoint titres on day 16 of infection (table 2 ). an igm-specifi c immunofl uorescence assay confi rmed recent infection in the same serum sample (table 2) . plaque-reduction neutralisation test confi rmed mers-cov specifi city of detected antibody titres (table 2) . these titres were somewhat lower than those recorded for serum samples from an unlinked non-fatal case of mers-cov treated in germany in 2012. 8 serum samples from this patient had been taken later than they were for our patient (table 2) . we tested two urine samples on day 12, one on day 13, and one on day 16. one of the two samples on day 12, and the sample from day 13 were positive, meaning that the virus was not present in urine after renal failure (day 14), with a maximum virus concentration of 2691 rna copies per ml on day 13. both stool samples obtained on day 12 and the fi ve on day 16 were positive, with up to 1031 rna copies per g, which is a concentration close to the lowest detection limit of the assay. we recorded a low virus concentration in one of two oronasal aspirate samples taken from the intubated patient on day 16 (5370 copies per ml). one dialysate sample and two serum samples on day 16 , and one serum sample on day 18 were negative. although we obtained several isolates for the herpes simplex virus type 1, repeated attempts to isolate mers-cov were unsuccessful. herpes simplex virus is a frequent bystander infection in intubated patients, and is known to not aff ect the cardiorespiratory prognosis and outcome. 15 we sequenced the full mers-cov genome directly from respiratory samples (genbank accession number kf192507). we subjected all available mers-cov genome sequences to phylogenetic analysis, including a correlation and regression analysis of known dates of virus isolation versus tree branch lengths (fi gure 3). we estimated the rate of evolution as 1·6 × 10 -³ substitutions per site per year. the time of the common ancestor of all fi ve viruses for which genomes are available was halfway through 2011 (fi gure 3). the virus in our patient clustered with a sequence from a virus imported into the uk from qatar. 16 to compare this sequence with that of another virus from the same region, we reanalysed a stored clinical sample from another case of mers-cov infection imported into germany in october, 2012. this sample contained low concentrations of rna, so the genome of the virus had not been successfully sequenced previously. 8 after many attempts to recover rt-pcr fragments from the available bronchoalveolar lavage sample, we could sequence 12 fragments, covering 4012 nucleotides of the mers-cov genome (genbank accession number kc875821). a concatenated alignment of homologous sequence portions of all available mers-cov sequences was subjected to phylogenetic analyses, confi rming a clustering of sequences from qatar and the united arab emirates (fi gure 3). a sequence from a patient with a history of travel to pakistan and saudi arabia branched next to this cluster. during and up to 10 days after the course of treatment, 14 health-care workers who had direct contact with our patient or patient-derived materials reported mild respiratory symptoms. samples were taken from the upper respiratory tract and tested by two diff erent rt-pcr assays for mers-cov. none yielded positive results. by contrast, one patient who had had direct contact with the patient with mers-cov was infected with hcov-nl63, a common human coronavirus, and four patients were infected with rhinoviruses. these rhinoviruses were not all mutually related, and none was related to the rhinovirus detected in the patient with mers-cov (appendix). follow-up of all contact patients, including investigation for subclinical infections, is in progress. we have outlined the chronological follow-up of a patient with mers-cov, in which we used quantitative virological diagnostic tests (panel). viral loads were highest in the lower-respiratory tract. the viral sequence from this patient clustered with sequences from nearby qatar. laboratory data are crucial for diagnostic recommendations, to make projections about prognosis, and to estimate infection risks. without quantitative laboratory data from well documented cases of mers-cov infection, most considerations had been made on the basis of an assumed analogy to severe acute respiratory syndrome (sars). 12, [18] [19] [20] however, elementary traits of the virus, such as its receptor usage and sensitivity to type i and type iii interferon, diff er substantially from that of the sars coronavirus, suggesting that diff erences in disease patterns (eg, in organ tropism or in virus shedding) might exist. [21] [22] [23] [24] [25] we focused on these aspects with quantitative virus testing in all relevant body compartments, including viral loads in non-respiratory samples. 5, 12 however, our patient-like most other cases anecdotally reported so far-had an underlying disease that could aff ect virus shedding patterns. only analysis of a large number of patients can yield general fi gures about qualitative virus data. faecal shedding was of particular interest, because patients with sars regularly showed high virus concentrations and prolonged virus excretion in stools that led to the use of stool samples, even for routine sars diagnostic tests. 18, 20, 26 diarrhoea was reported in two descriptions of mers-cov clusters, and it was speculated that faecal virus shedding might have occurred. 5, 12 however, no laboratory data for virus in stool samples were provided. our patient had low faecal virus concentrations that were close to the lowest detection limit on days 12 and 16 of illness. in the only other description so far, one stool sample from a patient with mers-cov had a negative result. 16 stool samples from many patients, including those with early stages of disease, should be tested to assess whether faecal sources could have a role in transmission, or whether mers-cov diff ers from sars in this aspect. another important fi nding was that we recorded low concentrations of virus in urine samples. this fi nding is surprising, because early kidney failure during the course of mers-cov infection has been reported, and kidney cells in laboratory models are highly permissive for mers-cov replication. 5, 12, 23 the fact that the virus was present in urine but not in the blood suggests autonomous virus replication in the kidneys, potentially without active secretion of virus into the urine. however, renal failure due to specifi c viral infection or immunopathogenesis is not necessarily indicated, because the patient had received several doses of potentially nephrotoxic antimicrobial agents in a setting of underlying multiple myeloma. post-mortem examinations are urgently needed to clarify whether kidney failure in mers-cov infection is a primary and preventable result of viral infection, or a secondary complication of severe systemic disease. 27, 28 quantitative virus data are needed to orient diagnostics and hospital infection control measures. the recorded viral load profi le, with highest rna concentrations in bronchoalveolar lavage and tracheobronchial aspirates, confi rms suggestions made in another report about the preferential use of lower-respiratory-tract samples for virus diagnostic tests. 5 notably, the reported overall stability of detectable virus rna in closed suction catheters indicates a straightforward and non-contagious way to collect diagnostic samples even from non-intubated patients. oronasal swabs should not be preferentially submitted for testing, especially in patients presenting late in their disease course with substantial lower respiratory involvement. 5 our data for stool, urine, and blood samples suggest a fairly low infection risk during non-respiratory care procedures. the absence of detectable virus in blood matches reports made in an earlier case of mers-cov infection. 16 however, guery and colleagues 5 reported low semi-quantitative virus measurements in the blood of one of their patients. moreover, initial experimental studies suggested that mers-cov infected vascular endothelial cells. 25 however, quantitative data suggest a low risk from general laboratory procedures involving blood. by contrast, the low virus concentrations and failure to isolate infectious virus from respiratory secretions should not be taken as a general indication of airway-associated infection risks. virological monitoring of the patient started only late in the disease course, at a time when the infectiousness of the virus could already have been reduced (as suggested by the occurrence of neutralising antibodies). the fact that we could not isolate mers-cov could have been due to the concomitant presence of herpes simplex virus type 1, which overgrew some of the diagnostic cell cultures. we searched pubmed for reports published in english at any time before june 7, 2013. we used the search term "mers-cov or hcov-emc". we identifi ed 29 reports linked to middle east respiratory syndrome coronavirus (mers-cov), starting with zaki and colleagues' initial report, 1 in which a previously unknown coronavirus isolated from the sputum of a 60-year-old man is described. when we used the search term "mers-cov", we identifi ed four reports, 2,5,12,17 none of which provided quantitative viral load profi les of infected patients. our report provides the fi rst complete viral load profi le in a case of mers-cov infection. the distribution of viral loads in the respiratory tract suggests lower-respiratory-tract samples should be taken preferentially. low concentrations of the virus in stool, urine, and blood samples suggests little virus excretion-at least in our patient-from body compartments other than the respiratory tract. furthermore, coronaviruses that infect people are generally diffi cult to isolate, particularly in late phases of disease. only two successful isolations of mers-cov have been reported worldwide so far. 1, 5 these isolations were done on day 4 of disease, 5 and day 7 of disease. 1 our samples were taken on days 12 and 14, and the sample from day 12 was stored for 3 days before cell culture. isolation success cannot provide any information about infectiousness of the patient. even a highly concentrated rhinovirus does not seem to have been transmitted from the patient, suggesting that eff ective protective measures were in place during treatment of the intubated patient in the intensive care unit. the sequence data from this and another patient treated in germany enable an extended analysis of phylogeny, hinting at a geographical structure of the mers-cov tree. specifi cally, viral sequences from the eastern part of the arabian peninsula cluster together and stem from one common ancestor whose date of existence is projected to be after that of viruses from jeddah and jordan. date estimates will probably be refi ned when more sequences become available. moreover, whether the reported geographical structure represents repeated transfer from a geographically structured viral reservoir population and limiting chains of person-to-person transmission or multiple sustained lineages of human infections is unclear. with only fi ve complete genome sequences available as yet, genetic data are urgently needed to establish the spatial and temporal distribution of cases, estimate the number of independent human chains of transmission, and thus better assess the threat that mers-cov poses to world health. four sequences from a continuing hospital outbreak in al-hasa, saudi arabia, have now been deposited in genbank. preliminary phylogenetic analyses confi rm the clustering of viruses from the eastern part of the arabian peninsula (qatar: strains england2 and essen; abu dhabi: strain munich; al-hasa: genbank accession numbers kf186564-kf186567). cd designed the virological studies, phylogenetic analysis, and virological data analysis. ms, whar, gs, ss, wg, hgu, tr, ms-w, fb, and c-mw contributed to clinical data generation and analysis. ms, vmc, rk, dm, sj, and ar contributed to fi gures. vmc, rk, dm, sj, mam, sa, and ud generated and analysed virological data. ar did phylogenetic analysis. cd, whaa, hgo, and c-mw interpreted data. cd, ms, vmc, fb, ar, and c-mw wrote the report. whaa advised the public health intervention. hgo and pg coordinated the public health intervention. we declare that we have no confl icts of interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov)-update severe respiratory illness caused by a novel coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections contact investigation of a case of human novel coronavirus infection treated in a german hospital detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction plaque assay for human coronavirus nl63 using human colon carcinoma cells a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood family cluster of middle east respiratory syndrome coronavirus infections detection of herpes simplex virus dna by real-time pcr real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses respiratory herpes simplex virus type 1 infection/colonisation in the critically ill: marker or mediator? severe respiratory illness caused by a novel coronavirus genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus the severe acute respiratory syndrome the aetiology, origins, and diagnosis of severe acute respiratory syndrome identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures severe acute respiratory syndrome multiple organ infection and the pathogenesis of sars acute renal impairment in coronavirus-associated severe acute respiratory syndrome this work was supported by a european research project on emerging diseases detection and response (emperie; contract no 223498). cd has received infrastructural support from the german centre for infection research, which included full funding of the position of vc. virological analyses were partly support by the german ministry for key: cord-354881-7o20cn1x authors: brown, rebecca c h; kelly, dominic; wilkinson, dominic; savulescu, julian title: the scientific and ethical feasibility of immunity passports date: 2020-10-16 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30766-0 sha: doc_id: 354881 cord_uid: 7o20cn1x there is much debate about the use of immunity passports in the response to the covid-19 pandemic. some have argued that immunity passports are unethical and impractical, pointing to uncertainties relating to covid-19 immunity, issues with testing, perverse incentives, doubtful economic benefits, privacy concerns, and the risk of discriminatory effects. we first review the scientific feasibility of immunity passports. considerable hurdles remain, but increasing understanding of the neutralising antibody response to covid-19 might make identifying members of the community at low risk of contracting and transmitting covid-19 possible. we respond to the ethical arguments against immunity passports and give the positive ethical arguments. first, a strong presumption should be in favour of preserving people's free movement if at all feasible. second, failing to recognise the reduced infection threat immune individuals pose risks punishing people for low-risk behaviour. finally, further individual and social benefits are likely to accrue from allowing people to engage in free movement. challenges relating to the implementation of immunity passports ought to be met with targeted solutions so as to maximise their benefit. at this point in the covid-19 pandemic, how or when our lives might return to normality is unclear. one strategy proposed to help this resumption is the identification and documentation of immunity: so-called immunity passports. these passports are a potential tool for recording and sharing the immune status of an individual. the introduction of immunity passports is being considered by several countries, including the uk, estonia, italy, and chile; although as yet, there is no information on the effects of their use. [1] [2] [3] health certification for public health purposes is already used in other contextseg, in the management of yellow fever. 4 passports could take different forms, such as a wristband, smartphone application, or certificate, and be used to confirm to others that a particular individual is at a low risk of acquiring or transmitting severe acute respiratory syndrome coronavirus 2 (sars-cov-2). during periods of lockdown, immunity passports could allow immune individuals to follow less stringent requirements around physical distancing and travel, perhaps permitting them to return to work, care for those at risk, visit friends and relatives, or undertake other activities that expose them to the virus. whether this strategy should be pursued depends on both scientific evidence and ethical reasoning. immunity passports could be implemented on the basis of either a laboratory test of immune response (a correlate of protection) or an immunising event (infection or vaccination), which would identify individuals less likely to get disease or transmit virus when exposed to sars-cov-2. important immunological issues for such passports are: (1) the degree of immunity induced (an immune response might only attenuate disease severity, or might prevent any symptomatic disease and even pathogen carriage, which is necessary for herd immunity) and (2) the duration of immunity. critics of immunity passports point to persisting uncertainties about the immune response to covid-19, claiming that "covid-19 immunity is a mystery" and that this uncertainty makes immunity passports unfeasible. 5 whether sars-cov-2 will generate a short-lived immune response similar to those produced by seasonal coronaviruses, or one more similar to those from sars and middle east respiratory syndrome coronaviruses, in which antibody responses persist for 2-3 years, is unclear. [6] [7] [8] [9] concerns also surround the sensitivity and specificity of the tests used to define immunity, especially in populations with a low incidence of previous infection, and the need for impractical numbers of tests to be done to ensure a population remains immune. 5,10-12 symptomatic infection with sars-cov-2 generates various t-cell, b-cell, and antibody responses against components of the virus, including the spike glycoprotein and the nucleoprotein, which could be assessed as potential correlates of protection. [13] [14] [15] [16] [17] [18] [19] [20] [21] for infections that have reliable correlates, these correlates have been based on antibody responses, which have the following advantages: these tests use serum or plasma that are easy to collect and store; the assays are more easily standardised and scaled for high throughput use than are cellular assays; and the assays provide a direct link to the protective immune response. current antibody assays for sars-cov-2 correlates already include sensitive and specific quantitative measurements of igg against various viral proteins, and complex viral neutralisation assays. [22] [23] [24] following symptomatic infection, most patients develop antibody responses, with the majority of these individuals having neutralising antibodies. [13] [14] [15] [16] [17] [18] [19] [20] [21] 25, 26 although, like all antibody responses to viral infections, responses to sars-cov-2 wane in the weeks after infection, increasing evidence suggests that these responses remain higher than preinfection levels for at least 4 months (the longest period that has been possible to study). 14, 15, 19, 25 as of september, 2020, there is not a recognised level of response for any assay that has been definitively shown to protect against disease or viral transmission. however, personal view some progress has been made: monoclonal antibodies in animal models prevented sars-cov-2 infection in a dose-dependent manner; [27] [28] [29] individuals with pre-existing neutralising antibodies were less likely to get infected during an outbreak on a fishing boat than were individuals with either no antibody response or antibody responses that were non-neutralising when tested preexposure; 30 and higher antibody concentrations were associated with lower viral loads during illness in a study of hospitalised patients in the uk. 31 in the absence of an immunological correlate of protection, confirmed infection itself could be used to certify immunity. in human challenge trials with seasonal coronaviruses and primate models of sars-cov-2 infection, disease severity was attenuated by previous infection. 32 with widespread testing available and ongoing trans mission occurring, there is likely to be rapid progress on quantifying the protective effect of previous infection and the duration of this effect. however, there are likely to be complexities to this approach because, for sars-cov-2 infection, antibody responses might be less marked in individuals with asymptomatic or mild disease than in those with severe disease. 17 furthermore, several individuals with evidence of reinfection within a short period of a first illness have been described, with at least one individual being more symptomatic with the second illness than with the first. 33, 34 given the scale of the pandemic and the research into covid-19, there is likely to be rapid progress in understanding the nature of infection and immunity such that clinical infection, with or without a measure ment of antibody response, might form the basis of a time-limited immunity passport. challenges for this approach include the heterologous nature of the initial infection, reflected in the variation in quality and duration of the subsequent immune response; the almost complete absence of information about an individual's ability to still transmit virus to others even if protected from disease; and the need to undergo the risk of infection to acquire immunity. a recurrent criticism of immunity passports based on correlates of infection is the use of serosurveys of populations with a low incidence of infection. for instance, a cochrane review suggested that the number of false positives produced in a setting with a 5% sars-cov-2 seroprevalence would be around 21%. 35 however, an assessment of individual immunity is not likely to be derived from serosurveys of whole populations with a low incidence of infection. such approaches are also unattractive given that asymptomatic infections might lead to less functional and less persistent antibody than might symptomatic infections. if a surveillance approach was considered in a group at high risk of infection, such as exposed health-care workers, a twostage testing process could be implemented whereby individuals with a positive antibody test undergo secondline testing with neutralisation assays. at least one study has shown that individuals positive for antibodies against sars-cov-2 but negative for neutralising antibodies were susceptible to sars-cov-2 infection, reinforcing the need for a two-stage approach in some settings, especially where there is no infection documented as the origin of the antibodies. 30 there has been rapid progress in vaccine development and emerging evidence that vaccines can provide protection from sars-cov-2 in animal models. 32, 36, 37 given the ongoing clinical efficacy trials that use widespread serological and cellular sampling, there will be more data emerging as to whether vaccination could form the basis of an immunity passport and, if so, whether there are any assays that provide correlates of protection. basing immunity passports on a vaccine has advantages: the stimulus is uniform and is therefore likely to have a more predictable pattern and duration of immunity than is infection, and vaccination makes immunity potentially available to the whole population. the ethical issue then becomes one of timely access to vaccination for everyone. in the setting of routine immunisation in a population, the duration of immunity can be estimated from efficacy studies, together with serological surveys, and these data can support booster doses rather than continually reassessing immunity in individuals. a neglected issue in discussions of immunity passports is that of individual protection versus community protection. perhaps the most important consideration for immunity passports is whether an individual can transmit the infection to others. evidence from previous work with seasonal coronaviruses and studies of sars-cov-2 vaccines in macaques suggests that previous infection or vaccination might protect from severe disease but an individual might nevertheless carry the virus at similar levels, and for a similar duration, to those previously uninfected, with an unchanged potential for transmission. 17, 32, 38, 39 this fact provides the greatest challenge to the assurance that individuals who carry immunity passports would have a reduced risk to others. there are considerable challenges in measuring and inferring immunity to sars-cov-2. however, many of those challenges could potentially be overcome in the coming months. as information continues to accumulate, it is important to consider whether immunity passports should be used to reliably identify immune individuals, if this identification were possible. there are several key ethical advantages to immunity passports. first, the justification behind requiring people to remain in lockdown is the risk their free movement poses to themselves and others as they could acquire and pass on the virus. individuals who are immune to sars-cov-2 are expected to be at a vastly reduced risk of getting and transmitting the virus, and so removing their civil liberties would be unjustified. it is unethical to require someone to avoid contact with others if they pose no or minimal risk of spreading the virus. second, people will know the reduced risks, and are likely to become less compliant with lockdown restrictions if they believe themselves to be immune anyway. 40 consider neil ferguson, who resigned from his role as an adviser to the uk government after breaking lockdown guidelines, stating "i acted in the belief that i was immune". 41 by refusing to formalise the permissibility of such actions, inevitable low-risk behaviour is classified as rule breaking, and could even subject people to fines and punishments that do not correspond to the harm their behaviour causes. finally, there will be benefits for immune individuals and broader benefits to society from allowing people to return to work and care obligations. lonely and isolated individuals could be visited by immune friends and relatives; small businesses can be reopened by staff who are immune and will not risk the health of colleagues and customers; immune health-care staff can care for patients with covid-19; and immune care workers can protect vulnerable people in residential homes. despite these advantages, some people have strongly opposed immunity passports. in numerous articles, ethicists natalie kofler and françoise baylis have claimed that immunity passports are "the height of folly" and should be fought against "tooth and nail". 42, 43 kofler and baylis have highlighted how, in 19th century new orleans, la, usa, presumed immunity to yellow fever "was weaponised to justify white supremacy", 5 and have warned that immunity passports could cause similar effects in modern day. who have also expressed concerns about immunity passports, 44 as has alexandra phelan writing in the lancet, 11 and numerous news outlets. [45] [46] [47] [48] [49] a rapid policy briefing by the nuffield council on bioethics emphasised the ethical risks of immunity passports, speculating that these passports could "create coercive and stigmatising work envi ronments" and are "more likely to compound than redress…structural disadvantages and…social stigma tisation". 50 however, the strength of much of this opposition does not seem justified by the strength of the arguments opposing immunity passports. critics warn that immunity passports create a "perverse incentive for individuals to seek out infection" or choose to fraudulently acquire passports. 11 how likely these scenarios would be is largely unknown. one survey suggested that people are very unlikely to intentionally seek infection, and gilad edelman, writing in wired, has proposed that the reporting of so-called covid parties might be overblown. 51, 52 this area is one for which additional evidence would greatly help to judge risk and how this risk can be traded off against the benefits of immunity passports. behavioural scientists have also highlighted research on the psychology of in-groups, social groups with which people identify, and out-groups, social groups with which people do not identify, suggesting that permitting immune individuals to exercise more free doms than those who are not immune would undermine the message that we are "all in this together". 12 we are not aware of any published research that presents clear and compelling evidence supporting this assertion. there has been some exploration as to how various theories and findings from social and behavioural science can be applied to the pandemic response. 53 at this stage, however, extrapolation from theories supported by evidence often based on laboratory experimentation or very different situational contexts is risky. several behavioural scientists have raised concerns about the robustness and generalisability of claims from behavioural science and caution against the use of these claims to inform major policy decisions. 54 the baseline prevalence of immunity varies from place to place. in some cities (ie, london [uk], new york [ny, usa], and stockholm [sweden]), the prevalence of immunity could be as high as a fifth; 55-59 elsewhere, prevalence is likely to be much lower. economic analyses are needed to find out how much economic benefit would be generated if some proportion of the population had fewer restrictions on their movement. we are not aware of any published work that informs this argument. however, it would be a mistake for ethicists, in the absence of such evidence or expertise, to dismiss immunity passports on the assumption that there will be "too few survivors to boost the economy". 5 in addition, immunity passports might deliver important non-economic benefits, such as regaining the ability to operate small buisnesses, to a small proportion of people. there is suspicion that immunity passports could provide a way in to more troublesome monitoring of people's movements and health statuses. 60 some have claimed that "the whole point of immunity passports is to control movement". 5 however, this claim is a gross mischaracterisation: the point of immunity passports is to facilitate movement when it is safe to do so. of course, steps must be taken to avoid the production of fraudulent immunity passports, and careful attention must be given to privacy concerns and information governance. these problems are not unique to immunity passports (conventional passports and contact tracing measures personal view also encounter such problems) and are not insurmountable. immunity passports have been frequently objected to on the basis that their introduction would exacerbate existing inequalities. 5, 11, 47, 50 the main concerns are that, if immunity passports were introduced, marginalised groups would be subject to more scrutiny because of existing inequities and racism (eg, police checks for lockdown regulations) and would be less likely to access testing (and establish immunity) than non-marginalised groups would be. furthermore, the advantages accruing to those with immunity (and immunity passports) would persist into the future. although we recognise the deep existing inequalities in all countries, and the ways in which covid-19 has increased the hardships for the worst off, we are surprised that the published responses 42 of some prominent critics of immunity passports do not include suggestions of ways to mitigate resultant inequalities. as frequently noted, such unequal experiences are not new. factors, such as race and socioeconomic status, influence the health care that people access and the treatment they receive. [61] [62] [63] yet, this issue is rarely interpreted as a reason to remove health-care treatments or refuse to introduce new ones, assuming these therapies are considered cost-effective and net beneficial. instead, such patterns point us to areas in which more effort must be made to improve the care of the most deprived, to look for solutions to the inequitable distribution of resources, and to tackle the upstream causes of inequality. this same reasoning should be applied to immunity passports. furthermore, as some have highlighted, the advantages of covid-19 immunity might not entrench existing inequalities in the way often assumed. 40 lockdown measures considerably curtail people's freedom. immunity passports would potentially allow some proportion of the population to access more freedoms during lockdown periods. it is unethical to restrict freedom unless there is a real risk to other people. if we have the technology to decide who is not a risk, we should use it. the specific scenarios in which immunity passports can be used will depend on the nature of the immunity generated. it might be desirable for immunity passports to record individuals, especially those who have pre-existing vulnerabilities, who have been identified as having a correlate of immunity that reliably indicates that they will not contract severe disease in the future. alternatively, if immunity passports certify that individuals can move around freely and interact for business or leisure without increasing the risk of transmission, we might wish to certify only those who are unlikely to transmit the virus. although there remains considerable uncertainty regarding the nature, degree, and duration of immunity to sars-cov-2, the world's intense research focus on this infection will potentially yield useful answers in a practicable timeframe that could be translated into some form of immunity passport. even after a correlate of protection is established, there will still be uncertainty around the duration of protection or whether the correlate can be applied across all ages and clinical scenarios, but complete certainty might not be necessary for policy in the medium term. 64 assuming that developing scientific evidence supports the use of immunity passports, safety nets must be put in place to protect individuals who remain in lockdown and are the most disadvantaged by lockdown (eg, those who are unable to work, socially isolated, or at risk from domestic violence). similarly, we must take seriously the need to ensure fair access to testing and to address inequality that arises in the context of covid-19 through targeted solutions. we must be clear about what the alternatives are when evaluating the merits of different ways of tackling this pandemic. the choice is not between returning to a normal life versus issuing immunity passports. instead, the choice is between periodic lockdowns, attempting to emerge from lockdowns with immunity passports, and attempting to emerge from lockdowns without immunity passports. immunity passports are a potentially valuable and ethical tool. as further evidence relating to the immune response to covid-19 accumulates, and the capacity to reliably identify immune individuals develops, immunity passports could be appropriately adopted. in such an event, the freedoms these passports confer must be subject to amendments and cancellations, and integrated with other measures, such as contact tracing and physical distancing, to keep people safe while maintaining quality of life. rchb produced the initial draft of the manuscript. dk drafted the sections relating to scientific knowledge of sars-cov-2 immunity and provided comments on the rest of the manuscript. dw and js provided input into the initial preparation of the manuscript and edited the full manuscript. we declare no competing interests. italy, going back to work may depend on having the right antibodies no 10 seeks to end coronavirus lockdown with 'immunity passports estonia starts testing digital immunity passport for workplaces ten reasons why immunity passports are a bad idea sars-cov-2 vaccines: status report lack of cross-neutralization by sars patient sera towards sars-cov-2 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications seasonal coronavirus protective immunity is short-lasting serology for sars-cov-2: apprehensions, opportunities, and the path forward covid-19 immunity passports and vaccination certificates: scientific, equitable, and legal challenges immunity passports aren't a good way out of the coronavirus crisis decline of humoral responses against sars-cov-2 spike in convalescent individuals dynamics of neutralizing antibody titers in the months after sars-cov-2 infection dynamics and significance of the antibody response to sars-cov-2 infection dynamics of anti-sars-cov-2 igm and igg antibodies among covid-19 patients longitudinal evaluation and decline of antibody responses in sars-cov-2 infection rapid generation of neutralizing antibody responses in covid-19 patients sars-cov-2 infection induces robust, neutralizing antibody responses that are stable for at least three months sars-cov-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic covid-19 neutralizing and binding antibody kinetics of covid-19 patients during hospital and convalescent phases antibody testing for covid-19: a report from the national covid scientific advisory panel a serological assay to detect sars-cov-2 seroconversion in humans performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in humoral immune response to sars-cov-2 in iceland high prevalence of sars-cov-2 antibodies in care homes affected by covid-19 potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model a human neutralizing antibody targets the receptor-binding site of sars-cov-2 neutralizing antibodies correlate with protection from sars-cov-2 in humans during a fishery vessel outbreak with high attack rate anti-sars-cov-2 igg antibodies are associated with reduced viral load chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques genomic evidence for reinfection with sars-cov-2: a case study covid-19 re-infection by a phylogenetically distinct sars-coronavirus-2 strain confirmed by whole genome sequencing antibody tests for identification of current and past infection with sars-cov-2 coronavirus vaccine trials have delivered their first results-but their promise is still unclear development of an inactivated vaccine candidate for sars-cov-2 the time course of the immune response to experimental coronavirus infection of man sars-cov-2 infection protects against rechallenge in rhesus macaques privileges and immunity certification during the covid-19 pandemic coronavirus: prof neil ferguson quits government role after 'undermining' lockdown why canadians should fight tooth and nail against proof-of-immunity cards covid-19 immunity testing: a passport to inequity immunity passports to ease lockdown might trigger discrimination the perverse world of immunity passports the dangerous history of immunoprivilege start-ups are racing to develop covid-19 'immunity passports'-but experts warn they're unethical immunity passports aren't a good way out of the coronavirus crisis covid-19 antibody testing and 'immunity certification united kingdom wave 2 on 16 covid parties' are not a thing using social and behavioural science to support covid-19 pandemic response is social and behavioural science evidence ready for application and dissemination? coronavirus has infected a 5th of new york city, testing suggests uppdaterad modellering av spridningen av covid-19 i stockholms län sero-surveillance of covid-19 seroconversion of a city: longitudinal monitoring of sars-cov-2 seroprevalence in covid-19-a very visible pandemic immunity passports are a threat to our privacy and information security fair society, healthy lives. the marmot review socioeconomic inequality of access to healthcare: does choice explain the gradient? race, socioeconomic status, and health-care access disparities in ovarian cancer treatment and mortality: systematic review and meta-analysis waiting for certainty on covid-19 antibody tests-at what cost? this research was supported by the wellcome trust (wt 104848/z/14/z and wt 203132/ z/16/z). js, through his involvement with the murdoch children's research institute, was supported by the victorian government's operational infrastructure support program. dk receives salary support from the national institute for health research (nihr) oxford biomedical research centre. the views expressed in this personal view are those of the authors and not necessarily those of the nihr or the department of health and social care. key: cord-279001-l5ogbl5p authors: wilder-smith, annelies; chiew, calvin j; lee, vernon j title: can we contain the covid-19 outbreak with the same measures as for sars? date: 2020-03-05 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30129-8 sha: doc_id: 279001 cord_uid: l5ogbl5p the severe acute respiratory syndrome (sars) outbreak in 2003 resulted in more than 8000 cases and 800 deaths. sars was eventually contained by means of syndromic surveillance, prompt isolation of patients, strict enforcement of quarantine of all contacts, and in some areas top-down enforcement of community quarantine. by interrupting all human-to-human transmission, sars was effectively eradicated. by contrast, by feb 28, 2020, within a matter of 2 months since the beginning of the outbreak of coronavirus disease 2019 (covid-19), more than 82 000 confirmed cases of covid-19 have been reported with more than 2800 deaths. although there are striking similarities between sars and covid-19, the differences in the virus characteristics will ultimately determine whether the same measures for sars will also be successful for covid-19. covid-19 differs from sars in terms of infectious period, transmissibility, clinical severity, and extent of community spread. even if traditional public health measures are not able to fully contain the outbreak of covid-19, they will still be effective in reducing peak incidence and global deaths. exportations to other countries need not result in rapid large-scale outbreaks, if countries have the political will to rapidly implement countermeasures. in november, 2002, the severe acute respiratory syn drome coronavirus (sarscov) emerged in china causing global anxiety as the outbreak rapidly spread, and by july, 2003, had resulted in over 8000 cases in 26 countries. in december, 2019, a novel coronavirus, named sarscov2, emerged in wuhan, china, and led to a rapidly spreading outbreak of coronavirus disease 2019 (covid19). by jan 30, 2020, covid19 was declared a public health emergency of international concern. the similarities between sarscov and sars cov2 are striking, not only in name. the whole genome of sarscov2 has a 86% similarity with sarscov. 1 both viruses share high degrees of homology to sars like coronaviruses isolated in bats, suggesting that bats are the probable origin of both sarscov and sars cov2. live animal markets selling multiple species of wild and domestic animals in proximity to large populations of densely housed humans are thought to be the source of both outbreaks. even in terms of disease dynamics there are apparent similarities. the main transmission route is thought to be respiratory droplets, although viral shedding via faeces has also been reported for both viruses. the angiotensinconverting enzyme 2 (ace2), found in the lower respiratory tract of humans, has been identified as the receptor used for cell entry for both sarscov and sarscov2. 2,3 covid19 and sars have a median incubation time of about 5 days. the mean serial interval of covid19 is 7·5 days (95% ci 5·3-19·0) and the initial estimate for the basic number (r₀) was 2·2 (95% ci 1·4-3·9), 4 similar to that reported for sars (mean serial interval 8·4 days, and basic r₀ range 2·2-3·6 for serial intervals of 8-12 days). 5 risk factors for severe disease outcomes are old age and comorbidities. the progression for patients with severe disease follows a similar pattern in both viruses, with progression to acute respiratory distress syndrome approximately 8-20 days after onset of first symptoms, whereby lung abnormalities on chest ct show greatest severity approximately 10 days after initial onset of symptoms. [6] [7] [8] [9] however, the similarities end here. successful and included active case detection, isolation of cases, contact tracing and quarantine of all contacts, social distancing, and community quarantine. • whether these measures will also be successful for covid-19 will not depend on the similarities but the differences between sars and covid-19. • clear differences are emerging, such as in transmissibility and severity pyramids; covid-19 has a higher transmissibility than sars, and many more patients with covid-19 rather than sars have mild symptoms that contribute to spread because these patients are often missed and not isolated. • because of the extent of community spread, traditional public health measures might not be able to halt all human-to-human transmission, and we need to consider moving from containment to mitigation. toronto, canada. sars was eventually contained by means of syndromic surveillance, prompt isolation of patients, strict enforcement of quarantine of all contacts, and in some areas communitylevel quarantine. by interrupting all humantohuman transmission, sars was effectively eradicated. by contrast, by feb 28, 2020, within a matter of 2 months since the beginning of the outbreak, more than 82 000 confirmed cases of covid19 have been reported with more than 2800 deaths, mostly in china. outside of china, 46 countries are reporting more than 3600 cases including at least 700 on several cruise ships. traditional public health measures were widely used to eradicate sars. can covid19 be controlled by these same measures? it is crucial to remember what measures were taken, and which lessons could be applicable to covid19. in the absence of vaccines and specific treatment, the only available public health tools to control persontoperson transmittable diseases are isolation and quarantine, social distancing, and community containment measures. 10 isolation is the separation of ill people from non infected people, and usually occurs in hospital settings, but could also be done at home for mild infections. 11 for isolation to be successful in preventing transmission, case detection should be early-ie, before the onset of viral shedding or at least before the onset of peak viral shedding. for sars, viral loads peaked at 6-11 days after onset of illness for nasopharyngeal aspirates, enabling early isolation before transmission. 12 the number of secondary cases from an infected patient was clearly reduced if the infected patient was isolated within 4 days after onset of symptoms. 13 for sars, a highly sensitive case definition was used with a focus on fever or respiratory symptoms, and an epidemiological link (contact or travel history). all suspected patients were isolated until sars was ruled out. implementing optimal isolation by itself was modelled to be more costeffective than implementing suboptimal isolation and quarantine together. 14 secondary household transmission was low in singapore (6·2%), indicative of rapid detection and isolation of patients. similarly, in toronto, secondary attack rate in households was 10%, with a linear association between secondary attack rate and the time that the index patient spent at home after symptom onset. 15 in singapore, healthcare associated transmission accounted for more than 90% of all cases. 16 once full measures were in place, almost all patients were promptly isolated before secondary transmission had occurred. the outbreak in singapore was propagated by five superspreading events, 16 including three super spreading events as a result of atypical clinical manifestations of sars. 17 quarantine involves movement restriction, ideally combined with medical observation during the quarantine period, of close contacts of infected patients during the incubation period. 18 the premise for successful quarantine is prompt and comprehensive contact tracing of each and every confirmed patient. quarantine can take place at home or in designated places such as hotels, and both of these options were used during the sars epidemic. quarantined contacts had to record their temperatures, and were visited or telephoned daily by a member of the public healthcare team. if the contact developed symptoms, they were investigated at a designated healthcare facility. the principle is that if the person under quarantine developed illness, that person would not have any close contacts to spread the disease, effectively reducing the r₀ of the outbreak to less than 1. as an example of the magnitude of efforts taken, toronto public health investigated 2132 potential cases of sars and identified 23 103 contacts as requiring quarantine. 19 legally enforceable quarantine orders were issued to contacts. police made spot checks in hong kong whereas video cameras were installed at the home of each contact in singapore. 16, 20 once it is no longer feasible to identify all infectious individuals and their contacts in the attempt to slow the spread of disease, a possible next step is to apply communitywide containment measures. community wide containment is an intervention that is applied to an entire community, city, or region, designed to reduce personal interactions. 10 these interventions range from measures to encourage personal responsibility to identify disease, increase social distancing among community members including cancellation of public gatherings, and finally implement community quarantine. 11 enforcement of communitywide contain ment measures is far more complex than isolation or quarantine because of the larger number of people involved. china best exemplified this largescale quarantine by declaring epidemic zones and placing people under collective quarantine in villages, cities, or institutions. 21 in april 2003, the chinese authorities gained full control of all activities to combat sars, with guidelines and control measures that were national, unambiguous, rational, and widely followed, under central guidance. the stringent control measures included school closures and closures of all universities and public places, as well as the cancellation of the public holiday in may. immediately, the r₀ decreased greatly and consistently. 22 in may, 2003, china locked down beijing and closed more than 3500 public places in an effort to curb community spread. singapore became the socalled country of thermo meters: temperature monitoring was made mandatory in schools and temperature screening was instituted at entry to public buildings. case detection was further improved by the opening of hundreds of fever clinics and use of the mass media to encourage people to check for fever several times a day. citizens with higher degrees of anxiety, better knowledge about sars, and greater risk perceptions than average were more likely to take comprehensive personal view precautionary measures against the infection. 23 in hong kong, 49% of the patients with sars were in clinics, hospitals, and elderly or nursing homes. the amoy gardens cluster accounted for another 18·8% of cases; these affected areas were put on community quarantine. 24 awareness about sars was very high, and there was an extraordinarily high willingness (90% of respondents in a psychobehavioural study in singapore and hong kong) to be quarantined if deemed necessary. 23 there was a strong political will in all affected countries with a topdown approach to enforce all public health measures in a short timeframe. in mainland china, the strong political commitment with a centrally coordinated response was considered the most important factor in the control of sars. 22 hospitalbased measures included isolation rooms with barrier nursing techniques, strict enforcement of personal protective equipment for staff, and restriction of visitors and movement of staff. negative pressure rooms were not used, or only used when available. infection control precautions were enhanced in all hospitals, and included the provision of separate triage facilities for patients with fever or respiratory symptoms. in toronto and singapore, workers were required to use gloves, gowns, eye protection, and n95 respirators for all contact with all patients, regardless of whether sars was suspected or not. 19 to reduce withinhospital spread, hospitals banned all visitors to patients with sars except on compassionate grounds. 19 healthcare workers or visitors exposed to facilities where sars transmission had occurred were not permitted to enter nonsars areas. 25 in singapore, temperature screening was mandated twice daily for all healthcare workers. 26 health care workers who developed a fever had to report to a designated healthcare facility, and were isolated until sars was ruled out. to accommodate the large number of patients with sars (both probable and suspect), beijing rapidly constructed the 1000bed xiaotangshan hospital within a week, which admitted a seventh of sars patients in the country within 2 months. following the who global alert, and a stronger emergency travel advisory issued by who on march 15, 2003 , almost all countries with imported cases were able to either prevent any further transmission or keep the number of additional cases small. exit screening via thermal scanners was done for all departing passengers at all airports of affected countries. many countries also implemented entry screening for all passengers arriving from affected areas. after all these measures were implemented, new imported cases of sars did not trigger new outbreaks as systems were in place to identify and isolate them early. 27 no travel bans were implemented at any time, but travel advisories to avoid nonessential travel to countries affected by sars were issued by several governments. the psychological effects of sars, coupled with travel restrictions imposed by various national and international authorities, resulted in a major economic loss for the airline industry and world economy in 2003, far beyond the main areas affected by sars. 28 17 years later, we can draw upon the lessons learnt from sars. the global community is much better prepared now, with many capacitybuilding initiatives including those under the who's international health regulations (2005). indeed, the time interval from the first case description to the virus sequencing and the availability of diagnostic assays was much faster for covid19 than for sars, and diagnostic tests were available globally within 2 weeks of reports of cases from china. organisations such as the global outbreak alert and response network, the coalition for epidemic preparedness innovations, and the global research collaboration for infectious disease preparedness, supported by the who research blueprint and its global coordinating mechanism, were able to accelerate the outbreak response and rapidly initiate technical platforms for the development of vaccines and therapeutics. 29 standardised data collection tools were distributed and used within a matter of weeks, and the first clinical trials for therapeutic interventions was initiated in january. 30 china has higher medical standards, a better educated healthcare workforce, and more technical and scientific expertise now than in 2003. china's current response contains much more transparency and decisive action, which was initiated much earlier in the current outbreak than in the 2003 outbreak. 31 so why have the case numbers of covid19 already surpassed those of sars by jan 30, 2020? there are several explanations. first, the situation is different. wuhan, the epicentre of covid19, combines multiple elements that make containment challenging. as the largest city (>11 million) in central china, wuhan is a major transport hub and centre for industry and commerce, home to the largest train station, biggest airport, and largest deepwater port in central china. 32 china's outward travel has more than doubled in the past decade, and its urban population densities possibly even tripled. the proximity of people in residential housing, during commute, and in work environments in a megacity such as wuhan amplifies persontoperson transmission. the sheer population size is the single biggest challenge. hospitals were initially overwhelmed by the number of patients, and many patients were not hospitalised because of a shortage of hospital beds, thereby contributing to seeding in the community. a new hospital was built within 10 days. a massive banquet with 40 000 guests took place just before the lockdown, further exacerbating community spread. worse, in the days just before wuhan was put under lockdown, more than 5 million people (many of whom might have been incubating the virus) had travelled out because of the personal view upcoming spring festival, thus spreading covid19 to other provinces in china. the high connectivity of wuhan to international airports further facilitated rapid spread to cities and countries with high air passenger volumes from china, such as singapore, japan, and thailand. 33, 34 a second explanation might be that the infectious period is different. isolation was effective for sars because peak viral shedding occurred after patients were already quite ill with respiratory symptoms and could be easily identified. although asymptomatic or mildly symptomatic patients have been reported for sars, 35 no known transmission occurred from these patients. by contrast, preliminary evidence from exported covid19 cases suggest that transmission during the early phase of illness also seems to contribute to overall transmission; 36, 37 therefore, isolation of more severely ill patients at the time of presentation to healthcare facilities will be too late. the effectiveness of isolation and contact tracing methods depends on the proportion of transmission that occurs before symptom onset. presymptomatic transmission will also make temperature screening less effective. 38 a third explanation could be that the trans missibility might be higher for covid19 than for sars. r₀ is a central concept in infectious disease epidemiology, indicating the risk of an infectious agent with respect to its epidemic potential. a recent review (published in february, 2020) found the average r₀ of covid19 to be 3·28 and median r₀ to be 2·79, higher than that of sars, although more accurate estimates can only be ascertained when the epidemic stablises. 39 the speed of spread of covid19from the first documented case in early december, 2019, to 80 000 cases by the end of february, 2020, despite massive con tainment efforts-is certainly much faster than that reported for sars between november, 2002, and march, 2003, before any forms of containment were even instituted. the high attack rates on the cruise ship diamond princess in japan, with more than 700 on board infected out of approximately 3700 cruise ship passengers and crew members by feb 28, 2020, despite public health measures, suggest a very high transmissibility. 40 a fourth explanation is that the clinical spectrum is different. china's initial case definition was focused on pneumonia, and initial case fatality rates (cfr) were reported at about 10% on the basis of this narrow case definition. 6 however, as the epidemic unfolds, it has become apparent that mild cases are common in covid19. patients with mild disease manifestations will be missed even if a more sensitive surveillance system were in place, and these patients might spread the disease silently, similar to influenza. on feb 18, 2020, the chinese center for disease control and prevention published their data of the first 72 314 patients including 44 672 patients with confirmed covid19. 41 as many as 81% of the patients with confirmed covid19 were reported to have a mild disease or less severe forms of pneumonia, whereas 13·8% had a severe condition and 4·7% were critically ill. the study also noted that nearly half (49%) of the critically ill patients have died. of the patients from the diamond princess cruise ship, which provides a captive cohort to study the virus, more than 10% were asymptomatic upon diagnosis. estimating cfrs remains elusive, as many patients are still in the early stages of disease, and more fatalities could occur given that death seems to occur 2-4 weeks after onset of symptoms. 6, 7 the current estimate by the chinese center for disease control and prevention is a cfr of 2·3%, with increasing cfrs for patients with advanced age or comorbidities. however, the report also shows that cfrs vary greatly between wuhan and other provinces in china; therefore some uncertainty remains around cfrs. with already more than 3600 cases imported into or acquired in other countries (or on cruise ships), more information on the full spectrum of disease and a better estimate of the cfr should be available in the next few weeks. of note is that even if the cfr of covid19 (possibly <2%) is far lower than that of sars (10%), this is not reassuring, as a highly transmissible disease with low cfr will result in many more cases, and therefore also ultimately more deaths than sars. a fifth explanation is that community spread is more prominent. whereas sars was mainly an outbreak propagated within hospitals, widespread community transmission is already evident for covid19. by feb 28, 2020, more than 82 000 cases had been reported. some models indicate that several hundred thousand infections might already exist in china. consequently, there will be more unknown contacts than known contacts in the community, which means that many contacts who will subsequently develop an infection are not quarantined and under proper medical observation. hence, china decided to enforce the most drastic of all classic public health measures: community containment with social distancing, community use of facemasks, and a lockdown of wuhan's public transportation, including buses, trains, ferries, and airport. as the communitybased outbreak spread, the lockdown was extended to more than 60 million residents in more than 20 cities by jan 30, 2020. china has essentially issued the largest quarantine in history to prevent its spread to the rest of the world. by jan 30, 2020, 113 579 close contacts have been tracked and a total of 102 427 people were receiving medical observation. this approach is an unprecedented gigantic effort that surpasses all efforts done during sars. the gigantic efforts come at a cost to travel and trade, to china's economy and beyond, let alone the mental health of millions of people under lockdown. these sacrifices are being made because the memories of sars fuel hope that containment is feasible. whether these rigorous measures will indeed result in the same success as for sars depends on the extent of transmissibility of personal view subclinical cases (asymptomatic or mildly symptomatic), including the timing of peak viral shedding during the course of disease, as well as on the role of fomites and other environmental contamination in propagating transmission. the answers to these questions will determine the success. until those answers are known, the political and medical community needs to persist with containment efforts with the tools that are available at hand for the time being. china should be commended for its political will in implementing what might appear to be extreme measures. undoubtedly, no other country could enact what china is currently doing. the daily decline in new cases by mid february suggests that china is on the right path, showing that containment could be feasible. other countries should be aware and reduce the spread of covid19. what is already known is that exportations to other countries need not result in rapid largescale outbreaks, if the countries have the political will to rapidly implement early case detection, prompt isolation of ill people, comprehensive contact tracing, and immediate quarantine of all contacts. if this approach is not feasible because of widespread community transmission, then community quarantine is also needed with rigorous implementation of social distancing. containment of covid19 should remain the focus at the moment. the shortterm cost of containment will be far lower than the longterm cost of noncontainment. however, closures of institutions and public places, and restrictions in travel and trade, cannot be maintained indefinitely. countries have to face the reality that individualcase containment might not be possible in the long run, and there might be the need to move from containment to mitigation, balancing the costs and benefits of public health measures. even if our public health measures are not able to fully contain the spread of covid19 because of the virus characteristics, they will still be effective in delaying the onset of widespread community trans mission, reducing peak incidence and its impact on public services, and decreasing the overall attack rate. in addition, minimising the size of the outbreak or suppressing its peak can reduce global deaths by providing health systems with the opportunity to scale up and respond, and to slow down the global spread until effective vaccines become available. aws initiated this personal view and wrote the first draft. all authors contributed equally to the final manuscript. we declare no competing interests. genomic characterization of the 2019 novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia transmission dynamics and control of severe acute respiratory syndrome 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 first case of 2019 novel coronavirus in the united states time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid19) pneumonia isolation, quarantine, social distancing and community containment: pivotal role for oldstyle public health measures in the novel coronavirus (2019ncov) outbreak battling 21stcentury scourges with a 14thcentury toolbox viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome predicting super spreading events during the 2003 severe acute respiratory syndrome epidemics in hong kong and singapore modelling strategies for controlling sars outbreaks household transmission of sars epidemiology and control of sars in singapore hospitalized patients with bacterial infections: a potential focus of sars transmission during an outbreak public health and ethical considerations in planning for quarantine public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto sars: public health measures in hong kong controlling sars: a review on china's response compared with other sarsaffected countries the impact of public health control measures during the sars epidemic in mainland china a tale of two cities: community psychobehavioral surveillance and related impact on outbreak control in hong kong and singapore during the severe acute respiratory syndrome epidemic the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients sars in healthcare facilities sars in singapore-key lessons from an epidemic experience of severe acute respiratory syndrome in singapore: importation of cases, and defense strategies at the airport the severe acute respiratory syndrome: impact on travel and tourism sars to novel coronavirus-old lessons and new lessons a novel coronavirus outbreak of global health concern china's response to a novel coronavirus stands in stark contrast to the 2002 sars outbreak response travelers give wings to novel coronavirus (2019ncov) potential for global spread of a novel coronavirus from china pneumonia of unknown etiology in wuhan, china: potential for international spread via commercial air travel asymptomatic sars coronavirus infection among healthcare workers transmission of 2019ncov infection from an asymptomatic contact in germany importation and humantohuman transmission of a novel coronavirus in vietnam effectiveness of airport screening at detecting travellers infected with novel coronavirus the reproductive number of covid19 is higher compared to sars coronavirus covid19 outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid19) -china, 2020. china cdc weekly 2020 key: cord-292749-f15h3315 authors: azman, andrew s; luquero, francisco j title: from china: hope and lessons for covid-19 control date: 2020-04-03 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30264-4 sha: doc_id: 292749 cord_uid: f15h3315 nan juanjuan zhang and colleagues 1 use detailed, publicly available data to explore key epidemiological features of the coronavirus disease 2019 (covid-19) pandemic in china. outside the original epicentre of hubei province, they found that the effective reproduction number dropped below the critical threshold of 1 by the end of january, 2020, for nine heavily affected chinese provinces or cities. this finding suggests significant slowing of local transmission. importantly, these reductions were achieved in a matter of weeks from the first signs of local transmission in most provinces. although the true causal nature of these transmission reductions is not addressed in zhang and colleagues' analyses, it is probably due to the strict governmentimposed restrictions on movement of people and social gatherings, widespread symptom screening, testing and quarantine programmes, and the strong emphasis on personal behaviour change (eg, hand hygiene, mask use, and physical distancing) to reduce the risk of transmission. the authors also found, as others have shown, 2 that the mean incubation period and serial interval were of similar length (5·2 days [95% ci 1·8-12·4] and 5·1 days [1·3-11·6], respectively), suggesting an important role of transmission before or soon after symptoms have developed. although this study has a number of limitations, it illustrates the power of rapid openly available data for providing important insights to guide complex policy decisions in the coming months. the authors used detailed, publicly available line lists, epidemiological reports, and case and contact investigation results from across china. although, in the past, china has been criticised for a lack of transparency related to epidemiological surveillance data, this rapid openness goes beyond what most countries are doing today. 3 rapid analyses, including computational modelling efforts, are vital to assist decision makers in these largely uncharted waters; however, these analyses are only as good as their data. our daily understanding of the pandemic is primarily based on the number of confirmed cases reported (eg, who daily reports and online dashboards 4 ), which can only be interpreted with an understanding of who is being tested (eg, only severe cases) and laboratory capacity. to correct the epidemic curves, data on testing capacity and test eligibility criteria over time across the globe are urgently needed. furthermore, insights to the frequency of asymptomatic and mildly symptomatic infections from individuals tested for the virus or antibody responses, irrespective of symptoms, will greatly improve real-time assessments. 5 the interventions implemented throughout china include complete lockdown of cities, active case surveillance, rapid investments in increased testing capacity, isolation of cases, treatment of severe cases, quarantine of cases and high-risk groups, and behavioural risk-reduction strategies, such as the compulsory use of masks in the general population. the trajectory of the epidemic curves in china alone suggest that these measures-some of them extreme-might have led to substantial reductions in transmission as of late march, 2020. china made difficult decisions with complex trade-offs between economic and social consequences and acute health effects on the basis of little historical data. these decisions pave the way for other countries to design responses to covid-19 on the basis of their experiences. the encouraging results from zhang and colleagues' study provide hope that rapid control might be possible, although with high economic and social costs. countries across the world are making some of the same policy decisions, effectively halting their economies in the hopes of avoiding a massive death toll, but such lockdowns cannot go on forever. in the search for a new sustainable normal, countries and municipalities will inevitably adopt a range of approaches adapted to local specificities in the coming months. through open documentation of these varying policy choices and timelines, and real-time assessments of their effects, we can and must generate evidence to minimise the acute and long-term consequences of this pandemic. we declare no competing interests. cdc deletes coronavirus testing numbers from website. the independent an interactive web-based dashboard to track covid-19 in real time substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) key: cord-314734-ai0hz4uq authors: hung, ivan fan-ngai; cheng, vincent chi-chung; li, xin; tam, anthony raymond; hung, derek ling-lung; chiu, kelvin hei-yeung; yip, cyril chik-yan; cai, jian-piao; ho, deborah tip-yin; wong, shuk-ching; leung, sally sau-man; chu, man-yee; tang, milky oi-yan; chen, jonathan hon-kwan; poon, rosana wing-shan; fung, agnes yim-fong; zhang, ricky ruiqi; yan, erica yuen-wing; chen, lin-lei; choi, charlotte yee-ki; leung, kit-hang; chung, tom wai-hin; lam, sonia hiu-yin; lam, tina poy-wing; chan, jasper fuk-woo; chan, kwok-hung; wu, tak-chiu; ho, pak-leung; chan, johnny wai-man; lau, chak-sing; to, kelvin kai-wang; yuen, kwok-yung title: sars-cov-2 shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series date: 2020-06-12 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30364-9 sha: doc_id: 314734 cord_uid: ai0hz4uq background: a cruise ship is a closed-off environment that simulates the basic functioning of a city in terms of living conditions and interpersonal interactions. thus, the diamond princess cruise ship, which was quarantined because of an onboard outbreak of covid-19 in february, 2020, provides an opportunity to define the shedding pattern of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and patient antibody responses before and after the onset of symptoms. methods: we recruited adult (≥18 years) passengers from hong kong who had been on board the diamond princess cruise ship docked in yokohama, japan in february, 2020. all participants had been found to be negative for sars-cov-2 by rt-pcr 4 days before disembarking and were transferred to further quarantine in a public estate in hong kong, where they were recruited. participants were prospectively screened by quantitative rt-pcr (rt-qpcr) of nasopharyngeal and throat swabs, and serum igg and igm against internal nucleoprotein and the surface spike receptor-binding protein (rbd) of sars-cov-2 at baseline (upon entering quarantine) and on days 4, 8, and 12 of quarantine. findings: on feb 22, 2020, 215 adults were recruited, of whom nine (4%; 95% ci 2–8) were positive for sars-cov-2 by rt-qpcr or serology and were hospitalised. of these nine patients, nasopharyngeal swab rt-qpcr was positive in eight patients (89%; 57–99) at baseline. all nine patients were positive for anti-rbd igg by day 8. eight (89%; 57–99) were simultaneously positive for nasopharyngeal swab rt-pcr and anti-rbd igg. one patient who was positive for anti-rbd igg and had a negative viral load had multifocal peripheral ground-glass changes on high-resolution ct that were typical of covid-19. five patients (56%; 27–81) with ground-glass changes on high-resolution ct were found to have higher anti-nucleoprotein-igg od values on day 8 and 12 and anti-rbd igg od value on day 12 than patients without ground-glass changes. six (67%; 35–88) patients remained asymptomatic throughout the 14-day quarantine period. interpretation: patients with covid-19 can develop asymptomatic lung infection with viral shedding and those with evidence of pneumonia on imaging tend to have an increased antibody response. positive igg or igm confirmed infection of covid-19 in both symptomatic and asymptomatic patients. a combination of rt-pcr and serology should be implemented for case finding and contact tracing to facilitate early diagnosis, prompt isolation, and treatment. funding: shaw foundation hong kong; sanming-project of medicine (shenzhen); high level-hospital program (guangdong health commission). a modern cruise ship is like a travelling city, with a common food and water supply, shared sanitation and airconditioning systems, and a large population confined together. 1 the individuals are often from a middle-toupper social class, with different cultures, immunisation backgrounds, and health statuses. the proximity of passen gers and crew members in a semi-enclosed environ ment, with interactions in the dining halls, recreational rooms, spas, and pools, creates a unique environment for the person-to-person transmission of microbes. contamination of commonly shared and frequently touched surfaces, food, or water supply or sanitation systems can occur and can cause considerable morbidity and mortality. cruise passengers tend to be older people, with the 60-69-year-old group accounting for the majority, 2 who are more likely to have underlying chronic medical comorbidities and thus be more susceptible to infection and associated complications than the general population. common reported out breaks on cruise ships have included respiratory infections with influenza a h1n1 and h3n2, legionnaires' disease, and acute gastroenteritis due to norovirus. 1, [3] [4] [5] [6] since the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in december, 2019, the virus has caused a pandemic affecting more than 4·7 million people, with more than 300 000 deaths in 229 countries. [7] [8] [9] [10] on jan 20, 2020, an 80-year-old man from hong kong boarded the diamond princess cruise ship in yokohama, japan, and disembarked in hong kong on jan 25, 2020 (figure 1). on feb 1, 2020, he was hospitalised with fever and tested positive for sars-cov-2. the cruise continued to vietnam and taiwan, then returned to yokohama, at which time ten passengers were diagnosed with coronavirus disease 2019 (covid-19), 11 and a 14-day quarantine order was imposed for all passengers and crew on board. between feb 5, and feb 17, 2020 , the japanese health ministry tested all passengers by throat swab rt-pcr; individuals who tested positive were isolated in local hospitals. individuals who tested negative were kept on board confined to their cabins and were only allowed out of the cabin for 1 h per day to exercise. the ship remained docked in the port of yokohama. by feb 17, countries were allowed to air-evacuate citizens back to their home nations. therefore, the hong kong government chartered two flights on feb 20 and feb 21 to transfer passengers who had tested negative to hong kong for a further 14 days of quarantine in a newly completed public estate. as of may 14, 2020, 712 (19%) of the 3711 passengers and crew had been infected with sars-cov-2, and 13 deaths had occurred among those with confirmed infection. 12 we investigated the sequential sars-cov-2 shedding and the specific antibody response in this cohort of passengers quarantined in hong kong. hong kong citizens were air-evacuated from the diamond princess cruise ship on feb 20 and feb 21, 2020, and entered quarantine in the chun yeung estate, a newly built and vacant public estate in hong kong, on feb 22. each unit had a bathroom and kitchen and housed one to two people. during the 14-day quarantine period, they were placed in complete isolation. only health-care workers from the hong kong department of health would visit daily to do health checks and to deliver meals. family visits and contact with the general public were not allowed and individuals were asked to handle their own laundry. with approval by the department of health, we visited and collected samples from the individuals together with the aforementioned health workers. all adult passengers aged 18 years and older were screened and given the choice to join the study. there were no exclusion criteria. all participants provided written informed consent and could withdraw from the study at any point. the institutional review board of the university of hong kong and hospital authority approved this study. participants were screened by quantitative rt-pcr (rt-qpcr) for sars-cov-2 in nasopharyngeal, throat, and rectal swabs at baseline (feb 22, 2020), day 4, day 8, and day 12, and tested for serum igg and igm against both internal nucleoprotein and the surface spike receptorbinding protein (rbd) of sars-cov-2, at baseline, day 4, day 8, and day 12. the following participant data were entered into a database: name, hong kong personal identification number, age, sex, medical history by completion of a questionnaire, presenting symptoms and signs, and laboratory, radiological, virological, and serological evidence before this study we searched pubmed on march 14, 2020, with no date restrictions, for articles in english, using the terms "covid-19", "coronavirus", "antibody", "viral load", "cruise ship", "quarantine", "shedding", and "seroconversion". our search did not identify any reports on the prospective follow-up of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) viral shedding and seroconversion in a cruise ship setting. we identified one case report on the clinical presentation of two individuals on the diamond princess cruise ship, and an article estimating the effectiveness of public health measures on controlling the covid-19 epidemic potential on a cruise ship. to our knowledge, this is the first cruise ship study of the clinical evolution and seroconversion from coronavirus disease 2019 (covid-19) from last possible exposure to the pathogen to the end of the suspected incubation period. the study showed that asymptomatic individuals might seroconvert while carrying a high viral load and continue to shed the virus. patients who had viral pneumonitis detected by high-resolution ct tended to have a higher antibody response. high-resolution ct also helped to establish a clinical diagnosis and detect cases of asymptomatic lung infection. asymptomatic covid-19 infection with continuous viral shedding makes infection control difficult. therefore, a combination of rt-pcr and serology should be implemented for case finding and contact tracing in a community outbreak of covid-19 to facilitate early diagnosis, prompt isolation, and treatment. high-resolution ct could also help to detect cases of asymptomatic infection. findings. participants who developed symptoms or who were found to have virological or serological evidence of sars-cov-2 infection at the time of screening were admitted to the queen mary hospital or the queen elizabeth hospital in hong kong for further investigation and treatment. all hospitalised participants under went chest radiograph and high-resolution ct examination. nucleic acid extraction and rt-qpcr were done as previously reported. 13 enzyme immunoassays for nucleoprotein and rbd and optical density (od) cutoff values at 450 nm and 620 nm were established as previously described. 14 the cutoff value for positivity was based on the mean value plus 3 sds of the negative control, using archived serum specimens collected in 2018 from 93 anonymous individuals without sars-cov-2 infection. 14 descriptive statistics are presented. findings of the participants who tested positive for sars-cov-2 are presented as a case series. all data were analysed with spss (version 26.0). 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 full access to all the data in the study and had final responsibility for the decision to submit for publication. of 3711 passengers and crew members on board the diamond princess cruise ship, 369 passengers (10%) were from hong kong (appendix). the passengers returned to yokohama on feb 1, 2020, and were quarantined onboard for a further 20 days (figure 1). by feb 20, 2020, 76 passengers from hong kong were hospitalised in japan after testing positive for sars-cov-2 by throat swab rt-pcr, of whom two individuals died from complications of the infection (appendix). 293 passengers from hong kong who tested negative were allowed to disembark, of whom 65 decided to stay in japan and 228 passengers (225 adults and three children) returned to hong kong for further quarantine. the three children were excluded from the study because their clinical data were not available for assessment. ten passengers declined to join the study and remained well until discharge. thus, 215 adult participants were enrolled. baseline charac teristics of participants who tested positive (n=9) and negative (n=206) for sars-cov-2 were well matched (table 1) . nine (4%; 95% ci 2-8) of 215 participants were found to have evidence of sars-cov-2 infection and were hospitalised. these nine individuals were not related to each other; they boarded the ship in hong kong on jan 25, 2020, when the index passenger had already disembarked and therefore had no contact with him (figure 1). at the last onboard covid-19 testing by throat swab on feb 17, 2020, all nine individuals tested negative. both parents and the grandfather of patient 7 had tested positive for sars-cov-2 at the initial governmental screen in japan. they were subsequently discharged from hospital in japan after testing negative, and they returned to hong kong in march, 2020. we have ordered the nine patients who tested positive for sars-cov-2 in the probable order of infection, according to their viral load and serological profile ( patients vs none). three (33%; 95% ci 12-65) of the nine patients with sars-cov-2 were men, and six (67%; 35-88) had underlying diseases, including hypertension, ischaemic heart disease, diabetes, hyperlipidaemia, old pulmonary tuberculosis, and non-toxic multinodular goitre. the median age was 58 years (iqr 57-65). the median (iqr) laboratory findings were: haemoglobin 12·5 g/dl (12·0-13·6), white cell count 6·49 × 10⁹ per l (5·82-7·20), neutrophil count 4·84 × 10⁹ per l (3·18-5·39), lymphocyte positive viral load could precede the onset of symptoms, as evidenced by patient 9. the median nasopharyngeal viral load at baseline was 4·31 log 10 copies per ml (iqr 3·79-6·65), on day 4 was 2·87 log 10 copies per ml (2·70-4·95), on day 8 was 2·84 log 10 copies per ml (2·70-4·61), and on day 12 was 2·7 log 10 copies per ml (2·70-3·44). three patients (33%; 95% ci 12-65) were found to be positive for sars-cov-2 on throat swab rt-qpcr (table 2). the median throat swab viral load at baseline was 2·70 log 10 copies per ml (iqr 2·70-5·50) and at day 4 was 2·70 log 10 copies per ml (2·70-3·66). the viral loads in the throat swabs were lower than those in the nasopharyngeal swabs at the same timepoints. rectal swab viral load was negative in all nine patients. the nasopharyngeal sars-cov-2 rt-qpcr median viral load was higher in symptomatic patients than in asymptomatic patients (7·62 log 10 copies per ml vs 3·86 log 10 copies per ml). chest radiographs showed changes consistent with interstitial pneumonia in four patients (44%; 95% ci 19-73), with right lower lobe changes in patient 2, right middle lobe changes in patient 6, left lower lobe changes in patient 7, and right lower lobe changes in patient 9 (table 2, figure 3 ). chest radiographs were normal in four patients (44%; 95% ci 19-73) and patient 4 had old granuloma from previous pulmonary tuberculosis. highresolution ct (table 2, figure 3 ) showed ground-glass changes typical of covid-19 in five patients (56%; 95% ci 27-81): patients 1 and 9 had right lower lobe ground-glass changes, patient 2 had right upper and lower lobe changes, patient 6 had right middle lobe changes, and patient 7 had left upper and lower lobe changes. patients 3, 5, and 8 had normal chest radiograph and ct results, but tested positive on nasopharyngeal swab rt-pcr (table 2) . patients 3 and 5 were asymp tomatic, whereas patient 8 developed fever and rhinorrhoea on day -2. three patients (patients 1, 2, and 6) had pulmonary ct changes but were asymptomatic. all nine patients were found to be positive for anti-rbd igg (100% seroconversion) on days 8 and 12 (table 2) . to our knowledge, this is the first study that investigates the real-time progression of a group of individuals who were initially negative for sars-cov-2 on rt-pcr after exposure to an outbreak of covid-19 on a cruise ship. nine individuals later tested positive on rt-pcr from nasopharyngeal swab, had high-resolution ct changes, or showed seroconversion, and were thus found to be infected with sars-cov-2. despite the positive clinical findings, six of the nine patients remained asymptomatic throughout the 14-day quarantine after leaving the ship (ratio of symptomatic to asymptomatic patients 1:2). if the cruise ship epidemic is a microcosm of the community outbreak scenario, then individuals with or without pneumonia could carry the virus for a long period but remain asymptomatic. 8, 9 asymptomatic patients with few or no comorbidities could spread the disease, whereas symptomatic patients represent only a small, but visible, proportion of total cases. 15 this finding is particularly important in densely populated cities like hong kong, with an average living space of 4·6 m² per person, equivalent to half a parking space. 16 viral shedding from asymptomatic individuals could be transmitting the virus to others, making it difficult to identify and isolate index patients by contact-tracing for early quarantine. trans mission from asymptomatic individuals could explain in part the rapid increases in numbers of new cases in high-incidence countries such as the usa, russia, the uk, brazil, and european nations. 10 sars-cov-2 could be spreading via respiratory droplets or through direct and indirect contact via the respiratory and gastrointestinal tracts. 17, 18 considering that most outbreaks on cruise ships are of respiratory and gastrointestinal infections, the risk of infection and associated morbidity and mortality in passengers increases with longer duration of travel and colder weather. 19, 20 the period of exposure on the diamond princess cruise ship was 3 weeks, during which the passengers were unable to disembark, which is consistent with the high number of passengers (n=712) who contracted the infection. 12 the situation was likely to have been made worse by infected but asymptomatic crew members who continued to work unprotected, but who could have been shedding the virus. a recent modelling study 11 estimated that the initial basic reproduction number (r 0 ) of the outbreak on the diamond princess cruise ship was 14·8, before any infection control measures had been implemented and while the passengers were a completely naive group. the r 0 then decreased to 1·78 after the quarantine and transfer interventions were initiated (feb 5-17, 2020). 11 this estimation of r 0 was lower than that of 2·2 (95% ci 1·4-3·9) that was suggested at the beginning of the outbreak in wuhan, 21 although a higher r 0 of more than 3 has also been reported. 22 an r 0 of 2·2 for covid-19 would be similar to that of sars and influenza. 23 nevertheless, evacuation of all passengers and crews early on in the outbreak would probably have prevented more infections, as suggested by the current findings. because the screening test was done between early february and mid-february, before the air evacuation, we assumed that passengers could have acquired and been incubating the infection shortly before the test, and therefore tested negative, or they could have acquired the infection after the test. based on this assumption, we screened 215 passengers by serial virological, serological, and radiological methods. our findings show a dynamic clinical presentation of sars-cov-2. of the three diagnostic methods we used, the immunoassay for igg and nasopharyngeal swab rt-qpcr were the most sensitive. throat swab rt-qpcr was less sensitive; 24 it identified only the two patients who consistently had the highest viral load in nasopharyngeal swab rt-qpcr and did not identify the other six patients identified by nasopharyngeal swab rt-qpcr. the apparent low sensitivity of throat swab rt-qpcr might also account for the negative screening test done on board the ship by the japanese health ministry. the three patients who were febrile felt transiently unwell, with concomitant symptoms of malaise, unproductive cough, and rhinorrhoea. the higher baseline nasopharyngeal swab viral load in symptomatic than asymptomatic patients in this study could be explained by the difference in time and degree of exposure to the virus, and might not be related to the symptoms. 25 nevertheless, the per sistent nasopharyngeal viral load in asymptomatic patients is a major concern for infection control. rectal swabs were negative in all nine patients, which was expected because none of the patients presented with diarrhoea. 18 important initial laboratory markers, including lymphocyte count, c-reactive protein, and lactate dehydrogenase, remained normal in these asym ptomatic patients. seroconversion occurred in all nine patients and it was the only diagnostic marker that was positive for one patient in this cohort who had high-resolution ct changes characteristic of a recent sars-cov-2 infection. for those patients who might have acquired the infection earliest and only shortly before the screening test, they might have seroconverted with a high igg titre and remained asymptomatic with a negative rt-qpcr, with high-resolution ct radiological evidence of sars-cov-2 pneumonitis with ground-glass changes. the immunological response could be explained by the lower respiratory tract infection consistent with pneumonia. similar to influenza viral infection, patients with severe disease and pneumonia had the highest antibody response. 26 this finding could be related to the more severe viral infection resulting in a stronger induction of the host's adaptive immunity mediated by b and t cells. 27 because sars-cov-2 and sars-cov have 90% amino acid homology for nucleoprotein and 73% amino acid homology for rbd, serum from patients with sars-cov might cross-react with our sars-cov-2 immunoassay, as shown by another group. 28 however, the cross-reactivity between sars-cov-2 and other human coronaviruses is expected to be lower, because the amino acid homology with nucleoprotein is less than 50%. based on our previous study, 14 the sensitivity of the serology assay was 94% for anti-nucleoprotein igg, 88% for anti-nucleoprotein igm, 100% for anti-rbd igg, and 94% for anti-rbd igm. as stated in the methods, the cutoff was based on a pre-pandemic general population cohort of 93 individuals without sars-cov-2 infection. therefore, we expected that 1% would be outliers. 14 the clinical presentation of the nine patients in this cohort ranged from mild to severe. among the mild cases, patients could remain asymptomatic (patients 3, 4, and 5) or symptomatic (patient 8), with no lower respiratory tract involvement, as suggested by normal chest radiograph and high-resolution ct. their antinucleoprotein and anti-rbd igg levels were low. patients with moderate infection had lower respiratory tract involvement, as suggested by the ground glass changes on high-resolution ct. nevertheless, they could still remain asymptomatic (patients 1, 2, and 6) or become symptomatic (patient 7). these patients had higher antinucleoprotein and anti-rbd igg than patients with mild disease. patient 9, who acquired the infection more recently, developed symptoms upon hospitalisation. the infection was more severe, involving the lower respiratory tract, with ground glass changes on high-resolution ct. seroconversion took place during the observation period, with anti-nucleoprotein and anti-rbd igg still increasing at the end of the observation period. overall, anti-rbd igg was the most sensitive serology marker, although the anti-nucleoprotein igg response was stronger. the difference in anti-rbd igg and antinucleoprotein igg could be related to the severity of the viral infection, in which anti-rbd igg was positive even in patients with upper respiratory tract infection, but at low od values. indeed, serology provides a feasible strategy to track sars-cov-2 infections. 29 we also investigated changes in igm/igg ratio for anti-nucleoprotein and anti-rbd and did not identify any trends that might reflect the timing of infection. similar to findings from one of the largest case series from china, 8 high-resolution ct provided a reasonable diagnostic tool, especially in asymptomatic patients. in this cohort, it was able to detect lung ground-glass opacities in five of nine patients, including the three patients with asymptomatic lung infection. similar to other studies, right-side changes were more common. 30 individuals with more severe radiological changes tended to have higher nasopharyngeal viral load. high-resolution ct could also detect an rt-qpcr-negative asymptomatic patient (patient 1) who had high igg and igm titres, suggestive of a recent infection. nevertheless, individuals who develop only an upper respiratory tract infection could still have a normal high-resolution ct. chest radiograph detected radiological changes in only four patients. this study has several limitations. we did not have access to the data of patients who were hospitalised in japan. we also did not have the cabin number and location of the participants when they were on board the ship, which could have provided some insight into how the infection was transmitted. ten passengers did not give consent to participate in the study, and therefore we did not have their virological or serological data. lastly, the study period was limited by the quarantine period and we could not follow up the participants beyond 14 days. the participants could have become rt-pcrpositive or antibody-positive after the quarantine period. the cruise ship setting provided an unusual opportunity to study the viral shedding and seroconversion of sars-cov-2, mimicking the start of a community outbreak. passengers who are exposed to the virus might seroconvert while remaining asymptomatic with a high viral load, and they could continue to shed the virus. highresolution ct also helped to establish a clinical diagnosis and to detect cases of asymptomatic lung infection. a combination of rt-qpcr and serology should be done to screen for community outbreaks and effectively perform contact tracing. we declare no competing interests. infections on cruise ships how old is the average cruise passenger? outbreaks of pandemic (h1n1) 2009 and seasonal influenza a (h3n2) on cruise ship a large outbreak of influenza a and b on a cruise ship causing widespread morbidity molecular characterization of influenza b virus outbreak on a cruise ship in brazil 2012 respiratory infections and gastrointestinal illness on a cruise ship: a three-year prospective study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of coronavirus disease 2019 in china who. coronavirus disease 2019 (covid-19) situation report-120 covid-19 outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures about new coronavirus infection improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19 rdrp/hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study covid-19 in 2 persons with mild upper respiratory symptoms on a cruise ship hong kong's poor live in homes smaller than prison cells-study can we contain the covid-19 outbreak with the same measures as for sars? gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis travellers and influenza: risk and prevention incidence and risk factors of diarrhoea in dutch travellers: consequences for priorities in pre-travel health advice early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia covid-19 outbreak progression in italian regions: approaching the peak by the end of march in northern italy and first week of april in southern italy pattern of early human-to-human transmission of wuhan audio interview: what clinicians need to know in diagnosing and treating covid-19 sars-cov-2 viral load in upper respiratory specimens of infected patients effect of clinical and virological parameters on the level of neutralizing antibody against pandemic influenza a virus h1n1 host immune response to influenza a infection severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients singapore claims first use of antibody test to track coronavirus infections radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study we thank all our health-care and laboratory colleagues from the queen mary key: cord-289744-suiqh3gv authors: lafolie, jérémy; labbé, andré; l'honneur, anne sophie; madhi, fouad; pereira, bruno; decobert, marion; adam, marie noelle; gouraud, françois; faibis, frédéric; arditty, francois; marque-juillet, stéphanie; guitteny, marie aline; lagathu, gisele; verdan, matthieu; rozenberg, flore; mirand, audrey; peigue-lafeuille, hélène; henquell, cécile; bailly, jean-luc; archimbaud, christine title: assessment of blood enterovirus pcr testing in paediatric populations with fever without source, sepsis-like disease, or suspected meningitis: a prospective, multicentre, observational cohort study date: 2018-10-30 journal: lancet infect dis doi: 10.1016/s1473-3099(18)30479-1 sha: doc_id: 289744 cord_uid: suiqh3gv background: enteroviruses are the most frequent cause of acute meningitis and are seen increasingly in sepsis-like disease and fever without source in the paediatric population. detection of enterovirus in cerebrospinal fluid (csf) specimens by pcr is the gold standard diagnostic test. our aim was to assess a method of detecting enterovirus in blood specimens by pcr. methods: we did a prospective, multicentre, observational study at 35 french paediatric and emergency departments in 16 hospitals. we recruited newborn babies (aged ≤28 days) and infants (aged >28 days to ≤2 years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged >2 years to ≤16 years) with suspected meningitis, who were admitted to a participating hospital. we used a standardised form to obtain demographic, clinical, and laboratory data, which were anonymised. enterovirus pcr testing was done in blood and csf specimens. findings: between june 1, 2015, and oct 31, 2015, and between june 1, 2016, and oct 31, 2016, we enrolled 822 patients, of whom 672 had enterovirus pcr testing done in blood and csf specimens. enterovirus was detected in 317 (47%) patients in either blood or csf, or both (71 newborn babies, 83 infants, and 163 children). detection of enterovirus was more frequent in blood samples than in csf specimens of newborn babies (70 [99%] of 71 vs 62 [87%] of 71; p=0·011) and infants (76 [92%] of 83 vs 62 [75%] of 83; p=0·008), and was less frequent in blood samples than in csf specimens of children (90 [55%] of 163 vs 148 [91%] of 163; p<0·0001). detection of enterovirus was more frequent in blood samples than in csf specimens of infants aged 2 years or younger with fever without source (55 [100%] of 55 vs 41 [75%] of 55; p=0·0002) or with sepsis-like disease (16 [100%] of 16 vs nine [56%] of 16; p=0·008). detection of enterovirus was less frequent in blood than in csf of patients with suspected meningitis (165 [67%] of 246 vs 222 [90%] of 246; p<0·0001). interpretation: testing for enterovirus in blood by pcr should be an integral part of clinical practice guidelines for infants aged 2 years or younger. this testing could decrease the length of hospital stay and reduce exposure to antibiotics for low-risk patients admitted to the emergency department with febrile illness. funding: university hospital clermont-ferrand. enteroviruses are the most frequent cause of paediatric aseptic meningitis and are attributed to more than 75% of viral meningitis cases in which a microorganism is identified. 1,2 detection of enterovirus by rt-pcr from cerebrospinal fluid (csf) specimens is recom mended for diagnosis of meningitis caused by entero virus. [3] [4] [5] paediatricians are also confronted frequently with young infants with fever without source or sepsis-like diseases. these febrile illnesses account for 3·4-13·6% of cases seen in emergency departments. 6 symptoms can result either from severe bacterial infection requiring admission to hospital and empirical antibiotic treatments or, most typically, from benign and spontaneously resolving viral infection; therefore, diagnosis is a challenge. additional molecular tests are needed to speed up diagnosis of conditions asso ciated with enterovirus infections. 5 several studies have evaluated testing blood specimens, [7] [8] [9] [10] [11] [12] but as yet no assessment has been done in a large cohort of paediatric patients. the aim of our multicentre study was to assess detection of enterovirus by pcr in blood specimens of newborn babies, infants, and children with fever without source, sepsis-like disease, or suspected meningitis. we did a prospective, multicentre, observational study at 35 paediatric and emergency departments in 16 french hospitals. we restricted enrolment of patients to the seasonal period of increased enterovirus circulation in countries with a temperate climate. 13 we enrolled newborn babies (aged ≤28 days) and infants (aged >28 days to ≤2 years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged >2 years to ≤16 years) with suspected meningitis. all participants were admitted to one of the participating hospitals. we required an edta blood sample (plasma) obtained by venepuncture for participation. we also did lumbar puncture when clinically indicated. we defined fever without source as a body temperature of 38°c or higher for less than 7 days in a child whose medical history and physical examination did not reveal the cause of the fever. 14 we defined sepsis as suspected or proven infection and at least two of another four criteria, one of which had to be abnormal temperature (>38·5°c or <36°c) or abnormal white-blood-cell count (elevated [>20 000 × 10⁹ per l] or depressed [<4000 × 10⁹ per l] for age), and the other criterion could be either tachycardia or bradycardia, or tachypnoea. further criteria for sepsis were a platelet count lower than 100 000 × 10⁹ per l and c-reactive protein greater than 15 mg/l. 15 we defined meningitis as either the presence of age-specific pleocytosis or the presence of at least two of these neurological signs or symptoms: headache, nuchal rigidity, photophobia, bulging fontanelle, irritability, lethargy, nausea, vomiting, or positive kernig's or brudzinsky's signs. we defined pleocytosis as a white-blood-cell count in the csf of more than 19 per µl for newborn babies (aged ≤28 days), ten per µl or more for infants (aged 29-56 days), and five per µl or more for older children (aged >56 days). 16 exclusion criteria were refusal of consent from parents, absence of or insufficient blood samples, and bacterial or other viral infections in blood or csf specimens. we also excluded patients (at a later stage) who were diagnosed evidence before this study we searched pubmed up to feb 7, 2018, for papers reporting paediatric enterovirus diseases and enterovirus pcr testing or molecular detection of viruses in cerebrospinal fluid (csf) or blood specimens of patients with aseptic meningitis, sepsis and sepsis-like disease, or fever without source. we used the search terms "enterovirus", "nonpolio enterovirus", "meningitis", "viral meningitis", "aseptic meningitis", "enterovirus meningitis", "acute meningitis", "sepsis", "sepsis-like disease", "fever", "fever without source", "genome detection", "enterovirus detection", "enterovirus rt-pcr", "molecular detection", "viremia", "viremic", "virus load", "blood", "plasma", and "cerebrospinal fluid". we also reviewed references from relevant articles not identified in the original search. our search identified 12 studies in which enterovirus detection was reported in blood and csf. most studies were retrospective, the number of patients recruited varied between 11 and 34, and blood samples were not obtained in all patients whose csf was tested. two studies of 80 and 122 patients aged 90 days or younger with enterovirus infection were referenced to discuss our enterovirus detection frequency in the blood and csf of febrile infants. in a study of 75 patients aged 16 years or younger with aseptic meningitis, 76% had enterovirus detected in blood samples by pcr. however, in that study, age groups were not analysed separately. in all these studies, symptom duration before lumbar puncture or venepuncture, and time between csf and blood collection, were not recorded. our study of 360 patients with laboratory findings of enterovirus infection is, as far as we are aware, the largest prospective, multicentre, observational study to assess pcr testing for enterovirus in both blood and csf samples from newborn babies (aged ≤28 days) and infants (aged >28 days to ≤2 years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged >2 years to ≤16 years) with suspected meningitis. to our knowledge, our study is the first to show that sensitivity of enterovirus detection in blood samples is related to patients' age and clinical presentation. detection of enterovirus was more frequent in blood samples than in csf specimens of newborn babies and infants with fever without source or sepsis-like diseases, and it was less frequent in blood samples of patients with suspected meningitis. furthermore, our study showed that enterovirus positivity in blood was related inversely to patient's age with meningitis. at present, blood samples from febrile patients in the paediatric emergency department are not sent routinely for enterovirus pcr testing, and only csf samples are sent for infants younger than 90 days or for patients with suspected meningitis. guidelines for biological management of patients aged 2 years or younger with febrile illness in the emergency department need to be reconsidered. when blood is sampled for a complete blood count, an additional blood tube should be obtained for enterovirus pcr testing, which can now be done sufficiently rapidly to have a real effect on infection management. pcr testing of blood samples done in routine practice could result in a more accurate assessment of the actual number of positive cases in patients with suspected meningitis, sepsis-like diseases, and fever without source. a positive enterovirus diagnosis could affect beneficially decisions about a patient's management, by reducing antibiotic or antiviral therapy, avoiding ancillary tests, lowering hospital-related costs, and allowing earlier discharge. with infections in other biological specimens (eg, urine, nasopharyngeal aspirate, or stool). the study was approved by the french ethics committee (au1180), under the institutional review board number 00008526. we obtained verbal consent for use of clinical samples for research from parents or guardians of children aged 8 years or younger and from children older than 8 years. within 24 h of admission, a doctor completed a standardised questionnaire for every patient, with details of the nature and duration of preadmission symptoms and signs and the results of a physical examination done at the time of admission. laboratory findings comprised the date and time at which biological specimens were taken, csf and full blood count characteristics, c-reactive protein assay, and the results of other bacteriological and virological testing of samples recorded by biologists. symptom duration was the interval between onset of symptoms and venepuncture (and lumbar puncture, if indicated). we estimated the onset of symptoms as either 8 am, 2 pm, 8 pm, or 2 am when symptoms were recorded in the morning, afternoon, evening, and night, respectively. csf protein concentration was classified as normal if it was 0·9 g/l or lower for newborn babies (aged ≤28 days) and 0·45 g/l or lower for older children (aged >28 days). investigation for urinary-tract infection in febrile patients was done with urine dipsticks and confirmed by culture of specimens obtained from urethral catheters. a urinary-tract infection was diagnosed as leucocytosis (≥10⁴ cells per ml) and clinically significant bacteria (≥10⁵ colony-forming units per ml) in urine culture. all samples were submitted for routine bacteriological and virological investigations at every participating hospital, according to local practice. a senior paediatrician and the study team reviewed the final diagnosis at discharge. we did pcr testing for enterovirus in blood and csf specimens at microbiology laboratories of five participating hospitals: centre hospitalier universitaire (chu) de clermont-ferrand (clermont-ferrand), cochin hospital (assistance publique-hôpitaux de paris, paris), grand hôpital de l'est francilien, site de meaux (meaux), centre hospitalier de versailles andré mignot (versailles), and chu de rennes (rennes). we used commercial (xpert ev, cepheid, sunnyvale, ca, usa [only used with csf samples]; enterovirus r-gene, biomérieux, marcy l'etoile, france; and o-diaent, diagenode, seraing, belgium) or in-house 17 rt-pcr assays. a diagnosis of enterovirus was established with positive pcr findings in either plasma or csf, or both. for specimens negative for enterovirus on pcr testing, and if the volume of sample remaining was sufficient, we did a specific rt-pcr parecho virus assay in blood and csf (parechovirus r-gene, biomérieux). we typed enterovirus-positive specimens at the national reference centre for enteroviruses and parechoviruses (clermont-ferrand, france) by amplification and sequencing of the vp1 capsid protein. 18 we did statistical analyses with stata 13. statistical tests were two-sided with a type i error set at an α of 0·05. we presented continuous data as median (iqr) for every age group (newborn babies, infants, and children). we estimated κ coefficients and sensitivity, specificity, and predictive values (with 95% cis) for blood enterovirus pcr testing and compared these with values in csf to ascertain validity. we judged κ values according to recommendations: less than 0·2 (negligible), 0·2-0·4 (low or weak consistency), 0·4-0·6 (moderate agreement), 0·6-0·8 (substantial or good agreement), and greater than 0·8 (excellent agreement). 19 for comparisons between groups, we used χ² or fisher's exact tests for categorical variables, then (when appropriate) we did marascuilo's procedure. for quantitative parameters, we used student's t test or the mann-whitney test when assumptions of the t test were not met. we did a regression model for newborn babies and infants to identify factors that were associated independently with viraemia, using a stepwise (backward and forward) approach. we ascertained covariates according to univariate results (entry at p=0·15) and relevant biological and clinical variables-eg, csf whiteblood-cell count, duration of symptoms, tachycardia, hypotonia, irritability, and seizure (adjustment factors). we paid attention to multicollinearity. we expressed results as odds ratios (ors) and 95% cis, and we represented findings using forest plots. we did a sensitivity analysis for multivariate analysis, which we applied to all groups. we also did a sensitivity analysis to assess the effect of any inaccurate dates or times of symptom onset recorded by parents. the funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. ca and al had full access to all data in the study and had final responsibility for the decision to submit for publication. csf=cerebrospinal fluid. *parents did not agree to participation of their child after reading the information leaflet. †pcr inhibitors were present either in blood (n=3) or in csf (n=3) samples. of those with pcr inhibitors in blood, two had negative csf (one infant and one child) and one had positive csf (newborn baby). of those with pcr inhibitors in csf, one had negative blood (infant) and two had positive blood (newborn baby and infant). three patients had enterovirus infection. all six patients were excluded from the analysis. 355 data are n (%) or median (iqr), unless otherwise indicated. csf=cerebrospinal fluid. +=enterovirus pcr positive. -=enterovirus pcr negative. *time between onset of symptoms and venepuncture or lumbar puncture was significant between children and infants and between children and newborn babies (p<0·0001). †time between onset of symptoms and lumbar puncture was significant between infants and newborn babies (p=0·045). a multivariate analysis that adjusted for age, duration of symptoms, hypotonia, irritability, and all factors judged significant in univariate analysis confirmed that patient's age, tachycardia, and exposure to a sick contact were associated significantly with viraemia in patients aged 2 years or younger (figure 2a). in a further multivariate analysis, including patients in all three age groups, the time between onset of symptoms and venepuncture (<24 h, or 2·96, 95% ci 1·57-5·58; p=0·001; 24 h to <48 h, 2·55, 1·27-5·11; p=0·008) and pleocytosis (7·60, 4·34-13·32; p<0·0001) were also associated with viraemia ( figure 2b) . data for patients in each age group with and without enterovirus infection, who had samples available for pcr testing, were compared for differences in symptoms and laboratory characteristics ( (p=0·042), hypotonia (p=0·012), and nausea or vomiting (p<0·0001) than were those without infection. patients of all ages infected with enterovirus were more likely to have been exposed to a sick contact than were those without infection (p≤0·003). newborn babies infected with enterovirus were more likely to show symptoms of irritability (p=0·001) and hypotonia (p=0·033) compared with those testing negative for enterovirus. tachycardia was more frequent in infants infected with enterovirus than in those not infected with enterovirus (p=0·005). all patients recovered from the enterovirus infection. pleocytosis was recorded in 192 (66%) of 292 patients infected with enterovirus compared with 59 (24%) of 246 who were not infected (p=0·0001). pleocytosis increased with patient's age, with 20 (35%) of 58 newborn babies, 33 (47%) of 71 infants, and 139 (85%) of 163 children having pleocytosis. amounts of protein in csf were similar in patients infected and not with enterovirus. blood lymphocyte counts were lower in patients of all ages infected with enterovirus than in those not infected (p≤0·044). prospective enterovirus typing was done for 311 (86%) of 360 patients whose csf or blood specimens, or both, were positive for enterovirus (appendix p 2). viral strains were assigned to 29 different types, eight within the entero virus a species (29 patients) and 21 within the enterovirus b species (282 patients). the enterovirus genotypes e9, e25, e7, and cvb5 were more frequent in newborn babies than in infants and children; patients with suspected sepsis were more likely to be infected with e25 genotype than were patients with other clinical presentations; and those with suspected meningitis were more frequently infected with e30, e6, or cvb5 genotypes. our study shows that, in newborn babies and infants, the sensitivity of enterovirus pcr testing is higher in blood samples than in csf specimens. this finding substantiates those of previous single-centre studies [7] [8] [9] and lends support to use of blood enterovirus testing as a diagnostic adjunct to rapidly identify newborn babies and infants admitted with fever without source, sepsislike disease, or suspected meningitis whose antibiotic treatment can be discontinued and who are eligible for discharge. our study also shows that blood enterovirus pcr testing in children with suspected meningitis has no additional benefit compared with pcr testing in csf. seizures tachycardia exposure to a sick contact hypotonia irritability time between onset of symptoms and blood sample ≥48 h time between onset of symptoms and blood sample 24-48h time between onset of symptoms and blood sample <24h white blood cell count pleocytosis c-reactive protein level (>15 mg/l) this lower sensitivity, compared with that recorded in newborn babies and infants, was attributable mainly to the long period between onset of symptoms and venepuncture. to our knowledge, we report here the largest, prospective, multicentre, observational study to show that positive detection of enterovirus in blood is associated with patient's age and clinical presentation. detection of enterovirus was significantly higher in blood samples than in csf specimens from newborn babies and infants and varied by clinical presentation, with detection higher in patients admitted with fever without source or sepsis-like diseases than in those with suspected meningitis. a positive result for enterovirus in blood samples from patients with suspected meningitis was related inversely to age, with higher detection in newborn babies (97%), then infants (87%), then children (55%). compared with previous reports, enterovirus was detected in blood samples from newborn babies and infants more frequently in our study. in a study of 122 infants (aged ≤90 days) with fever who were infected with enterovirus, pcr yielded equally positive results in blood samples and csf specimens (77% and 83%, respectively). 7 in another study of 80 infants with suspected sepsis, detection of enterovirus was similar in blood and csf samples (69% vs 78%). 8 our study followed guidelines from the uk national institute for health and care excellence (nice) for clinical management in emergency departments of newborn babies, infants, and children with febrile illness, 20 . these data suggest that viraemia occurs early and is of short duration, a finding rarely noted in other reports and only with small patient populations. 21, 22 in some biological diagnostic practices, csf pcr testing is done only in patients with pleocytosis. in our study, we did lumbar puncture in 675 patients, of whom 371 did not have pleocytosis (data not shown). in these patients, however, enterovirus was detected in csf (n=70) and blood (n=98). without detection of enterovirus in blood or csf samples, these patients without pleocytosis would have been discharged without aetiological diagnosis. thus, the diagnostic practice to do enterovirus testing solely in patients with pleocytosis can lead to enterovirus infections being missed. moreover, 144 patients had enterovirus pcr testing done only in blood samples, of whom 40 (28%) had enterovirus infection-mostly newborn babies or infants with fever without source. ahmad and colleagues detected enterovirus in blood samples from 34 (24%) of 139 neonates with sepsis-like illness. 11 the diversity of prevailing enterovirus genotypes during the two seasons of the present study and differences in the distribution of genotypes among age white-blood-cell count (× 10⁹ per l) 23 harvala and colleagues 24 reported higher or similar viral loads in csf compared with plasma in 11 children younger than 3 years with cns disease. they found low or undetectable csf viral loads and high plasma viral loads in 14 children with sepsis. a previous study by our group 25 showed that among 156 patients with acute meningitis, enterovirus viral loads in csf were higher in newborn babies than in infants and adults and that genotypes were associated with different viral loads. the analysis of clinical and biological characteristics in patients aged 2 years or younger showed that viraemia was detected more frequently in younger infants (median age 28 days [iqr 17-41]) with acute-stage disease, as suggested in earlier retrospective studies of smaller patient populations. 21, 22 clinically, tachycardia was present in 62 (50%) of 125 young patients with viraemia and fever without source, sepsis, or suspected meningitis. other reports have cited fever, irritability, lethargy, and poor feeding in patients with enterovirus viraemia. 11, 21 in our study, patients with enterovirus viraemia were also more likely to have been exposed to a sick contact. all patients with viraemia in our study were febrile. amounts of c-reactive protein in serum greater than 15 mg/l were detected more frequently in patients without viraemia. pleocytosis was noted in 44 (38%) of 116 patients with enterovirus viraemia, mainly those with suspected meningitis (data not shown). dagan and colleagues 21 reported an inverse relation between viraemia and pleocytosis. the number of lymphocytes and monocytes was significantly lower in patients with viraemia than in those without viraemia. this finding and earlier data 21,26 suggest a detrimental effect of enterovirus infection on populations of mononuclear leucocytes during viraemia. this effect can be caused by virus replication in mononuclear leucocytes, because some enterovirus genotypes can replicate in vitro in human peripheral blood mononuclear cells. 27, 28 accordingly, the concentrations of circulating mono nuclear cells at different ages and the ability of entero virus genotypes to replicate in these cells can affect the sensitivity of blood enterovirus detection. concomitant bacterial infections, mostly urinary-tract infections, occurred in a small proportion (22 [6%] of 360) of patients with fever and enterovirus infection. amounts of c-reactive protein in serum greater than 15 mg/l were noted in five (23%) of these patients (data not shown). the frequency of concomitant enterovirus and bacterial infections was consistent with that in other studies. 7, 8, 29 no enterovirus and bacterial coinfections were identified in csf of patients with meningitis and in blood samples of patients with sepsislike disease. our study has three limitations. first, the date and time of symptom onset were recorded less reliably by parents of children than by those of newborn babies and infants. a sensitivity analysis (data not shown) excluding the inaccurate dates and times of symptom onset for 190 (23%) of 822 patients did not affect our results. second, virology management (including detection of viruses other than enterovirus) and bacteriology management varied between the 16 hospitals taking part. in 340 (41%) of 822 patients, no diagnosis was established, a figure similar to that reported by ahmad and colleagues (43%). 11 third, four different rt-pcr assays were used in our multicentre study. it is unlikely that this variability affected the overall results, because methods used are assessed annually by an external quality assess ment programme, and all yielded correct results. in conclusion, detection of enterovirus in blood improved diagnostic yield in newborn babies and infants admitted for fever without source, sepsis-like disease, or suspected meningitis, compared with detection in csf. the high frequency of detection of enterovirus in blood samples from very young patients with fever without source and sepsis-like disease suggests that enterovirus febrile illnesses are underdiagnosed. it is important to reconsider the guidelines for biological management of patients aged 2 years or younger with febrile illness in emergency departments, to obtain-at the time of blood sampling-an additional tube for enterovirus pcr testing, which can be done sufficiently rapidly to have a real effect on management. a positive enterovirus blood diagnosis could beneficially affect patient management decisions by reducing antibiotic or antiviral therapy, avoiding ancillary tests, lowering hospital-related costs, and allowing earlier discharge. blood enterovirus genome can also be used as an alternative biomarker in case of contraindications for csf sampling and failure of lumbar puncture. service urgences pédiatriques inserm, cic 1405 service de microbiologie clinique des hupssd service de microbiologie clinique des hupssd service pédiatrie générale et urgences pédiatriques service de microbiologie et hygiène, hôpitaux universitaires service de pediatrie-néonatologie laboratoire de biologie médicale viral infections of the central nervous system in spain: a prospective study hospital admissions for viral meningitis in children in england over five decades: a population-based observational study impact of rapid enterovirus molecular diagnosis on the management of infants, children, and adults with aseptic meningitis improvement of the management of infants, children and adults with a molecular diagnosis of enterovirus meningitis during two observational study periods recommendations for enterovirus diagnostics and characterisation within and beyond europe epidemiology of a pediatric emergency medicine research network: the pecarn core data project diagnosis and outcomes of enterovirus infections in young infants a polymerase chain reaction-based epidemiologic investigation of the incidence of nonpolio enteroviral infections in febrile and afebrile infants 90 days and younger prospective comparison of the detection rates of human enterovirus and parechovirus rt-qpcr and viral culture in different pediatric specimens epidemiology of sepsis-like illness in young infants: major role of enterovirus and human parechovirus frequency of enterovirus detection in blood samples of neonates admitted to hospital with sepsis-like illness in kuwait enterovirus and parechovirus viraemia in young children presenting to the emergency room: unrecognised and frequent surveillance of enteroviruses in france management of fever without source in infants and children report on the expert meeting on neonatal and paediatric sepsis defining cerebrospinal fluid white blood cell count reference values in neonates and young infants rapid routine detection of enterovirus rna in cerebrospinal fluid by a one-step real-time rt-pcr assay prospective identification of enteroviruses involved in meningitis in 2006 through direct genotyping in cerebrospinal fluid quality criteria were proposed for measurement properties of health status questionnaires fever in under 5s: assessment and initial management-clinical guideline (cg160) viremia in hospitalized children with enterovirus infections the correlation between the presence of viremia and clinical severity in patients with enterovirus 71 infection: a multi-center cohort study point sur les infections à entérovirus au 9 décembre distinct systemic and central nervous system disease patterns in enterovirus and parechovirus infected children variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus characteristics of young infants in whom human parechovirus, enterovirus or neither were detected in cerebrospinal fluid during sepsis evaluations replication of enteroviruses in human mononuclear cells enterovirus receptors and virus replication in human leukocytes enterovirus neurological disease and bacterial coinfection in very young infants with fever this study was supported by a grant from the university hospital of clermont-ferrand aoi (appel d'offre interne) 2015. we gratefully acknowledge the blood enterovirus diagnosis infection (bledi) in paediatric population study team, who contributed to data collection from 16 french participating hospitals. we thank emilie leroy and nathalie rodde for assistance with virus genotyping; and jeffrey watts for help preparing the article (funded with a grant from the university hospital of clermont-ferrand aoi 2015). key: cord-261133-m00gcci4 authors: eccles, ron title: understanding the symptoms of the common cold and influenza date: 2005-10-25 journal: lancet infect dis doi: 10.1016/s1473-3099(05)70270-x sha: doc_id: 261133 cord_uid: m00gcci4 the common cold and influenza (flu) are the most common syndromes of infection in human beings. these diseases are diagnosed on symptomatology, and treatments are mainly symptomatic, yet our understanding of the mechanisms that generate the familiar symptoms is poor compared with the amount of knowledge available on the molecular biology of the viruses involved. new knowledge of the effects of cytokines in human beings now helps to explain some of the symptoms of colds and flu that were previously in the realm of folklore rather than medicine—eg, fever, anorexia, malaise, chilliness, headache, and muscle aches and pains. the mechanisms of symptoms of sore throat, rhinorrhoea, sneezing, nasal congestion, cough, watery eyes, and sinus pain are discussed, since these mechanisms are not dealt with in any detail in standard medical textbooks. acute upper respiratory tract viral infections (urtis) are the most common diseases of human beings, with adults having two to five common colds each year and school children having from seven to ten colds per year. 1 the symptoms of urtis are so common that selfdiagnosis of common cold or influenza (flu) is normal among the general public and clinical diagnosis is usually the only diagnosis used by the physician. over 200 serologically different viral types are responsible for human urtis, with the rhinoviruses being the most common cause. 1 there is a large amount of information available about the molecular biology of the viruses associated with urtis but relatively little information on the origins of the symptoms associated with them. an understanding of the pathophysiology of symptoms of urtis is important, as most treatments for urtis are symptomatic and clinical trials on the efficacy of new treatments usually focus on changes in symptom scores as the main parameter of efficacy rather than changes in viral titres in the airway or viral shedding. clinical trials on any new antiviral treatment for urtis aimed at the general population will need to demonstrate changes in symptom severity or duration of symptoms, since these parameters are the key benefits for most patients. differences in clinical presentation are not so useful in identifying the causative agent of an urti but there has been increasing interest in improving the accuracy of symptomatic diagnosis of emerging viral infections such as pandemic influenza 2 and severe acute respiratory syndrome (sars) 3 because early diagnosis is essential for any antiviral therapy and for the initiation of publichealth measures in the community (eg, isolation of infected cases). here, i discuss the mechanisms that generate symptoms associated with urtis, especially common cold and flu, but will not review virology in any detail except as regards relevance to symptoms. the clinical expression of urtis is variable and is partly influenced by the nature of the infecting virus but to a greater extent is modulated by the age, physiological state, and immunological experience of the host. 4 depending on these factors, urtis may occur without symptoms, may kill, or most commonly will be associated with an acute self-limiting illness. "common cold" and "flu" are syndromes of familiar symptoms caused by viral infection of the upper respiratory tract. it is difficult to define the syndromes exactly because of great variation in the severity, duration, and types of symptom. rhinoviruses account for 30-50% of all colds, and coronaviruses are the second most common agent, accounting for 10-15% of colds. 5 influenza viruses account for 5-15% of colds, and cold viruses such as respiratory syncytial virus are responsible for much flu-like illness, 6 demonstrating that there is much overlap in aetiology and symptomatology of common cold and flu syndromes. the common cold syndrome has been defined in terms of experimental colds as a short mild illness with early symptoms of headache, sneezing, chilliness, and sore throat and later symptoms of nasal discharge, nasal obstruction, cough, and malaise. 7 generally the severity of symptoms increases rapidly, peaking 2-3 days after infection, with a mean duration of symptoms of 7-10 days but with some symptoms persisting for more than 3 weeks. 5 experimental colds in the adult are rarely associated with fever, and some subjects have a transient depression of oral temperature during the early phases of a cold. 7 studies on the symptoms generated by different common cold viruses indicate that it is not possible to identify the virus on the basis of the symptoms, since similar symptoms are caused by different viruses. 8 the influenza syndrome is typically of sudden onset and is characterised by fever, headache, cough, sore throat, myalgia, nasal congestion, weakness, and loss of appetite. 2 antiviral agents are available for the treatment of influenza but they are ineffective against any other causes of urtis and therefore there is considerable interest in the early clinical diagnosis of influenza as opposed to common cold. the best predictors for influenza are cough and fever, since this combination of symptoms has been shown to have a positive predictive value of around 80% in differentiating influenza from a population suffering from flu-like symptoms. 2 the symptoms of urtis are triggered in response to the viral infection of the upper airway and the immune response to infection may be the main factor in generating the symptoms, rather than damage to the airway. 9,10 histological surveys of the nasal epithelium during experimental rhinovirus infections have not been able to find any morphological changes in the nasal epithelium of infected volunteers, apart from a substantial increase in polymorphonuclear leucocytes early in the course of the infection. 11 the major cell monitoring the host for the invasion of pathogens is the macrophage, which has the ability to trigger an acute phase response when stimulated with components of viruses or bacteria-eg, viral rna and bacterial cell wall components. 12 the surface of the macrophage exhibits toll-like receptors that combine with the components of viral and bacterial pathogens and trigger the production of cytokines. 12 the cytokines act to recruit other immune cells, trigger inflammation, and generate systemic symptoms such as fever. 13 a complex mix of proinflammatory cytokines and mediators generates the symptoms of urtis. 14 the inflammatory mediator bradykinin is believed to have a major role in generating the local symptoms of urtis (eg, sore throat and nasal congestion), 15, 16 and cytokines are believed to be responsible for the systemic symptoms (eg, fever). 17 a discussion of the mechanisms that generate the urti symptoms is the main topic of this review and each symptom will be discussed in turn. a scratchy sensation of throat irritation is often the first symptom of an urti. this symptom may be related to early viral infection of the nasopharynx rather than the nasal epithelium. 18 point inoculation of rhinovirus on the inferior turbinate caused early infection of the nasopharynx with subsequent spread of infection anteriorly into the nose. 18 the sensation of throat irritation may be caused by the formation of bradykinin in the airway in response to infection, since intranasal administration of bradykinin causes symptoms of rhinitis and a sore throat. 15, 19 the sensation of throat irritation as an early urti symptom may develop into sore throat pain associated with nasopharyngitis, pharyngitis, or tonsillitis and these conditions may also be associated with bacterial infection. 20 the symptom of sore throat is most likely caused by the actions of prostaglandins and bradykinin on sensory nerve endings in the airway and the sensation of pain is mediated by the cranial nerves supplying the nasopharynx and pharynx. sneezing, like sore throat, is a prominent early symptom associated with urtis. 7 sneezing is mediated solely by the trigeminal nerves, which supply the nasal epithelium and the anterior part of the nasopharynx with sensory fibres. 21, 22 sneezing is related to inflammatory responses in the nose and nasopharynx that stimulate the trigeminal nerves. the sneeze response may be mediated via histamine receptors on the trigeminal nerves, since intranasal administration of histamine causes sneezing. 23 the trigeminal nerves relay information to the sneeze centre in the brainstem and cause reflex activation of motor and parasympathetic branches of the facial nerve and activate respiratory muscles. a model of the sneeze reflex is illustrated in figure 1 . the sneeze centre coordinates the inspiratory and expiratory actions of sneezing via respiratory muscles, and lacrimation and nasal congestion via parasympathetic branches of the facial nerve. the eyes are always closed during sneezing by activation of facial muscles, indicating a close relation between the protective reflexes of the nose and eyes. a common phenomenon is the "photic sneeze", caused by a sudden increase in light intensity, again highlighting the overlap of protective nasal and eye reflexes. 24 sneezing activates parasympathetic pathways to nasal glands and there appears to be some cholinergic control of sneezing, since anticholinergics such as ipratropium and first generation antihistamines have been shown to inhibit sneezing. 25, 26 the nasal discharge associated with urtis is a complex mix of elements derived from glands, goblet cells, plasma cells, and plasma exudates from capillaries, with the relative contributions from these different sources varying with the time course of the infection and the the trigeminal nerves provide a sensory input to the "sneeze centre" in the medulla that triggers reflex activation of nasal and lacrimal glands to cause a rhinorrhoea, facial muscles to cause closure of the eyes and grimace, and respiratory muscles to cause inspiration followed by an explosive expiration. facial muscles nasal lacrimal glands trigeminal nerves sneeze centre in medulla severity of the inflammatory response. 27 a watery nasal secretion is an early urti symptom and is often accompanied by sneezing. this early phase of nasal secretion is a reflex glandular secretion that is caused by stimulation of trigeminal nerves in the airway, similar to sneezing. support for the glandular origin of the early nasal secretions comes from studies on anticholinergic medicines such as ipratropium. these studies have demonstrated that nasal secretions in the first 4 days of a common cold are inhibited by intranasal administration of ipratropium. 25 the nasal discharge also consists of a protein-rich plasma exudate derived from subepithelial capillaries, 28 which may explain why anticholinergics only partly inhibit nasal discharge associated with urtis. 27 the colour of nasal discharge and sputum is often used as a clinical marker to determine whether or not to prescribe antibiotics but there is no evidence from the literature that supports this concept, 29 since colour changes in nasal discharge or sputum reflect the severity of the inflammatory response 30 rather than the nature of the infection. much of the literature relates to colour changes in sputum and the lower airways but the same concepts apply to the upper airways and nasal discharge. the colour of nasal discharge may change from clear to yellow to green during the course of an urti. this colour change is related to the recruitment of leucocytes into the airway lumen and is a hallmark of airway disease. 30 neutrophils and proinflammatroy monocytes have azurophil granules that are green because of the green protein myeloperoxidase. nasal discharge with few leucocytes is white or clear, with increasing numbers of leucocytes the nasal discharge appears yellow (pale green), and with large numbers of leucocytes the colour becomes green. 30 nasal congestion is a later symptom of urtis that increases in severity over the first week of symptoms. 7 nasal congestion is caused by the dilation of large veins in the nasal epithelium (venous sinuses) in response to the generation of vasodilator mediators of inflammation such as bradykinin. 31, 32 these sinuses are well developed at the anterior end of the inferior turbinate and nasal septum where congestion of the sinuses in the narrow nasal valve region causes obstruction of the nasal airway. 33 the nasal venous sinuses exhibit phases of congestion and decongestion under the influence of the sympathetic vasoconstrictor nerves, causing reciprocal changes in nasal airflow (often termed the "nasal cycle"). 33 the asymmetry of nasal airflow associated with the nasal cycle is increased with an urti, and this may result in one nasal passage being patent while the other is completely obstructed. 34 figure 2 illustrates the changes in nasal airflow associated with the nasal cycle in health and with an urti. the paranasal sinuses surround the nasal airway and any infection of the airway usually involves the sinuses, causing inflammation and the accumulation of secretions in the sinuses. 35 the origin of sinus pain may be related to several factors-eg, pressure changes in the sinus air space and pressure changes in the blood vessels draining the sinus. 36 the ostia of the paranasal sinuses are often occluded because the nasal epithelium becomes inflamed and congested with an urti; this may result in gas absorption from the sinus and "vacuum maxillary sinusitis". 37 however, sinuses with patent ostia may also be painful, indicating that the generation of inflammatory mediators within the sinus may be sufficient to trigger the sensation of pain either by direct stimulation of pain nerve fibres or via distension of blood vessels that are also served by sensory nerves. 36 changes in posture from sitting to supine cause an increase in sinus pain that may be related to dilation of the blood vessels draining the sinus caused by an increase in venous pressure. pressure changes in the sinus may also cause pain by stimulation of branches of the trigeminal nerve that course in and around the sinuses. 37 watery eyes (epiphora) is a common symptom associated with allergic and infectious rhinitis. 38, 39 in children aged 7 years, 70% of cases of epiphora are related to allergic disease or urtis. 40 the nasolacrimal duct may be obstructed at its opening into the nose by inflammation and congestion of blood vessels in the nasal epithelium around the opening of the duct, causing an accumulation of tears and the symptom of watery eyes. the nasolacrimal duct has been shown to have a vascular plexus of veins (cavernous tissue) similar to the venous sinuses of the nasal epithelium, and congestion of this plexus causes obstruction of the duct. 41 the nasolacrimal cavernous tissue is innervated by parasympathetic and sympathetic nerves that may have a role in controlling the outflow of tears by regulating the congestion and decongestion of the cavernous tissue. 42 cough is a common symptom associated with urtis that may persist for 3 weeks or more, 43 and it represents the largest single cause of consultation in primary care. 44 cough is mediated exclusively by the vagus nerve, meaning that cough is initiated in the airway by stimulation of sensory nerves at the level of the larynx or below. 45 a model of cough control is illustrated in figure 3 . nasal stimulation and inflammation causes sneezing and not cough, indicating that the airway inflammation associated with rhinitis must reach the level of the larynx to trigger cough. the vagus nerve also supplies the external ear, oesophagus, and abdominal organs and cough can be elicited from these areas as with cough associated with gastroesophageal reflux. 44 cough is normally a protective reflex that prevents the aspiration of food and fluid into the airway and also aids in the expulsion of mucus and foreign objects from the lower airway. the first days of an urti are often associated with a dry, unproductive cough that serves no useful function and may cause loss of sleep and exhaustion. the unproductive cough may be caused by the inflammatory response in the upper airways spreading to the larynx. cough associated with urtis is believed to be caused by a hyper-reactivity of the cough reflex that may be due to the effects of inflammatory mediators on airway sensory nerve endings. 46, 47 in health, cough is readily induced by mechanical stimulation of the larynx, but when the larynx is inflamed and hyper-reactive, cough may occur spontaneously or in response to stimuli that would not normally cause cough-eg, the mildly irritating effects of cold air. cough occurs spontaneously with an urti, and some cough may be voluntary rather than reflex; this voluntary cough may be related to a sensation of airway irritation. 48 productive cough usually occurs later in the course of urti and may be related to the inflammation spreading to the lower airways and triggering mucus production. common cold viruses usually do not cause any substantial damage to the airway epithelium, 11 whereas influenza may cause substantial cellular damage to the respiratory epithelium; this difference may be why influenza infection is usually associated with cough 2 whereas common cold often occurs as a "head cold" with little, if any, symptom of cough. headache is a common early symptom associated with urtis. 7 in a clinical trial that recruited patients with sore throat associated with urtis, over 60% of patients experienced headache. 49 the mechanism of headache associated with urtis is unknown but a hypothesis has been proposed that headache associated with infections is caused by cytokines released from immune cells in response to viral infection. 50 administration of cytokines involved in the immune response to infection-eg, tumour necrosis factor and interferons-has been shown to cause headache in human beings. 50 headache is a common side-effect of administration of interferon beta-1a for the treatment of multiple sclerosis; 51 similarly headache is associated with therapy with pegylated interferon alpha-2b for treatment of hepatitis. 52 the mechanism of headache caused by cytokines is unknown but it is interesting that headache induced by cytokines is accompanied by symptoms such as fatigue, anorexia, malaise, nausea, and depression, and these symptoms are commonly associated with urtis. a sensation of chilliness is an early symptom of common cold, 7 and is sometimes explained as an initial stage of fever, since vasoconstriction of skin blood vessels may cause a fall in skin temperature that is perceived as chilliness. common cold in the adult is rarely accompanied by fever and some subjects have a transient fall in body temperature during the early stages of common cold. in a study of 272 patients with sore throat associated with urtis, the mean aural temperature was 36·8ºc and around 35% of these patients said they were suffering from "chills" and "feverish discomfort". 49 the sensation of chilliness may be unrelated to any change in skin or body temperature. in a study of human volunteers, a sensation of chill still develops on administration of exogenous pyrogen even though the volunteers are immersed in a water bath that maintains a neutral skin temperature (34·5ºc). 53 the sensation of chilliness occurred after visible signs of shivering in the volunteers. chilliness and shivering occurred even though there was no change in skin temperature and body temperature was actually rising in response to skin vasoconstriction. this finding indicates that the sensation of chilliness may be a central sensation closely linked to control of shivering. chilliness and shivering are most likely induced by the effects of cytokines on the temperature regulating centres of the hypothalamus and perceived at the level of the cerebral cortex. fever in response to infection is found in a wide range of animals and is believed to be beneficial as regards the host response to infection. 54 fever is usually associated with novel or severe viral infections, especially emerging viral infections where the virus is novel to the host, as in influenza epidemics and sars. 2, 3 as discussed, fever is uncommon in adult cases of common cold, but is common in infant cases, presumably because the adult has been exposed to numerous common cold viruses and subsequent infections do not trigger a strong immune response, whereas the viruses are novel to the infant. cytokines have been implicated as endogenous pyrogens that are released from macrophages and other leucocytes in response to infection, and there is considerable evidence for pyretic and antipyretic effects of cytokines. 17 the proinflammatory cytokines interleukin 1, interleukin 6, and tumour necrosis factor alpha, as well as the anti-inflammatory cytokines interleukin-1 receptor antagonist and interleukin 10 have been investigated for their pyrogenic or antipyretic action. 17 interleukin 1 and interleukin 6 are believed to be the most important cytokines that induce fever. 55 cytokines are believed to cross the blood-brain barrier or interact with the vagus nerve endings to signal the temperature control centre of the hypothalamus to increase the thermal set point. 55, 56 the hypothalamus then initiates shivering, constriction of skin blood vessels, and a sensation of chilliness (figure 4). the presence of physical features of urtis-eg, nasal congestion, rhinorrhoea, and cough-may cause discomfort, attention deficit, and mood changes but there is increasing evidence that the psychological changes associated with urtis may also be caused by the effects of cytokines on the central nervous system. 57 urtis have been shown to lead to a reduction in subjective alertness and impaired psychomotor functioning 58 but the relative contribution of cytokines to these changes is poorly understood. exogenous administration of interferon alpha is used as a therapy for chronic viral diseases such as hepatitis b and c, and therapy is associated with flulike side-effects similar to those observed with urtiseg, fatigue, fever, chills myalgia, nausea, and mood changes. 59 psychiatric side-effects such as depression, irritability, lack of motivation, impaired concentration, psychoses, and confusional states have been reported to occur in some patients after 2-3 months of therapy with interferon alpha. 59 the present knowledge on the effects of interferon alpha on the brain indicates that there at least two distinct syndromes related to therapy: an early neurovegetative syndrome characterised by psychomotor slowing and fatigue, and a later mood/cognitive syndrome that involves depression. 60 cytokinesincluding tumour necrosis factor and interleukins 1, 2, and 6-have been reported to induce the syndrome of "sickness behaviour" with anhedonia, cognitive dysfunction, anxiety/irritability, psychomotor slowing, anergia/fatigue, anorexia, sleep alterations, and increased sensitivity to pain. 60 these cytokines are also associated with urtis and may mediate mood changes associated with these infections. anorexia is a common behavioural response to urtis, and this response has entered the folklore as advice to the cytokines may act on vagal nerve endings or enter the brain to cause a resetting of the temperature control centre in the hypothalamus. the hypothalamus causes shivering and constriction of skin blood vessels and also initiates a sensation of chilliness that is perceived at the level of the cerebral cortex. il=interleukin; tnf=tumour necrosis factor. "feed a cold and starve a fever". 61 in association with fever, decreased food and water consumption are the most common signs of infection. 13 there is growing evidence that anorexia associated with infections such as urtis is mediated by cytokines that are released from leucocytes in response to infection, and that these cytokines cause inhibition of feeding by effects on the feeding centre in the hypothalamus. 62 the cytokines implicated in anorexia are those involved in the acute phase response to infection-eg, interleukins, tumour necrosis factor, and interferons. 62 in support of the folklore advice to starve a fever, evidence indicates that acute anorexia in response to infection is beneficial and that it is an important behavioural response to help overcome infection. anorexia may aid in eliminating infection by saving energy that would be otherwise used in finding food, reducing heat loss from the body that would be lost by convection, reducing the availability of micronutrients such as iron and zinc that are essential for the growth of pathogens, and enhancement of immune function by enhancing monocyte and macrophage activity. 13, 62 muscle aches and pains (myalgia) are a common symptom of urtis, with around 50% of patients with common cold experiencing these symptoms. 49 myalgia is a symptom of the acute phase response to infection and there is evidence that the symptom is caused by the effects of cytokines on skeletal muscle. 63 proinflammatory cytokines have been implicated as inducing the breakdown of muscle proteins, and tumour necrosis factor was initially referred to as cachetin because of its role in causing muscle wasting or cachexia. 64 the breakdown of muscle protein in response to urti can be viewed as beneficial because it mobilises proteins and aminoacids that can be converted in the liver to opsonins and other components of the immune response. 64 fever associated with urtis is usually accompanied by other systemic symptoms such as myalgia and there is much evidence that indicates that both these symptoms are caused by the production of prostaglandin e2 in response to circulating cytokines. 63 the cytokine-induced generation of prostaglandin e2 and the breakdown of skeletal muscle in vitro is inhibited by indomethacin, 63 and similarly myalgia associated with urtis is relieved with acetylsalicylic acid. 49 prostaglandin e2 is a mediator of pain by its effects on peripheral pain receptors. 65 the cytokine stimulation of prostaglandin e2 production in skeletal muscle, and the effects of prostaglandin e2 on sensory nerves in muscle, may explain the myalgia associated with urtis. in a study of common cold symptoms induced by challenge with infected nasal secretions, urti symptoms were classified as either "early" or "later" symptoms. 7 the early symptoms were headache, sneezing, chilliness, and malaise, which developed quickly and also declined rapidly after 1-2 days, whereas the later symptoms-malaise, nasal discharge, nasal obstruction, and cough-developed slowly over several days and were still present 1 week after challenge. the time course of an early symptom (sneezing) is compared with that of a later symptom (cough) in figure 5 . the early development of sneezing compared with cough in cases of common cold may be explained on the basis that urtis develops in the upper airways first and subsequently spread to the lower airways. the upper airways are innervated by the trigeminal nerves that mediate sneezing whereas the airways below the larynx are innervated by the vagus nerves that mediate cough. fever is usually an early symptom of influenza but is of short duration (3-4 days) . the systemic symptoms of fever, headache, malaise, myalgia, and anorexia are related to the effects of cytokines released from immune cells and these responses develop rapidly in the first days of infection when the virus is detected by the immune system. the local symptoms of nasal congestion and rhinorrhoea are dependent on the generation of inflammatory mediators such as prostaglandins and bradykinin. the inflammatory mediator response may have a slower onset and longer duration than the cytokine response, which may explain the time course of local symptoms such as congestion and rhinorrhoea. our understanding of the generation of urti symptoms has been helped by the discovery of cytokines and new knowledge about their roles in the acute phase response. urti symptoms-eg, anorexia-that previously were in the realm of folklore now have a physiological explanation in terms of the effects of cytokines on the hypothalamus. the present rationale for the treatment of urtis is for symptom relief, since cough sneezing the symptoms of urti are perceived as a nuisance, but this review suggests that some of the symptoms are an integral part of the acute phase response and may aid in recovery from infection. the unpleasant symptoms of fever, malaise, and anorexia help to overcome infection and it is debatable whether elimination of these symptoms with non-steroidal anti-inflammatory drugs is beneficial. at present there is no evidence that symptomatic treatment of urtis interferes with the course of the common cold or influenza but this is an area that is worthy of more research. i declare that i have no conflicts of interest. epidemiology of viral respiratory infections clinical signs and symptoms predicting influenza infection clinical features and short-term outcomes of 144 patients with sars in the greater toronto area influenza in man the common cold contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study transmission of the common cold to volunteers under controlled conditions. 1 the common cold as a clinical entity signs and symptoms in common colds epidemiology, pathogenesis, and treatment of the common cold the host response, not the virus, causes the symptoms of the common cold: comment histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds science review: key inflammatory and stress pathways in critical illness-the central role of the toll-like receptors infection-induced anorexia: active host defence strategy pathophysiology of nasal symptoms search strategy and selection criteria data for this review were identified by searches of pubmed, isi web of science, and references from relevant articles; articles were also identified through searches of the files of the author. the main search terms were nasal provocation with bradykinin induces symptoms of rhinitis and a sore throat bradykinin levels during experimental nasal infection with rhinovirus and attenuated influenza virus sites of rhinovirus recovery after point innoculation of the upper airway sore throat following nasal and oropharyngeal bradykinin challenge nasopharyngitis, pharyngitis, and tonsillitis upper airway reflexes and involvement of the lower airway role of histamine and antihistamines in the nose the photic sneeze: literature review and discussion effectiveness and safety of intranasal ipratropium bromide in common colds. a randomized, double-blind, placebo-controlled trial a clinical study to evaluate the efficacy of the antihistamine doxylamine succinate in the relief of runny nose and sneezing associated with upper respiratory-tract infection physiology of nasal secretion mucosal exudation of fibrinogen in coronavirusinduced common colds predictors of an antibiotic prescription by gps for respiratory tract infections: a pilot assessment of airway neutrophils by sputum colour: correlation with airways inflammation microvascular anatomy of the nose anatomy and physiology of the nose and control of nasal airflow nasal airflow in health and disease changes in the amplitude of the nasal cycle associated with symptoms of acute upper respiratory tract infection computed tomographic study of the common cold the relationship between body posture and pressure in occluded maxillary sinus of man infraorbital nerve dehiscence: the anatomic cause of maxillary sinus "vacuum headache epiphora: the role of rhinitis an association between acquired epiphora and the signs and symptoms of chronic rhinosinusitis: a prospective case-control study the natural history of epiphora in childhood the cavernous body of the human efferent tear ducts contributes to regulation of tear outflow innervation of the cavernous body of the human efferent tear ducts and function in tear outflow mechanism duration of cough in acute upper respiratory tract infections epidemiology of cough neurophysiology of the cough reflex pathophysiology of airway viral infections cough induced by airway vibration as a model of airway hyperreactivity in patients with acute upper respiratory tract infection voluntary control of cough effects of acetylsalicylic acid on sore throat pain and other pain symptoms associated with acute upper respiratory tract infection the cytokine theory of headache the long-term safety and tolerability of high-dose interferon beta-1a in relapsingremitting multiple sclerosis: 4-year data from the prisms study the safety of pegylated interferon alpha-2b in the treatment of chronic hepatitis b: predictive factors for dose reduction and treatment discontinuation the chill sensation in fever phylogeny of fever invited review: cytokine regulation of fever: studies using gene knockout mice circulating cytokines as mediators of fever common respiratory tract infections as psychological entities: a review of the mood and performance effects of being ill effects of the common cold on mood and performance interferon-alpha, cytokines and possible implications for mood disorders cytokines and psychopathology: lessons from interferon-alpha feed a cold, starve a fever" folk models of infection in an english suburban community, and their relation to medical treatment anorexia of infection: current prospects stimulation of muscle protein degradation and prostaglandin e2 release by leukocytic pyrogen (interleukin-1). a mechanism for the increased degradation of muscle proteins during fever prostaglandins, pain, and inflammation key: cord-254478-scc9wee0 authors: to, kelvin kai-wang; tsang, owen tak-yin; leung, wai-shing; tam, anthony raymond; wu, tak-chiu; lung, david christopher; yip, cyril chik-yan; cai, jian-piao; chan, jacky man-chun; chik, thomas shiu-hong; lau, daphne pui-ling; choi, chris yau-chung; chen, lin-lei; chan, wan-mui; chan, kwok-hung; ip, jonathan daniel; ng, anthony chin-ki; poon, rosana wing-shan; luo, cui-ting; cheng, vincent chi-chung; chan, jasper fuk-woo; hung, ivan fan-ngai; chen, zhiwei; chen, honglin; yuen, kwok-yung title: temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study date: 2020-03-23 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30196-1 sha: doc_id: 254478 cord_uid: scc9wee0 background: coronavirus disease 2019 (covid-19) causes severe community and nosocomial outbreaks. comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) are not yet available. nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. we aimed to ascertain the serial respiratory viral load of sars-cov-2 in posterior oropharyngeal (deep throat) saliva samples from patients with covid-19, and serum antibody responses. methods: we did a cohort study at two hospitals in hong kong. we included patients with laboratory-confirmed covid-19. we obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. serial viral load was ascertained by reverse transcriptase quantitative pcr (rt-qpcr). antibody levels against the sars-cov-2 internal nucleoprotein (np) and surface spike protein receptor binding domain (rbd) were measured using eia. whole-genome sequencing was done to identify possible mutations arising during infection. findings: between jan 22, 2020, and feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37–75]). the median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log(10) copies per ml (iqr 4·1–7·0). salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope −0·15, 95% ci −0·19 to −0·11; r(2)=0·71). in one patient, viral rna was detected 25 days after symptom onset. older age was correlated with higher viral load (spearman's ρ=0·48, 95% ci 0·074–0·75; p=0·020). for 16 patients with serum samples available 14 days or longer after symptom onset, rates of seropositivity were 94% for anti-np igg (n=15), 88% for anti-np igm (n=14), 100% for anti-rbd igg (n=16), and 94% for anti-rbd igm (n=15). anti-sars-cov-2-np or anti-sars-cov-2-rbd igg levels correlated with virus neutralisation titre (r(2)>0·9). no genome mutations were detected on serial samples. interpretation: posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. unlike severe acute respiratory syndrome, patients with covid-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. this finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. serological assay can complement rt-qpcr for diagnosis. funding: richard and carol yu, may tam mak mei yin, the shaw foundation hong kong, michael tong, marina lee, government consultancy service, and sanming project of medicine. coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first reported from china in december, 2019. 1 although middle east respiratory syndrome coronavirus (mers-cov) 2 and severe acute respiratory syndrome coronavirus (sars-cov) 3 infections have a higher mortality rate than does covid-19, sars-cov-2 spreads much more rapidly than mers-cov and sars-cov. reliable data for profiles of serial viral load and serum antibody responses are needed urgently to guide antiviral treatment, infection control, epidemiological measures, and vaccination. the peak viral load of patients with mers-cov and sars-cov infections occurs at around 7-10 days after symptom onset, which could be associa ted with nosocomial outbreaks involving healthcare workers. 2, 4 clinical studies of antiviral agents for sars showed that the viral load decreased significantly with treatment success. 5 no systematic study of these two important variables with statistical analysis has been done for sars-cov-2, although preliminary descriptive studies have been reported. [6] [7] [8] in most studies of respiratory virus infections, serial sampling of nasopharyngeal or throat swabs is used for viral load monitoring. however, collection of nasopharyngeal or throat swab specimens can induce coughing and sneezing, which generates aerosol and is a potential health hazard for health-care workers. collection of throat swabs also requires direct inspection of the patient's posterior pharynx and tonsils. furthermore, collection of nasopharyngeal specimens is a relatively invasive procedure, which is uncomfortable and can even induce bleeding. a patient's reluctance to provide a sample can account for the paucity of timepoints in viral load studies of respiratory virus infections. findings of previous studies have shown high concordance between saliva and nasopharyngeal aspirate as specimens for laboratory diagnosis of respi ratory viruses. 9 we have reported use of posterior oro pharyngeal (deep throat) saliva for diagnosis and viral load monitoring in a cohort of 12 patients with covid-19. 6 here, we report use of self-collected posterior oro pharyn geal saliva samples from patients with covid-19, which avoids close contact between healthcare workers and patients, for viral load monitoring. we also monitored serial serum antibody levels of patients. we included consecutive patients with laboratory confirmed covid-19 who were admitted to princess margaret hospital and queen mary hospital in hong kong. in hong kong, patients were tested for sars-cov-2 based on clinical and epidemiological criteria as outlined and updated by the hospital authority. initial laboratory confirmation was done using nasopharyngeal or sputum specimens at the public health laboratory centre of hong kong. we excluded patients if archived saliva or serum samples were insufficient for testing. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw 13-372). since archived specimens were used, written informed consent was waived. 12 of 23 patients included in this study have been reported previously, 6 but their clinical information, viral load by single copy rna-dependent rna polymerasehelicase gene, antibody response, or viral genome analysis has not been reported before. for viral load monitoring, all patients were asked to produce an early morning saliva sample from the posterior oropharynx (ie, coughed up by clearing the throat) before toothbrushing and breakfast, because naso pharyn geal secretions move posteriorly and broncho pulmonary secretions move by ciliary activity to the posterior oro phar yn geal area while the patients are in a supine position during sleep. patients were instructed and supervised by nurses. viral transport medium was added to the saliva specimen. if patients were intubated, we obtained endotracheal aspirate instead of posterior oropharynx saliva. 6, [9] [10] [11] our initial experience showed that such saliva samples are promising in viral load monitoring in patients with covid-19. 6 we also retrieved serum remnant from blood samples taken for routine bio chemical testing, and refrigerated these samples at -20°c until antibody testing could be done. we recorded clinical findings in a predesigned database, including a patient's history and physical examination and findings of haematological, biochemical, radiological, and microbiological investigations. we defined severe disease as the need for supplemental oxygen, admission to the intensive care unit (icu), or death. evidence before this study we searched pubmed on feb 24, 2020, with no limitations by starting date, with the terms "covid-19", "coronavirus", "antibody", and "viral load"; we restricted our search to articles published in english. our search did not retrieve any reports on clinical progression of coronavirus disease 2019 (covid19) with respect to temporal viral load and concomitant serum antibody profiles. we identified one correspondence piece on viral load with no statistical analysis, and another article with a few cases of antibody response. we present findings of an observational cohort study of the temporal profile of viral load of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from posterior oropharyngeal saliva samples and serum antibody responses, dated by symptom onset and correlated with clinical findings. salivary viral load was highest during the first week after symptom onset and subsequently declined with time. eia of igg and igm against internal viral nucleoprotein (np) and surface spike protein receptor binding domain (rbd) showed correlation between antibody response and neutralising antibody titre. posterior oropharyngeal saliva specimens are non-invasive and acceptable to patients and can be used for initial diagnosis and subsequent viral load monitoring of covid-19. the early peaking of viral load has important implications for transmission of sars-cov-2 in the community and hospital settings. eia of igg and igm against internal viral np and surface spike protein rbd can be used for those with delayed presentation or retrospective diagnosis of mild cases. as the positive eia antibody level correlates well with neutralising antibody titre, further studies on its role in immunopathology or antiviral therapy are warranted. we did in-house reverse transcriptase quanti tative pcr (rt-qpcr) targeting the sars-cov-2 rna-dependent-rna-polymerase-helicase gene region, as described (appendix p 1). 12 we did eias for sars-cov-2 nucleo protein (np) and spike protein receptor binding domain (rbd), as described but with modifications. 13 recombinant np and spike protein rbd of sars-cov-2 were used for the eias. we assessed the purity of np and rbd by sodium dodecyl sulphate polyacrylamide gel electro phor esis and western blotting (figure 1a, b; appendix pp 2-3). a positive sample was included in each run as a positive control. we used an archived anonymous sample from 2018 as a negative control. 14 the cutoff for seropositivity was set as the mean value of 93 anonymous archived serum specimens from 2018, plus 3 sds. we verified the validity of eias by competitive eia (appendix p 6) and by western blotting, using patients' serum samples (figure 1c, d; appendix p 9). we did microneutralisation assays and virus culture, as described (appendix pp 1-5). 6, 15 we did whole-genome sequencing using the oxford nanopore minion device (oxford nanopore technologies, oxford, uk), as described (appendix p 4). 1 we did statistical analyses using spss version 26.0 or prism version 6.0. we compared categorical variables using fisher's exact test and continuous variables with the mann-whitney u test. we used spearman's correlation to assess the relation between age and viral load. a p value less than 0·05 was judged statistically significant. the funders 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 data in the study and had final responsibility for the decision to submit for publication. between jan 22, 2020, and feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (13 male and ten female). ten patients had severe covid-19, of whom all required oxygen supplementation, and 13 patients had mild disease. the median age of patients was 62 years (range 37-75). 11 (48%) of 23 patients had chronic medical illnesses, and the most common under lying diseases were hypertension in six (26%) patients and diabetes in four (17%). chronic comorbidities were more common among patients with severe covid-19 (seven [70%] patients with severe disease had chronic comorbidities vs four [31%] with mild disease), although this difference was not significant (table). five patients were admitted to the icu, including three who required intubation. two patients died. the median interval between symptom onset and hospitalisation was 4 days (range 0-13). on presentation, the most common symptom was fever in 22 patients (96%), followed by cough in five (22%), chills in four (17%), and dyspnoea in four (17%; table). dyspnoea was significantly more frequent among the ten patients with severe disease than among those with mild disease (four [40%] of ten vs none [0%] of 13; p=0·024). serum alkaline phosphatase was significantly higher among patients with severe disease than among those with mild disease (74 u/l [range 56-149] vs 60 u/l [38-118]; p=0·026). the lymphocyte count was lower among patients with severe disease than among those with mild disease (0·65 × 10⁹ cells per l [range 0·30-1·90] vs 1·03 [0·57-2·25]), but this difference was not significant (p=0·088). lymphopenia and neutrophilia were present in a higher proportion of patients with severe disease than in those with mild disease, but the differences were not significant (p=0·090 and p=0·068, respectively). chest radiographic abnormalities were seen in 15 (65%) patients. multifocal ground-glass lung opacities were seen in 17 (74%) patients on ct. sars-cov-2 rna was detected in blood samples in five (22%) patients and rectal swabs in four (27%), but the detection rate between severe and mild cases did not differ (p=0·62 and p=0·57, respectively; table). sars-cov-2 rna was not detected in any urine specimens. lopinavir-ritonavir with or without ribavirin or interferon beta 1b was given in 18 (78%) patients at different timepoints after symptom onset. in total, 173 respiratory specimens were obtained from 23 pati ents (mean 7·5 respiratory specimens per patient). the median viral load at presentation was 5·2 log 10 copies per ml (iqr 4·1-7·0). sars-cov-2 rna was not detected in the saliva of three (13%) patients. specimens with undetectable viral load were assigned a value of 1 log 10 copies per ml. no correlation was noted between days after symptom onset and initial viral load (spearman's ρ=0·48; p=0·97). the viral load in posterior oropharyngeal saliva samples was highest during the first week of symptom onset then gradually declined (slope -0·15, 95% ci -0·19 to -0·11; r²=0·71; figure 2). endotracheal aspirate viral load was available from day 8 after symptom onset and showed a non-significant decline (slope -0·13, 95% ci -0·31 to 0·04; r²=0·15). of the 21 patients who survived, seven (33%) had viral rna detected for 20 days or longer after symptom onset. no association was seen between prolonged detection of viral rna (≥20 days after symptom onset) and severity of illness (p=0·35). one patient had viral rna detected for up to 25 days after symptom onset; another patient had undetectable viral load on days 21 and 22 after symptom onset, with rebound of viral load on days 23 and 24, followed by 5 days of undetectable viral load. a significant positive correlation between age and peak viral load was noted (spearman's ρ=0·48, 95% ci 0·074-0·75; p=0·020; figure 3a). the median initial (p=0·56) and peak (p=0·52) viral loads in severe cases were about 1 log 10 higher than those in mild cases, although the difference was not significant (figure 3b, c). the initial (p=0·49) and peak (p=0·29) viral loads did not differ between patients without comorbidities and those with comorbidities (figure 3d, e). for patients with both viral load and antibody results available in week 1 or week 3, median viral load was 6·70 log 10 copies per ml (range 4·17-8·64), and the concomitant median optical density (od) for anti-np igg was 0·13 (range 0·10-1·83) in week 1, whereas in week 3, median viral load was 4·91 log 10 copies per ml (range 3·99-8·94) and the concomitant median od for anti-np igg was 2·59 (range 2·12-2·65). 108 serum specimens were obtained from 23 patients (mean 4·7 serum specimens per patient). an increase was noted in igg or igm antibody levels against np or rbd for most patients at 10 days or later after symptom to assess for host factors that affect the antibody titre, the correlation was analysed between the highest od value during the convalescent period (third week after symptom onset) and age or comorbidities. patients with comorbidities had a lower anti-rbd igg od than did those without comorbidities, although the difference was not significant (median od, 0·65 vs 1·32; p=0·15; appendix p 7). no association was seen between comorbidity and anti-np igg or igm od values, or between age and anti-np igm or igg or anti-rbd igm or igg od values (appendix p 8). specimens with microneutralisation assay titres less than 10 were assigned a value of 5, and specimens with microneutralisation assay titres greater than 320 were assigned a value of 640. for one patient, a microneutralisation antibody assay was done with ten serial samples. the correlation between micro neutralisation assay titres and anti-np igg (r²=0·99) or anti-rbd igg (r²=0·96) was better than those between microneutralisation assay titres and anti-np igm (r²=0·88) or anti-rbd igm (r²=0·87; figure 5 ). nanopore sequencing was successful for paired samples from four patients. the interval between the first and second specimens was 1-3 days. no viral mutations were identified between paired samples from individual patients. we analysed the serial viral load, antibody kinetics, and viral genome of patients with covid-19 in hong kong. for most patients, the viral load of sars-cov-2 was very high at presentation and declined steadily. despite develop ment of antibodies against surface and internal proteins of sars-cov-2, viral rna could still be detected in posterior oropharyngeal (deep throat) saliva samples from a third of patients for 20 days or longer. peak viral load correlated positively with age. most patients had an antibody response at 10 days or later after onset of symptoms. viral whole-genome sequencing of paired samples from four patients did not identify any single nucleotide poly morphisms. a high viral load on presentation of covid-19 was recorded in our cohort, even for patients who were hospitalised shortly after symptom onset. using nasal swab and throat swab, zou and colleagues 8 have also reported a high viral load shortly after symptom onset. however, in that study, only cycle threshold values (not exact viral loads) were reported, and no statistical or correlative analysis was done with clinical variables such as age, comorbidities, disease severity, and antibody response. the viral load profile of sars-cov-2 is similar to that of influenza, which peaks at around the time of symptom onset, but contrasts with that of sars-cov at around 10 days and that of mers-cov at the second week after symptom onset. 4, 16, 17 the high viral load on presentation suggests that sars-cov-2 can be transmitted easily, even when symptoms are relatively mild. this finding could account for the efficient person-to-person transmission noted in community and health-care settings. clusters in families, workplaces, religious gatherings, and food premises have been widely reported. 18 the viral load profile is important for guiding antiviral treatment. since viral load had already peaked around the time of hospital admission, the risk of emergence of antiviral resistance could be similar to that of single-drug treatment of influenza by adamantanes, acid polymerase inhibitors, and neuraminidase inhibitors. however, our previous clinical trial of influenza treatment showed that a triple antiviral combination could significantly improve the clinical outcome and viral load profile and could reduce emergence of resistant virus quasispecies. 19 currently, no standard treatment is available for figure 3 : relation between viral load and age or disease severity correlation between age and peak viral load (a). comparison of initial (b) and peak (c) viral load between severe and mild cases. comparison of initial (d) and peak (e) viral load between patients with comorbidities and those without comorbidities. covid-19. for sars-cov infection, our previous treatment study showed that a combination of lopinavirritonavir and ribavirin led to significantly fewer compli cations (eg, acute respiratory distress syndrome) or deaths than reported with historical controls treated with ribavirin. 5 lopinavir-ritonavir or interferon beta 1b also reduced lung damage and decreased viral load in a nonhuman primate model of mers-cov. 20 lopinavir is a protease inhibitor with in-vitro activity against sars-cov and mers-cov. however, the idea that sars-cov 3c-like protease was the antiviral target of lopinavir was based purely on binding in computational modelling. 21 other protease inhibitors and nucleotide analogues (eg, remdesivir [gilead sciences, foster city, ca, usa]) are potential candidates for treatment. combination treatment with virus-targeting and host-targeting agents to improve clinical outcome should be investigated. studies for sars-cov have shown that a high initial viral load was associated with death. 22 however, our study only showed that the median viral load was 1 log 10 higher in severe cases than in mild cases, and the difference was not significant. but, older age was associated with a higher peak viral load. in a previous study of patients infected with sars-cov, older age was an independent factor associated with higher viral load, 23 as expected for immunosenescence, which impairs our innate and adaptive immune responses. sars-cov-2 rna could be detected for 20 days or longer in a third of patients who survived in our cohort, and one patient had sars-cov-2 rna detected for 25 days. prolonged detection of viral rna of 20 days or longer was also commonly seen for patients with mers-cov or sars-cov infections. 4, 16 prolonged detection of viral rna represents a challenge for the limited availability of hospital isolation facilities because patients might not be discharged until viral rna is undetectable in respiratory specimens. further studies are warranted to ascertain whether patients are shedding live virus, by viral culture of the prolonged rt-pcr-positive specimens obtained from patients with concomitant seropositivity when shedded virions are coated with host antibodies which render them non-infectious. a criterion for discontinuation of transmission-based precautions is a negative rt-qpcr result from two sets of nasopharyngeal and throat swab specimens. in the current study, one patient with complete symptom resolution tested positive for sars-cov-2 again after 2 days of negative findings. our results suggest that sars-cov-2 might be excreted at low levels despite clinical recovery. thus, both serial viral load monitoring and antibody response should be considered when making decisions about infection control measures, because viral load seemed to be related inversely to serum antibody response in this study. the antibody profile is vital for timing requests for serological assays and interpretation of antibody test results. serological diagnosis is important for patients who present late with a very low viral load, below the detection limit of rt-pcr assays. because most patients have rising antibody titres 10 days after symptom onset, collection of serial serum samples in the convalescent phase would be more useful. serum igg amounts can rise at the same time or earlier than those of igm against sars-cov-2. by comparison with findings of a study on igm and igg eia, in which more patients were seropositive for igg than igm at day 0 and day 5 of hospital admission, 24 a higher anti-np igg anti-np igm proportion of patients in the current study also had earlier igg than igm seroconversion. however, this finding could also be accounted for by a lower sensitivity of the igm eia, which warrants investi gations with more patients. serum antibody levels were not correlated with clinical severity. notably, one patient with severe disease had an early antibody response 6 days after symptom onset. deceased patients infected with sars-cov developed faster peak anti-spike antibody responses when compared with patients who recovered 25 and had subsequent reduced b-cell immunity with impaired neutralising ability. in a sars-cov macaque model, anti-spike igg stimulated pulmonary proinflammatory responses and caused acute lung injury. 26 the detrimental effect of antispike igg was attributable to the effect on wound-healing macrophages, which was mediated via the fcγ receptor. our findings showing correlation between antibody level detected by eia and virus neutralisation titre are especially important for design of vaccine studies, and use of convalescent plasma or therapeutic monoclonal antibodies, which could improve clinical outcome or paradoxically cause immuno pathological damage to the recipient. whole-genome sequencing on paired samples from four patients was successful and showed no differences in individually paired genome sequences. however, single nucleotide polymorphisms were shown to emerge during hospitalisation for mers-cov infection, using a targeted sequencing approach. 27 further studies in more patients with samples obtained at longer intervals could be more informative. a high viral load in throat wash and saliva (up to 10⁸ copies per ml of sars-cov rna) was reported in 17 patients with sars. 28 in a chinese macaque model of sars-cov, salivary gland ducts were early targets of sars-cov and, therefore, were a likely source of the virions found in patients' saliva, particularly early in infection. 29 because of these important findings, our study used posterior oropharyngeal saliva brought up by a throat-clearing manoeuvre to ascertain the temporal viral load profile. the posterior oropharynx is the meeting point between secretions coming from the posterior nasopharynx and the salivary glands and respiratory secretions swept up from the tracheal-bronchial tree. testing of saliva could show viral shedding from both the salivary glands and the upper and lower respiratory tract. moreover, because of greater patient acceptability for posterior oropharyngeal saliva samples than for nasopharyngeal or throat swabs, we obtained 7·5 respiratory specimens per patient for testing. thus, our temporal viral load profile can be analysed by statistics, unlike previous clinical studies of viral kinetics of infections by highly pathogenic betacoronaviruses. 8, 16 further studies are needed to ascertain whether the salivary glands can be infected by sars-cov-2. our study has several limitations. first, we could only include a few patients, and viral load and antibody titre data were not available everyday. this limitation is a common problem in studies of emerging infections such as sars-cov and mers-cov. 16 the few patients enrolled does not allow for adjustment for potential confounding factors that could affect viral load or antibody response. second, 48% of patients enrolled had chronic medical illness, which is a higher proportion than that reported in a large clinical series (24%). 30 although a lower anti-rbd igg level was noted among patients with comorbidities, further studies are warranted with more patients. third, posterior oropharyngeal saliva samples cannot differentiate whether the virus is coming from the nasopharynx or from secretions from the lower respiratory tract; thus, our study cannot indicate whether sars-cov-2 has a predilection for both upper and lower respiratory tract. moreover, some patients might not clear the throat effectively to cough out saliva from deep in the throat, which could decrease test sensitivity when compared with that of nasopharyngeal swabs, particularly in patients with predominant upper respiratory involvement or mild symptoms. finally, the most abundantly expressed internal np might have some cross-antigenicity between sars-cov-2 and sars-cov (90% amino acid homology) and, occasionally, oc43-cov (38% amino acid homology). 31 thus, the less abundantly expressed surface spike protein rbd, which is specific for sars-cov-2 and is the direct target for neutralising antibodies, was used to guard the specificity of our dual antibody assays. 32 covid-19 is an emerging infection with many unknowns. this study has shed light on viral kinetics and antibody response in patients and provides scientific evidence for guiding infection control policies and therapeu tics. further virological and immunological studies are needed to understand sars-cov-2 infection; infection control measures should be reviewed with the rapidly evolving epidemiology of covid-19. kk-wt and k-yy contributed to study design, data collection, data analysis, data interpretation, the literature search, and writing of the report. ot-yt, w-sl, art, t-cw, dcl, jm-cc, ts-hc, dp-ll, cy-cc, vc-cc, jf-wc, and if-nh contributed to patients' recruitment, data collection, and clinical management. cc-yy, j-pc, l-lc, w-mc, k-hc, jdi, ac-kn, rw-sp, c-tl, zc, and hc contributed to the experiments, data collection, data analysis, and data interpretation. all authors reviewed and approved the final version of the report. we declare no competing interests. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings consistent detection of 2019 novel coronavirus in saliva a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 viral load in upper respiratory specimens of infected patients 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 additional molecular testing of saliva specimens improves the detection of respiratory viruses improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia assessment of population susceptibility to upcoming seasonal influenza epidemic strain using interepidemic emerging influenza virus strains serological responses in patients with severe acute respiratory syndrome coronavirus infection and cross-reactivity with human coronaviruses 229e, oc43, and nl63 viral load kinetics of mers coronavirus infection use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china efficacy of clarithromycin-naproxen-oseltamivir combination in the treatment of patients hospitalized for influenza a(h3n2) infection: an open-label randomized, controlled, phase iib/iii trial treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset molecular dynamic simulations analysis of ritonavir and lopinavir as sars-cov 3cl(pro) inhibitors initial viral load and the outcomes of sars nasopharyngeal shedding of severe acute respiratory syndrome-associated coronavirus is associated with genetic polymorphisms molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes antibody responses against sars coronavirus are correlated with disease outcome of infected individuals anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection analysis of intrapatient heterogeneity uncovers the microevolution of middle east respiratory syndrome coronavirus detection of sars-associated coronavirus in throat wash and saliva in early diagnosis 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 clinical characteristics of coronavirus disease 2019 in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 we thank wallace wong, charlotte yee-ki choi, and travis law for technical assistance and data collection. key: cord-269264-ebqq8x8a authors: winter, amy k; hegde, sonia t title: the important role of serology for covid-19 control date: 2020-04-21 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30322-4 sha: doc_id: 269264 cord_uid: ebqq8x8a nan as of april 14, 2020, just under 2 million cases of corona virus disease 2019 (covid19) have been reported worldwide. 1 with the pandemic growing at an alarming rate and national governments struggling to control local epidemics because of scant diagnostics and impermanent nonpharmaceutical interventions, we should look to additional epidemiological solutions. locations such as singapore and taiwan have been successful in slowing epidemic growth by using intensive surveillance with broader testing strategies to identify and contain cases. 2, 3 in the lancet infectious diseases, sarah ee fang yong and colleagues 4 report three clusters of covid19 cases identified in singapore in early 2020 by active casefinding and contact tracing and confirmed with rtpcr. one cluster from a church (church a) was previously identified 5 and linked to two imported cases from wuhan, china. the two additional clusters (church b and a family gathering) were attributable to community transmission of severe acute respiratory syndrome coronavirus 2 (sarscov2) by one individual interacting with both clusters. serological platforms were developed and assessed for confirmation of sarscov2specific antibody responses to capture past infections. by serological analysis, yong and colleagues identified the missing link between the church a cluster and the other two clusters-an individual who had twice tested negative by rtpcr. by linking all three clusters, yong and colleagues highlight the success of such surveillance measures to capture many cases and effectively slow the spread of covid19 in singapore. this investigation exemplifies the failings of rtpcr as a sole diagnostic method in surveillance, because of its inability to detect past infection, and the added value of serological testing, which if captured within the correct timeframe after disease onset can detect both active and past infections. 6, 7 in public health practice, serological analysis can be useful for rapid caseidentification and the subsequent chain of events to actively identify close contacts, recommend quarantine, and define clusters of cases. contact tracing, which is a necessary but insufficient means of disease control, needs careful effort and is sensitive to timing to be effective, particularly in highly dense populations. as shown in singapore, serological analysis can be useful for contact tracing in urban environments and linking clusters of cases retrospectively to delineate transmission chains and ascertain how long transmission has been ongoing or to estimate the proportion of asymptomatic individuals in the population. beyond the immediate use of serological data to identify and contain cases, these data can also be used to set control policies. population serological testing (specifically measuring sarscov2specific igg antibody titres) can estimate the total number of infections by assessing the number of individuals who have mounted an immune response, regardless of whether an infection was subclinical or happened in the recent past (current data suggest antibodies persist for at least 4 weeks). 8 by providing estimates of who is and is not immune to sarscov2, serological data can be used in at least four ways. first, to estimate epidemiological variables, such as the attack rate or casefatality rate, which are necessary to assess how much community transmission has occurred and its burden. second, to strategically deploy immune healthcare workers to reduce exposure of the virus to susceptible individuals. third, to assess the effect of nonpharmaceutical interventions at the population level and inform policy changes to release such measures, fourth, to identify individuals who mounted a strong immunological response to the virus and whose antibody isolates can be used to treat patients via plasma therapy. 9 although the potential for serological assays to help control the covid19 pandemic is substantial, the complexity of developing and validating a diagnostic test is not fully elucidated by yong and colleagues. 4 serological assays are currently being developed for widespread use. 10 yet, several challenges remain: first, assessing the sensitivity and specificity of tests, particularly for determining disease during the acute phase of infection; second, verifying the test is not detecting crossreactivity with other viral pathogens that result in falsepositive results; third, understanding antibody kinetics over time to distinguish thresholds of immunity, because we do not know how long immunity to this novel coronavirus might last; and finally, ensuring the test is reliable for distribution and is costefficient. although rtpcr diagnostics will still be covid19 dashboard by the center for systems science and engineering response to covid19 in taiwan: big data analytics, new technology, and proactive testing interrupting transmission of covid19: lessons from containment efforts in singapore connecting clusters of covid19: an epidemiological and serological investigation investigation of three clusters of covid19 in singapore: implications for surveillance and response measures serology characteristics of sarscov2 infection since the exposure and post symptoms onset serological and molecular findings during sarscov2 infection: the first case study in finland reinfection could not occur in sarscov2 infected rhesus macaques convalescent plasma as a potential therapy for covid19 a serological assay to detect sarscov2 seroconversion in humans key: cord-267436-mivxm8oh authors: groneberg, david a; poutanen, susan m; low, donald e; lode, hartmut; welte, tobias; zabel, peter title: treatment and vaccines for severe acute respiratory syndrome date: 2005-03-10 journal: lancet infect dis doi: 10.1016/s1473-3099(05)01307-1 sha: doc_id: 267436 cord_uid: mivxm8oh the causative agent of severe acute respiratory syndrome (sars), which affected over 8000 individuals worldwide and was responsible for over 700 deaths in the 2002–2003 outbreak, is a coronavirus that was unknown before the outbreak. although many different treatments were used during the outbreak, none were implemented in a controlled fashion. thus, the optimal treatment for sars is unknown. since the outbreak, much work has been done testing new agents against sars using in-vitro methods and animal models. in addition, global research efforts have focused on the development of vaccines against sars. efforts should be made to evaluate the most promising treatments and vaccines in controlled clinical trials, should another sars outbreak occur. severe acute respiratory syndrome (sars) is an infectious disease characterised by substantial morbidity and mortality, first recognised after an outbreak in 2002-2003. 1, 2 the who issued a global alert on sars on march 12, 2003 after receiving reports from china's guangdong province, hong kong, and vietnam regarding clusters of respiratory illness of unknown aetiology. one of the first reports was made by who scientist carlo urbani, who was called to investigate cases of pneumonia of unclear aetiology in a hospital in hanoi; he later died of sars. 3 following the who alert, probable sars cases were also reported from other regions in china, and other asian countries including singapore, taiwan, indonesia, thailand, and the philippines. other countries, including canada, the usa, and germany also identified cases. in retrospect, sars originated in guangdong at the end of 2002. it first spread to other regions in asia and then international travel facilitated its spread to other continents. a cumulative total of 8096 probable cases of sars were recorded during the period from november 1, 2002 to july 31, 2003 , with 774 deaths in 29 countries. 4 soon after sars was identified as a new disease, the who initiated a collaborative global network striving to work together to identify the aetiological agent of sars. 5 in unprecedented time, a novel coronavirus-sars coronavirus-was identified as the probable causative agent of sars 6-8 (figure 1) and koch's principles were demonstrated to be fulfilled by this agent. 10, 11 this virus belongs to the coronavirus family-enveloped, positive-sense rna viruses 12 associated with respiratory disease in human beings and animals. 13, 14 evidence suggests that sars coronavirus originated from sars-like viruses in animals in the southern chinese province of guangdong; the most frequently implicated animal is the himalayan palm civet, an animal found in food markets and eaten as a delicacy. 15, 16 sars coronavirus is organised into 13-15 open reading frames (orfs) containing approximately 30 000 nucleotides. 8, 17, 18 in total, 61 sars-coronavirus sequences derived from different sars epidemic phases have been analysed and genotypes characteristic of each phase have been identified. 19, 20 the different sars-coronavirus orfs represent typical viral genes such as protease and replicase, spike, envelope, membrane, and nucleocapsid, all of which may represent potential therapeutic targets (figure 2). in common with all infections caused by coronaviruses, after infection sars coronavirus induces the synthesis of 3ј coterminal sets of subgenomic mrnas in target the causative agent of severe acute respiratory syndrome (sars), which affected over 8000 individuals worldwide and was responsible for over 700 deaths in the 2002-2003 outbreak, is a coronavirus that was unknown before the outbreak. although many different treatments were used during the outbreak, none were implemented in a controlled fashion. thus, the optimal treatment for sars is unknown. since the outbreak, much work has been done testing new agents against sars using in-vitro methods and animal models. in addition, global research efforts have focused on the development of vaccines against sars. efforts should be made to evaluate the most promising treatments and vaccines in controlled clinical trials, should another sars outbreak occur. cells. 21 in laboratory settings, sars coronavirus is able to infect macaque monkeys, 11 mice, 22 ferrets, and domestic cats. 23 clinically, sars is characterised by systemic symptoms such as fever and myalgia, followed by respiratory symptoms including a non-productive cough and dyspnea. laboratory findings include lymphopenia, and chest radiographs commonly exhibit unilateral or bilateral infiltrates. approximately 15% of cases deteriorate, requiring intubation and mechanical ventilation. 24 the overall mortality rate has been reported to be about 10%. however, sars mortality rates in those over 60 years old have been reported to be as high as 50%. 25 affected children seemed to have milder symptoms with no reports of death. 26 at the time of the 2002-2003 outbreak, physicians shared their personal experiences supporting or rejecting various treatments for sars. because of the rapid progression of the outbreak, multicentre, randomised, controlled interventional trials were not possible, and the success of various treatments remains largely anecdotal. thus, a consensus on therapeutic strategies has not yet been reached. since the outbreak, global research efforts have focused on testing new agents against sars with in-vitro methods and animal models. in addition, much effort has been placed on developing effective vaccines against sars. this review summarises the clinical experience of the use of various treatments during the outbreak and provides an overview of the data, both in vitro and in vivo, supporting, or otherwise, the effectiveness of these treatments and those that have been proposed since the outbreak. in addition, we summarise the progress made to date regarding sars vaccines. a summary of the pharmacological agents that have been used or proposed for the treatment of sars is shown in figure 3 . during the 2002-2003 outbreak, suspected sars cases were usually treated initially with broad-spectrum antibacterial drugs effective against typical bacterial causes of acute community-acquired pneumonia. the administration of broad-spectrum antibiotics-eg, respiratory fluoroquinolones, second-generation cephalosporins, or third-generation cephalosporins-plus a macrolide is recommended at the first signs of the sars, because the initial features of the disease are nonspecific. however, after sars coronavirus is identified as the causative agent, antibiotics may be withdrawn, because there is no evidence that antibiotics are clinically beneficial in the treatment of sars. even before the causative agent of sars was discovered, treatment with ribavirin was used empirically to treat patients with sars. 27 ribavirin is a synthetic nucleoside with broad-spectrum antiviral activity. 28 clinical studies that have assessed the effectiveness of ribavirin in sars range from anecdotal case reports and retrospective case series to one randomised clinical trial with multiple treatment arms. however, none of these studies definitively determine whether or not ribavirin is effective against sars. case reports and case series suggest that combined treatments including ribavirin may be beneficial to some extent; however, in all of these studies, the effect of ribavirin is confounded by the concomitant use of other agents. for example, studies describe clinical and radiological improvements in patients treated with ribavirin and steroids, 29,30 but, without a control group, it is difficult to determine whether the improvements result from ribavirin, steroids, the combination of both, or the natural course of the illness. one study showed that the delayed initiation of combined therapy with ribavirin and steroids was among the risk factors associated with severe complicated disease, suggesting that ribavirin might be beneficial, but it is difficult to delineate the role of delaying the use of ribavirin from the delay in the use of steroids. 31 the results of a randomised clinical study in guangdong, involving multiple different treatment arms, suggest that ribavirin given at a low dose (400-600 mg/day) was less effective compared with an early and aggressive use of steroids with interferon alfa. 32 however, the lack of a control arm in this study does not allow for one to make definitive conclusions about whether or not ribavirin has any positive effect on sars compared with no treatment. invitro testing showed that ribavirin was not able to inhibit sars-coronavirus replication at clinically achievable concentrations. 33, 34 this finding, combined with postmortem findings demonstrating high viral loads in most patients despite treatment with ribavirin, 35 suggests that if ribavirin has any effect against sars coronavirus, it is likely to have only a small beneficial effect at best. this is important when the side-effects that have been associated with ribavirin use are considered. knowles and co-workers reported common adverse events in 110 people with suspected or probable sars who were treated with ribavirin. 36 61% of these people had evidence of haemolytic anaemia; hypocalcaemia and hypomagnesaemia were reported in 58% and 46% of the people, respectively. the combination of the protease inhibitors lopinavir and ritonavir was used less frequently during the sars outbreak compared with ribavirin. the lopinavir/ ritonavir combination was first considered a potentially useful treatment after in-vitro studies showed it had antiviral activity against sars coronavirus. 37, 38 chan and colleagues 38 compared outcomes in people who received lopinavir/ritonavir as initial treatment, and as rescue therapy, with matched controls; all patients were given ribavirin and steroids according to a standardised protocol. the addition of lopinavir/ritonavir as initial treatment was associated with a statistically significant reduction in the overall death rate and intubation rate compared with matched controls (pͻ0·05). however, the subgroup that received lopinavir/ritonavir as rescue therapy did not show a significant difference in these endpoints. chu and co-workers 37 also assessed treatment with lopinavir/ritonavir compared with historic controls; all patients were also treated with ribavirin and steroids in a similar protocol to that of chan and collegues. adverse events (development of acute respiratory distress syndrome [ards] or death within 21 days) were significantly lower in the lopinavir/ritonavir group than in the historic controls (pͻ0·001). in addition, a significant reduction in the need for rescue pulsed steroids for severe respiratory deterioration (pͻ0·001) and significantly lower nosocomial infections were also noted in those treated with lopinavir/ritonavir, compared with controls (pͻ0·043). by multivariate analysis, it was demonstrated that the lack of treatment with lopinavir/ritonavir, age 60 years old or greater, and positive hepatitis b carrier status were independent predictors of an adverse outcome including death or the development of ards requiring intensive care within 21 days of onset of illness. 37 based on these studies, lopinavir/ritonavir appears to be a promising anti-sarscoronavirus agent. other protease inhibitors have been studied in vitro for potential antiviral effects in sars. for example, yamamoto and colleagues 39 screened a set of compounds that included antiviral drugs already widely used, and found that nelfinavir strongly inhibited sarscoronavirus replication. nelfinavir inhibited the cytopathic effect induced by sars-coronavirus infection, and the expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated, infected cells. in addition, barnard and colleagues 40 found that two protease inhibitors-calpain inhibitor vi (val-leu-cho) and calpain inhibitor iii (z-val-phe-ala-cho)-inhibited sars coronavirus, suggesting that other protease inhibitors may also be useful in the treatment of sars. the membrane-associated carboxypeptidase angiotensinconverting enzyme 2 (ace2), is a cellular receptor for sars coronavirus, interacting with the s1 domain of the spike protein. 41 thus, peptides and small compounds that bind to ace2 42, 43 are possible agents for the treatment and prevention of sars. in addition, a soluble form of the receptor, antibodies to it, or the receptorbinding domain of the spike protein, may be candidate treatments. indeed, sui and co-workers 44 searched a nonimmune human antibody library and successfully identified an anti-s1 human monoclonal antibody, 80r, that potently neutralises sars-coronavirus infection and efficiently inhibits syncytium formation by blocking binding to ace2. 80r was shown to compete with soluble ace2 for association with the s1 domain of the spike protein and bound to it with high affinity. theoretical reasoning and in-vitro evidence suggest that fusion inhibitors are promising treatment candidates for sars. 45, 46 peptides derived from the heptad repeat regions 1 and 2 of hiv-1 gp41-a transmembrane protein involved in the fusion of hiv and target cellsare the basis for anti-hiv fusion inhibitors. based on similarities between the heptad repeat regions of gp41 in hiv-1 and the heptad regions in the spike protein of sars coronavirus, a common mechanism mediating fusion between each virus and target-cell membranes was postulated. 45, 46 liu and colleagues 45 tested two sets of peptides corresponding to the heptad regions in the spike protein for inhibitory activity against sars coronavirus, and found that one peptide-cp1inhibited sars-coronavirus infection in vitro. it has been postulated that cp1 binds to heptad region 1 of the spike protein and interferes with the conformational changes needed to allow fusion with target cells. rna interference (rnai) treatment is a process by which small interfering rnas (sirna) are administered, leading to degradation of mrna with identical sequence specificity. 47 this technology has been used to silence genes in cultured cells and in animals, and to target hiv, hepatitis b, and hepatitis c viral infections. [48] [49] [50] to explore the possibility of interrupting sars-coronavirus replication with sirnas, specific sirnas targeting the spike gene in sars coronavirus were synthesised. these sirnas effectively and specifically inhibited gene expression of the spike protein in sars-coronavirusinfected cells. 51 another study assessed the in-vitro efficiacy of six sirna molecules targeting different sites of the replicase 1a region of the sars-coronavirus genome. 52 judged by morphological changes, three of the molecules markedly inhibited the cytopathic effects caused by viral infection and replication. the three sirnas also inhibited the infection and replication of different strains of sars coronavirus, indicating that sirnas targeting the replicase 1a region may be an option for future clinical use. 52 in-vitro studies have shown that glycyrrhizin, a component of liquorice roots, is able to inhibit sarscoronavirus replication. 34 glycyrrhizin inhibits hiv replication in vitro 53 and has been used clinically in the treatment of hepatitis c 54 and hepatitis b 55 with some success. the mechanism of glycyrrhizin-induced inhibition of viral replication-and specifically sarscoronavirus replication-is unclear, but possibly involves inhibition of replication through an antiviral effect of nitric oxide (no). glycyrrhizin upregulates expression of inducible no synthase and production of no in mouse macrophages. 56 in addition, preliminary results by cinatl and colleagues 34 show that glycyrrhizin induces no synthase in vero cells used to cultivate sars coronavirus. cinatl and colleagues 34 showed that sars-coronavirus replication is inhibited when deta nonoate-a no donor compound-is added to the culture medium. this finding has been further corroborated by keyaerts and co-workers 57 using a different no donor compound, s-nitroso-n-acetyl-penicillamine. keyaerts and colleagues also report their findings on the use of inhaled no gas to treat a number of people with sars. their results suggest an associated immediate improvement in oxygenation and a lasting effect after termination of inhalation of no, which is known to be a potent mediator of airway inflammation. 58, 59 niclosamide wu and colleagues 60 screened a set of marketed drugs that were not registered for antiviral use to determine if any had in-vitro activity against sars coronavirus. they found that niclosamide, an existing antihelmintic drug, was able to inhibit replication of sars coronavirus. the underlying mechanism by which the drug exerts this effect is unclear, but the study shows that niclosamide does not interfere with the virion's attachment to, or entry into, cells, nor does it appear to inhibit the protease activity. new compounds continue to be tested, with the goal of finding more potential candidate treatments for sars. for example, from over 10 000 agents tested, wu and colleagues 61 found 15 compounds with potent anti-sars-coronavirus activity. more compounds are likely to be discovered in the future. during the 2002-2003 sars outbreak, systemic steroids became a mainstay of sars therapy in many centres. 62 the rationale for their use was based on the paradoxical finding that, despite a fall in sars-coronavirus viral load and a rise in sars-specific igg typically seen during the 3rd week of illness, a clinical deterioration was observed in some people. 31 in addition, pathological findings consistent with bronchiolitis obliterans organising pneumonia and ards led to the hypothesis that immune hyperactivity resulting from cytokine dysregulation may be a component of sars that could be reduced by steroid treatment. 63 in most cases, steroids were administered as adjunctive therapy to ribavirin treatment. if the patient's review respiratory condition worsened clinically, pulsed, highdose steroids were added. however, most studies were confounded by the concomitant use of other agents, and none of the studies contained a control group. thus, whether or not steroids have a beneficial effect in the treatment of sars cannot be readily determined. in some studies, treatment regimens containing steroids seemed to be associated with chest radiographic improvements, fever defervescence, and improvement in oxygenation rates earlier than patients not treated with steroids. 32, 64, 65 however, in a study by hsu and colleagues, 65 adding steroids was not associated with clinical improvement, although the dose of steroids in this study was lower than in those where benefit was seen. ho and co-workers 66 retrospectively compared the clinical and radiographic outcomes of people with probable sars who received ribavirin, 17 of whom initially received pulsed, highdose steroids and 55 of whom initially received lowdose steroids. pulsed, high-dose steroids were also given to any patient as rescue therapy in the presence of deteriorating respiratory status. the cumulative steroid dose, intensive care unit admission rate, need for mechanical ventilation, and mortality rates were similar in both groups after 21 days. however, those people initially given pulsed steroids required less oxygen and had earlier radiographic improvement. in addition, they required substantially less rescue pulsed steroids. this study suggests that early initiation of pulsed steroids may have a role in the treatment of sars. however, definitive studies are needed and the potential benefits of steroids must be compared with the associated risks, such as the development of avascular necrosis, secondary sepsis, and fatal aspergillosis, some of which have been described in people with sars. 67, 68 in beijing, hong and du 69 evaluated 67 people with sars who had received steroids and ribavirin, and who presented with largejoint pain, potentially caused by avascular necrosis, between march and may 2003. both plain radiographs and magnetic resonance imaging examination were completed on the same day. 28 people were identified with avascular necrosis. the mean time to diagnosis of avascular necrosis was 119 days after the onset of sars, or 116 days after steroid use. interferons type 1 interferons have been shown to inhibit sarscoronavirus replication in in-vitro studies. 33, [70] [71] [72] because of initial reports describing these in-vitro results, interferons were used clinically during the latter part of the outbreak. loutfy and colleagues 73 described their clinical experience with interferon alfacon 1-a recombinant, non-naturally occurring type 1 interferon containing common aminoacids from several natural interferon alfa subtypes-in 22 people with probable sars treated in an open-label study in toronto. 13 people with sars who received treatment with steroids alone were compared with nine people who received steroids plus interferon alfacon 1. the group treated with interferon alfacon 1 had significantly improved oxygen saturation levels (p=0·02) and a more rapid resolution of radiographic lesions. in addition, this group exhibited substantially less elevation in creatine kinase levels and a trend towards a more rapid normalisation of lactate dehydrogenase levels. 73 however, this group also received higher doses of steroids, so it is difficult to determine whether or not the beneficial effects were due to the interferon alfacon 1. haagmans and co-workers 74 investigated the prophylactic use of interferons in a macaque model. 3 days before inoculation with sars coronavirus, macaques were given pegylated interferon alfa. substantially reduced viral replication, viral excretion, viral antigen expression by type 1 pneumocytes, and pulmonary damage were noted in the treated macaques compared with untreated macaques. post-exposure treatment with pegylated interferon alfa yielded intermediate results. these results suggest that interferons have a role in the treatment of sars. because most patients develop antibodies against sars coronavirus and survive the disease, passive and active immunisation are viewed as possible effective means to prevent and/or treat sars. 75 indeed, the development of various vaccines is one of the most important goals of ongoing sars research. one of the initial proposals to treat sars was to use sera from people convalescing from sars as passive immunotherapy. this passive immunisation was attempted with anecdotal success. 76 since then, prior infection and passive transfer of murine neutralising antibodies have been shown to prevent replication of sars coronavirus in the respiratory tract in mice. 77 technological advances enabling the development and purification of human monoclonal antibodies can be exploited to create specific monoclonal antibodies in large-scale production. indeed, monoclonal antibodies obtained from immortalised b lymphocytes isolated during convalescence from people with sars have been shown to neutralise virus infection in vitro and to prevent virus replication in a mouse model of sarscoronavirus infection. 78 in addition, ter meulen and colleagues 79 showed that prophylactic administration of a human igg monoclonal antibody reactive with whole inactivated sars coronavirus was able to reduce replication of sars coronavirus in the lungs of infected ferrets, completely prevent the development of sars-coronavirus-induced macroscopic lung pathology, and stop the shedding of virus in pharyngeal secretions. although passive immunisation strategies appear promising, the ideal approach to ensure rapid control of future outbreaks of sars is to generate an effective and safe vaccine. there are numerous teams worldwide working on the creation of vaccines using inactivated sars coronavirus, recombinant subunits, recombinant dna, and viral vectors. 80 given the potential for antibody-directed viral enhancement and disease exacerbation, as reported for vaccines directed against another coronavirus (feline infectious peritonitis coronavirus), 81 it is important that all vaccines created be carefully evaluated before being used clinically. of all of the vaccines in development, most work relates to viralvectored vaccines and dna vaccines. to date, three different viral-vectored vaccines have been described with successful results reported in animal models. [82] [83] [84] gao and colleagues 82 reported using three adenoviralbased vectors expressing codon-optimised sarscoronavirus spike, membrane, and nucleocapsid proteins. intramuscular vaccination with all three vaccines at day 0 and day 28 was shown to induce broad, virus-specific immunity in rhesus macaques. all six vaccinated macaques had antibody responses against the spike protein and t-cell responses against the nucleocapsid protein. in addition, all vaccinated animals showed strong neutralising-antibody responses to sars-coronavirus infection in vitro. challenge tests to determine whether or not this immune response was able to prevent, or reduce the severity of, infection with sars coronavirus were not completed. bisht and co-workers 84 constructed recombinant forms of the highly attenuated modified vaccinia virus ankara (mva) containing the gene encoding the full-length sars-coronavirus spike protein and assessed whether expression of the spike protein alone in mva could raise neutralising antibodies and protectively immunise mice. 84 both intranasal and intramuscular administration of the vaccine to balb/c mice at 0 and 4 weeks led to the production of serum antibodies against the spike protein that neutralised sars coronavirus in vitro. 4 weeks after the second immunisation, vaccinated animals and control animals were challenged with sars coronavirus. those given the vaccine had reduced titres of sars coronavirus in the respiratory tract. likewise, the passive transfer of serum from mice immunised with the vaccine to naive mice led to a reduction in sars-coronavirus replication. these findings suggest that this mva-based vaccine is a promising sars-coronavirus vaccine candidate. bukreyev and colleagues 83 reported their successful experience with the mucosal immunisation of african green monkeys with an attenuated parainfluenza virus expressing the sars-coronavirus spike protein. the complete sars-coronavirus spike protein gene was incorporated into a recombinant attenuated parainfluenza virus that is being developed as a live attenuated, intranasal paediatric vaccine against human parainfluenza virus type 3. four african green monkeys were vaccinated with a single dose of the vaccine, administered via the respiratory tract, and four other monkeys were vaccinated with a control. all monkeys were challenged with sars coronavirus 28 days after immunisation. neutralising serum antibodies were noted in all of the vaccinated animals. after sarscoronavirus challenge, viral shedding was documented in all of the control animals but not in any of the vaccinated animals. 83 the authors concluded that a vectored mucosal vaccine expressing the sarscoronavirus spike protein alone may be highly effective for the prevention of sars in a single-dose format. dna vaccines are also an attractive option for sars vaccines. 85 thus far, three experimental studies have been published addressing dna vaccination in sars. [86] [87] [88] yang and colleagues 86 showed that giving mice a sars-coronavirus dna vaccine encoding the spike glycoprotein induced t-cell responses, neutralisingantibody responses, and protective immunity. alternative forms of the spike protein were assessed and all were found to induce substantial neutralisingantibody titres and strong immune responses mediated by cd8 and cd4 cells. in addition, a reduction in viral replication in the lungs by more than six orders of magnitude was noted after sars-coronavirus challenge; the protection was shown to be mediated by a humoral, but not a t-cell-dependent, immune mechanism. these findings show that dna vaccines based on the spike glycoprotein may lead to effective immune responses with protective immunity in animal models. kim and co-workers 87 reported the generation and characterisation of dna vaccines targeting the nucleocapsid protein of sars coronavirus by antigen linkage to calreticulin, which has been shown to enhance mhc class i presentation to cd8(+) t cells. with a murine model, it was shown that the vaccination with this dna vaccine leads to the generation of a more potent nucleocapsid-specific humoral and t-cellmediated immune responses, compared with nucleocapsid dna alone. in addition, mice vaccinated with the dna vaccine were capable of substantially reducing the titre of challenging vaccinia virus expressing sars-coronavirus nucleocapsid protein. in a similar study by zhu and colleagues, 88 immunisation of mice with a nucleocapsid-based dna vaccine led to nucleocapsid-specific antibodies and specific cytotoxic t-cell activity. challenge tests were not completed. together, the data presented on potential vaccines reflect enormous international efforts. because a vaccine usually takes 6-8 years of clinical development after review entering phase i clinical trials before being licensed, it is not expected that any of these vaccines will be available for clinical use in the near future. however, given the pace and amount of progress to date, the period of time before clinical production of a sars vaccine may be substantially shortened compared with other vaccines. whether or not sars will re-emerge is a matter of debate. 89, 90 however, in the event that sars does recur, the most promising-and immediately available-agents for the treatment of the syndrome seem to be type 1 interferons, steroids, and lopinavir/ritonavir, based on the available data on agents already clinically approved. however, by the time another outbreak arrives, many of the other promising agents-eg, sars-coronavirusspecific receptor-binding inhibitors, fusion inhibitors, and sirnas-may have been approved for clinical use. the choice of agents will need to be determined based on the available data at that time. ideally, the most promising agents would be given in a controlled clinical trial. the difficulties in designing and implementing controlled clinical trials-which limited the ability of researchers to do such trials during the past sars outbreak, and which will continue to pose problems in the event of future outbreaks of sars or other novel pathogens-have been summarised. [91] [92] [93] the best solution to facilitate the implementation of clinical trials in future outbreaks would be the establishment of an international collaborative clinical-trials group with access to appropriate contingency funds, and an internationally accepted ethics review board. until then, research based on in-vitro studies and in-vivo animal models should be continued to determine the best agent, or combination of agents, worthy of further clinical consideration. we declare that we have no conflicts of interest. data for this review were identified by searches of medline, current contents, and references from relevant articles; numerous articles were identified through searches of the extensive files of the authors. search terms were "severe acute respiratory syndrome", "sars", "treatment", "coronavirus", "infection", "sars coronavirus", "vaccination", and "antiviral". english language papers were reviewed. severe acute respiratory syndrome: global initiatives for disease diagnosis the aetiology, origins, and diagnosis of severe acute respiratory syndrome summary of probable sars cases with onset of illness from 1 world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of 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 a novel coronavirus associated with severe acute respiratory syndrome morphology and morphogenesis of severe acute respiratory syndrome (sars)-associated virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome koch's postulates fulfilled for sars virus nidovirales: a new order comprising coronaviridae and arteriviridae the structure and replication of coronaviruses the biology and pathogenesis of coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china wild animals could be source of sars the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome molecular evolution of the sars coronavirus during the course of the sars epidemic in china epidemiological and genetic analysis of severe acute respiratory syndrome the molecular biology of coronaviruses mice susceptible to sars coronavirus virology: sars virus infection of cats and ferrets severe acute respiratory syndrome: clinical outcome and prognostic correlates epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong children hospitalized with severe acute respiratory syndrome-related illness in toronto antiviral treatment of sars: can we draw any conclusions? the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china severe acute respiratory syndromerelated coronavirus is inhibited by interferon-alpha glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus severe acute respiratory syndrome-associated coronavirus in lung tissue common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus inhibition of severe acute respiratory syndrome-associated coronavirus (sarscov) by calpain inhibitors and beta-d-n4-hydroxycytidine angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus novel peptide inhibitors of angiotensin-converting enzyme 2 substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace2) inhibitors potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors cloaked similarity between hiv-1 and sars-cov suggests an anti-sars strategy inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering rna modulation of hiv-1 replication by rna interference interference of hepatitis c virus rna replication by short interfering rnas short interfering rna-directed inhibition of hepatitis b virus replication silencing sars-cov spike protein expression in cultured cells by rna interference inhibition of sars-associated coronavirus infection and replication by rna interference effect of glycyrrhizin, an active component of licorice roots, on hiv replication in cultures of peripheral blood mononuclear cells from hiv-seropositive patients long-term treatment of chronic hepatitis c with glycyrrhizin [stronger neo-minophagen c (snmc)] for preventing liver cirrhosis and hepatocellular carcinoma lamivudine and glycyrrhizin for treatment of chemotherapy-induced hepatitis b virus (hbv) hepatitis in a chronic hbv carrier with non-hodgkin lymphoma induction of inducible nitric oxide synthase and proinflammatory cytokines expression by o,pј-ddt in macrophages inhibition of sars-cov infection in vitro by s-nitroso-nacetylpenicillamine, a nitric oxide donor compound role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness role of nitric oxide in chronic allergen-induced airway cell proliferation and inflammation inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide small molecules targeting severe acute respiratory syndrome human coronavirus pro/con clinical debate: steroids are a key component in the treatment of sars lung pathology of fatal severe acute respiratory syndrome clinical features and short-term outcomes of 144 patients with sars in the greater toronto area severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome fatal aspergillosis in a patient with sars who was treated with corticosteroids sars: prognosis, outcome and sequelae avascular necrosis of bone in severe acute respiratory syndrome role of interferons in the treatment of severe acute respiratory syndrome inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs interferon-beta 1a and sars coronavirus replication interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques how the sars vaccine effort can learn from hivspeeding towards the future, learning from the past treatment of severe acute respiratory syndrome with convalescent plasma prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets caution urged on sars vaccines a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination effects of a sars-associated coronavirus vaccine in monkeys mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice nucleic acid vaccines: an overview a dna vaccine induces sars coronavirus neutralization and protective immunity in mice generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus induction of sars-nucleoproteinspecific immune response by use of dna vaccine sars-one year later seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us clinical trials and novel pathogens: lessons learned from sars preparing to prevent severe acute respiratory syndrome and other respiratory infections collateral damage: the unforeseen effects of emergency outbreak policies support from the deutsche atemwegsliga and the german research foundation (dfg gr2014/2-1) to dag is gratefully acknowledged. key: cord-269838-1943g1ha authors: haffizulla, jason; hartman, aaron; hoppers, melanie; resnick, harvey; samudrala, steve; ginocchio, christine; bardin, matthew; rossignol, jean-françois title: effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial date: 2014-05-19 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70717-0 sha: doc_id: 269838 cord_uid: 1943g1ha background: influenza is an important cause of morbidity and mortality worldwide. treatment options are scarce, and new drugs with novel mechanisms of action are needed. we aimed to assess the efficacy and safety of nitazoxanide, a thiazolide anti-infective, for treatment of acute uncomplicated influenza. methods: we did a double-blind, randomised, placebo-controlled, phase 2b/3 trial in 74 primary care clinics in the usa between dec 27, 2010, and april 30, 2011. we enrolled participants aged 12–65 years with fever, at least one respiratory symptom, and one constitutional symptom of influenza within 48 h of symptom onset. we randomly assigned participants to receive either nitazoxanide 600 mg, nitazoxanide 300 mg, or placebo twice daily for 5 days, (ratio 1:1:1) and followed them up for 28 days. randomisation lists were computer generated and done in blocks of three. sponsor, investigators, study monitors, patients, and laboratory personnel were all masked to treatment allocation in the study. the primary endpoint was the time from first dose to alleviation of symptoms. the primary analysis was by intention-to-treat for participants with influenza infection confirmed by rt-pcr or culture at baseline. this trial is registered with clinicaltrials.gov, number nct01227421. findings: of 650 participants screened, 624 (96%) were enrolled. of these, 212 were randomly assigned to receive placebo twice a day, 201 to receive nitazoxanide 300 mg twice a day, and 211 to receive nitazoxanide 600 mg a day. the median duration of symptoms for participants receiving placebo was 116·7 h (95% ci 108·1–122·1) compared with 95·5 h (84·0–108·0; p=0·0084) for those receiving 600 mg nitazoxanide and 109·1 h (96·1–129·5, p=0·52) for those receiving 300 mg nitazoxanide. adverse events were similar between the three groups, the most common being headache reported by 24 (11%) of 212 patients enrolled in placebo group, 12 (6%) of 201 patients in the low-dose group, and 17 (8%) of 211 patients in the high-dose group, or diarrhoea, reported by seven (3%) patients in the placebo group, four (2%) patients enrolled in the low-dose group, and 17 (8%) patients in the high-dose group. interpretation: treatment with nitazoxanide 600 mg twice daily for 5 days was associated with a reduction of the duration of symptoms in participants with acute uncomplicated influenza. further studies are warranted to confirm these findings and to assess efficacy of the drug alone or in combination with existing drugs in seriously ill patients and those at risk of influenza complications. funding: romark laboratories lc. infl uenza, a contagious respiratory illness caused by infl uenza a, b, or c viruses, is an infection of global concern resulting in about 3-5 million cases of severe disease and 250 000-500 000 deaths annually. 1 in the usa, seasonal infl uenza aff ects-on average-5-20% of the population per year leading to roughly 200 000 hospital admissions and 3000-49 000 deaths. 2, 3 the threat of pandemic infl uenza caused by emerging viruses such as the avian ah5n1 or more recently ah7n9 is a major concern because of potential eff ects on human life, economy, national security, and functioning of society. because of widespread adamantine resistance, the treatment of infl uenza is at present limited to the neuraminidase inhibitors, oseltamivir and zanamivir. new drugs with novel mechanisms of action that can be used alone or in combination with neuraminidase inhibitors are urgently needed to improve treatment outcomes and mitigate risks of resistance. nitazoxanide, a fi rst-in-class thiazolide anti-infective, inhibits replication of a broad range of infl uenza viruses, including neuraminidase inhibitor-resistant strains, blocking the maturation of viral haemagglutin at the posttranslational level. 4, 5 in cell culture studies, nitazoxanide acts synergistically with neuraminidase inhibitors. 5 repeated passage of infl uenza viruses in subinhibitory concentrations of the drug have proven unsuccessful in selecting resistant strains suggesting a high barrier to resistance. 5 nitazoxanide also inhibits replication of respiratory viruses including parainfl uenza virus, coronavirus, and respiratory syncytial virus in cell cultures. 5 nitazoxanide (alinia, romark laboratories lc) is licensed in the usa for treatment of diarrhoea caused by cryptosporidium parvum and giardia lamblia, 6 and has been widely used throughout latin america for treatment of intestinal parasitic infections. a 300 mg controlled-release tablet was developed to deliver plasma con centrations suitable for treatment of viral respiratory infections. we aimed to assess safety and effi cacy of two diff erent doses of nitazoxanide in reducing the duration of symptoms of acute un complicated infl uenza. we designed this phase 2b/3 randomised clinical trial in consultation with us food and drug administration (fda) guidance for industry 7 and published clinical trials of oseltamivir and zanamivir. [8] [9] [10] [11] [12] effi cacy of nitazoxanide in treatment of acute uncomplicated infl uenza had to be established before doing studies in seriously ill patients or patients at high risk of complications of infl uenza. because of the variability of disease and diffi culties defi ning margins of noninferiority to existing drugs, we used a placebo to clearly assess the activity of the drug. the 300 mg and 600 mg doses of nitazoxanide used in this study were selected on the basis of past experience with the pharmacokinetics and tolerability. 13 we recruited patients from 74 primary care clinics in the usa. the main eligibility criteria were participants aged 12-65 years, oral temperature greater than 38°c (>100·4°f), at least one respiratory symptom (cough, sore throat, nasal discharge, nasal congestion, sneezing) and one constitutional symptom (headache, myalgia, sweats or chills, fatigue), symptom duration of 48 h or less, and confi rmation of infl uenza by a laboratory in the local community (30 mile radius of the site). we excluded because of severity of illness requiring admission to hospital, high risk of infl uenza-related complications according to infectious diseases society of america guidelines 14 or present us centers for disease control and prevention (cdc) criteria, 15 vaccination for seasonal infl uenza on or after aug 1, 2010, treatment with any dose of oseltamivir, zanamivir, amantadine, rimantadine, nitazoxanide, or any investi gational drug therapy within 30 days before screening, and active respiratory allergies or pre-existing illnesses that could place the participant at an unreasonably increased risk. the central institutional review board (irb) or a local irb at each centre approved the protocol. the trial was done under an investigational new drug application with the fda and done in accordance with guidelines set by the world medical assembly (declaration of helsinki, last amendment in seoul, 2008). every participant or their guardian gave written informed consent. participants younger than 18 years of age provided written assent. randomisation lists were computer generated. every participant was randomly assigned (ratio 1:1:1) to receive either two bottles containing nitazoxanide 300 mg tablets (nitazoxanide 600 mg group), one bottle containing nitazoxanide 300 mg tablets plus one bottle containing placebo tablets (nitazoxanide 300 mg group), or two bottles of placebo (placebo group). nitazoxanide tablets and placebo tablets were packaged in white high-density polyethylene bottles, each containing ten tablets. participants were instructed to take two tablets, one from each bottle, twice daily with food for 5 days. the randomisation lists and medication packages were prepared by an independent third party who maintained the blinding of the trial until the database was locked. randomisation was done in blocks of three. masked study medication (three treatment kits per block) were assigned to every investigator who sequentially assigned treatment numbers to participants as they were enrolled. the randomisation list was masked to study participants such as sponsor, investigators, study monitors, patients, and laboratory personnel. immediately after obtaining informed consent and verifying eligibility, every patient had a baseline (day 1) physical examination and medical history recorded. we obtained two nasopharyngeal swabs (nylon fl ocked dry swabs, copan diagnostics, murrieta, ca, usa), and blood and urine samples for laboratory safety testing and calculation of infl uenza antibody titres. study drugs were dispensed, and participants were instructed to use a diary twice daily (roughly every 12 h) to record the time and amount of every dose of study drug, symptom severity, concomitant medications, and adverse events. at every diary entry, the participants graded all nine symptoms (runny nose, nasal congestion, sore throat, cough, headache, muscle aches, tiredness or fatigue, feverish, and sweats or chills) on a scale of 0 to 3 as absent (0), mild (1), moderate (2), or severe (3). participants completed the diaries up to at least study day 7, or until all symptoms were either absent or mild and had remained so for at least 24 h. a study nurse visited or telephoned every participant daily on study days 2-5 to review symptoms and check for infl uenza-related complications. participants returned to the clinic for physical examination and diary review on day 7 and day 28. we obtained blood and urine samples for laboratory safety tests and nasopharyngeal swabs for virology testing on day 7. we obtained a blood sample for measurement of infl uenza antibody titres on day 28. at ten sites, a nurse visited participants on days 2, 3, 4, and 5 to collect two nasopharyngeal swabs. we selected 24 participants for 12 h assessment of pharmacokinetics immediately after their fi rst dose on the morning of day 2 (12 h after their last dose on day 1). antiviral drugs for infl uenza, over-the-counter cough or cold medications, and anti-allergy drugs for respiratory allergies were prohibited during the study. paracetamol was allowed as necessary for fever (oral temperature ≥38°c). to monitor compliance, participants returned the bottles in which the medication was dispensed along with any unused medication, and pill counts were recorded. laboratory safety tests were haematology tests (complete blood count with diff erential), blood chemistry tests (comprehensive metabolic panel with lipid profi le), and urinalysis at baseline and on day 7. north shore-lij health system laboratories in collaboration with barc usa (lake success, ny, usa) did virology tests on nasopharyngeal swab, which were placed into 3 ml of viral transport medium and transported refrigerated to a central laboratory within 24 h. the swab samples were eluted, divided into aliquots, and immediately frozen at -70°c. naso pharyngeal swabs at baseline and on day 7 were cultured for infl uenza viruses and analysed by rt-pcr with the prodesseprofl u+ (gen-probe prodesse, waukesha, wi, usa) for infl uenza a and b and the luminex xtag rvp v1 assay (luminex corporation, austin, tx, usa) to detect infl uenza a (non-specifi c for subtype), infl uenza a subtypes seasonal h1 and h3, infl uenza b, respiratory syncytial virus a, respiratory syncytial virus b, adenovirus, human meta-pneumovirus, enterovirus or rhinovirus, parainfl uenza viruses 1, 2, 3, and 4, and coronaviruses 229e, oc43, nl63, and hku1. samples positive for infl uenza a were also analysed by real-time rt-pcr for 2009 infl uenza a h1n1 on the basis of cdc protocol. 16 for participants positive for infl uenza a or b by culture or rt-pcr at baseline, viral titres were measured at baseline and all subsequent collection points. we calculated titres as log 10 tissue culture infective dose 50 (tcid 50 )/0·2 ml and as log 10 rna copies with a quantitative rt-pcr method developed and validated by north shore-lij health system laboratories. for participants diagnosed with infl uenza by culture or rt-pcr at baseline, we measured infl uenza antibody titres by haemagglutination inhibition assay from serum samples obtained at baseline and at day 28 to assess eff ect of treatment on humoral immune response. reference antigens used for measurement of antibody titres were h1/a/ california/7/2009, h3/a/perth/16/2009, and b/ brisbane/60/2008. to assess potential for resistance, we tested the viruses cultured from nasopharyngeal swab samples of nitazoxanide-treated participants on day 5 or day 7 and the corresponding virus sample from baseline (day 1) for susceptibility to tizoxanide as previously described. 5 the primary endpoint for the clinical trial was time from fi rst dose to alleviation of symptoms based on patientreported symptom data. this method has been validated by clinical trials of oseltamivir and zanamivir. [8] [9] [10] [11] [12] we deemed the symptoms of the participants as alleviated at the beginning of the fi rst 24 h period throughout which each symptom was graded as either absent or mild (0 or 1). in addition to the seven symptoms (cough, nasal obstruction, sore throat, fatigue, headache, myalgia, and feverishness) monitored in the oseltamivir and zanamivir studies, participants enrolled in this study also monitored runny nose and sweats or chills. secondary endpoints were change in infl uenza virus titre with time, time to cessation of viral shedding, time to alleviation of individual symptoms, symptom severity, complications of infl uenza, time to return to normal activity, time lost from work, and infl uenza antibody response. we analysed data according to a statistical analysis plan developed before the start of the clinical trial. the plan called for two primary effi cacy analyses, each comparing the time from fi rst dose to alleviation of symptoms for nitazoxanide 300 mg versus placebo, and nitazoxanide 600 mg versus placebo using a kaplan-meier survival analysis, and a cox proportional hazards test including geographic location as a factor, two-sided α=0·025. we checked the proportional hazards assumption by including interaction terms of all covariates with (log)time and by visual inspection of the graph of the log(-log(survival)) versus the log of time. if hazards were not proportional, we used a prentice-wilcoxon test. we did analyses for a population consisting of participants who received at least one dose of study drug and had laboratory-confi rmed infl uenza infection by rt-pcr or culture at baseline. we did sensitivity analyses to assess eff ects of paracetamol use (repeating the primary analysis including paracetamol use and its interaction with the treatment group) and censored data (assuming participants with censored data had 28 days to alleviation of symptoms). sample size calculations assumed a median time to symptom alleviation for placebo group of 4·3 days, a diff erence in median time to symptom alleviation between placebo and nitazoxanide treatment groups of 1·5 days, and a dropout rate of 5%. with a log-rank test, with α=0·025 (due to two primary effi cacy analyses) and a power of 80%, a sample size of 146 randomly assigned and treated participants with laboratory-confi rmed infl uenza per group was calculated. we also did secondary effi cacy analyses of time to alleviation of symptoms for all participants who received study drug irrespective of laboratory evidence of infection and for participants with or without confi rmed respiratory virus infections. we did statistical comparisons of changes in viral titre with a mixed model for repeated measures including baseline viral titre, treatment group, and geographic location. the safety population included all participants who received at least one dose of study medication. all analyses were done with sas software (version 6.12; sas institute, cary, nc, usa). this trial is registered with clinicaltrials.gov, number nct01227421. the sponsor designed the protocol and engaged a contract research organisation to select, initiate, monitor, and close out study sites and collect and analyse data. the sponsor wrote the study report. all authors participated in the interpretation of data, writing of the manuscript, and decision to submit the manuscript for publication. all authors had full access to the data. between dec 27, 2010, and april 30, 2011, we enrolled 624 participants with infl uenza-like illness of 650 people screened at 74 centres in the usa (fi gure 1). the study was terminated with about 60% of the planned number of patients infected with infl uenza because of the end of the infl uenza season. after randomisation, patients' character istics were well balanced between groups (table 1). 386 (62%) of 624 participants enrolled were diagnosed with a respiratory virus infection at baseline including 257 (41%) with infl uenza a or b (table 2) . demographic and disease-related characteristics of participants were similar for each of the three treatment groups (tables 1-3). treatment with nitazoxanide 600 mg twice daily for 5 days signifi cantly decreased the time from fi rst dose to alleviation of symptoms compared with placebo in participants with confi rmed infl uenza (p=0·0084; fi gure 2, table 4). time to symptom alleviation also decreased in patients given nitazoxanide 300 mg compared with those given placebo, but the diff erence was not signifi cant (p=0·52). we used prentice-wilcoxon test for these analyses because the proportional hazards assumption required for use of the cox model was violated (interaction of the 600 mg nitazoxanide treatment group with log(time) was signifi cant [p=0·0294]). at the time of symptom alleviation, no participants were still receiving symptom relief medication (paracetamol). we did sensitivity analyses in the population with confi rmed infl uenza to assess the eff ects of censored data and use of paracetamol on the primary endpoint. for participants with censored data (dropouts or without symptom alleviation at last diary assessment, n=16), we assumed time to alleviation of symptoms of 28 days. in this analysis, results were similar to the primary effi cacy analysis favouring nitazoxanide 600 mg compared with placebo (p=0·0062 [data not shown). 212 (82%) of 257 participants enrolled in the population with confi rmed infl uenza reported taking paracetamol during the study as allowed by the study protocol. proportions of participants taking paracetamol were similar in the three treatment groups: 73 (82%) of 89 participants in the placebo group, 74 (83%) of 89 in the nitazoxanide 300 mg group, and 65 (82%) of 79 in the nitazoxanide 600 mg group. when we repeated the primary analysis including paracetamol use and its interaction with the treatment group, participants receiving nitazoxanide 600 mg had a shorter time to symptom alleviation than did those receiving placebo (p=0·0085). in this study, we enrolled participants within 48 h of symptom onset, whereas in studies of neuraminidase inhibitors participants were enrolled within 36 h. 8, 9, 11 to assess the eff ect of this variable, we analysed participants enrolled within 36 h of symptom onset. in these post-hoc analyses, diff erences (nitazoxanide 600 mg vs placebo) in responses for subgroups infected with infl uenza a or b were similar (table 4). in analyses of 624 participants treated and 238 (38%) participants with no virus identifi ed at baseline, time from fi rst dose to alleviation of symptoms was signifi cantly lower in the 600 mg group than in the placebo group (table 4). we investigated too few participants infected with other individual viral infections to make meaningful analyses. another diff erence between the design of this study and earlier studies of the neuraminidase inhibitors was the scoring of nine symptoms instead of seven. 8, 9 use of two additional symptoms, runny nose and sweats or chills, only aff ected the time to alleviation of symptoms for six participants (three given placebo, one given nitazoxanide 300 mg, and two given nitazoxanide 600 mg). analysis based on the seven symptoms used in the oseltamivir studies showed median times to alleviation of symptoms of 115·9 h (95% ci 108-121) for the placebo group, 109·1 h (96-130, p=0·58) for the nitazoxanide 300 mg treatment group, and 95·5 h (84-108, p=0·0074) for the nitazoxanide 600 mg treatment group. at baseline, cough and nasal congestion were the most persistent symptoms in participants infected with infl uenza. the median time to alleviation in the placebo group for cough was 104 h (95% ci 84-111) and for nasal congestion was 81 h (67-92). treatment with nitazoxanide 600 mg was associated with a decrease in the median duration of these symptoms to 84 h (95% ci 60-104) for cough and to 60 h (48-74) for nasal congestion. for nine symptoms, in participants infected with infl uenza, mean symptom score-hours (symptom severity score multiplied by hours) summed for every participant from baseline to alleviation of symptoms were 1221 (592 sd) for the placebo group, 1305 (765) for the nitazoxanide 300 mg treatment group, and 1125 (681) for the nitazoxanide 600 mg treatment group, whereas in all treated participants, the score-hours were 1232 (1028) for the placebo group, 1171 (972) for the 300 mg treatment group, and 1045 (661) for the 600 mg treatment group. complications of infl uenza (bronchitis, sinusitis, otitis, pneumonia, and pleurisy) were fairly uncommon in this population. the frequency was not signifi cantly diff erent between treatment groups: 15 events (7%) for placebo, compared with 20 (10%) for the nitazoxanide 300 mg treatment group (p=0·38), and 11 (5%) for the nitazoxanide 600 mg treatment group (p=0·54). we obtained daily nasopharyngeal swabs at baseline and on days 2-5 for 113 participants (41 in the placebo group, 33 in the nitazoxanide 300 mg treatment group, and 39 in the nitazoxanide 600 mg treatment group). tcid 50 viral titres signifi cantly decreased during treatment in the nitazoxanide 600 mg group compared with placebo group (p=0·0006; fi gure 3 roughly 1 log 10 were apparent within 24 h after initiation of treatment for the nitazoxanide 600 mg group and continued until day 5 when viral shedding stopped for almost all participants in the placebo group. reductions in tcid 50 viral titres were also recorded for the nitazoxanide 300 mg treatment group compared with the placebo group, although these diff erences were not as large as for the nitazoxanide 600 mg treatment group and were not signifi cant (fi gure 3). reductions in viral titres detected by tcid 50 were similar to those dectected by rt-pcr except that the magnitude of reductions were somewhat smaller when measured by rt-pcr, which also detects non-infectious virus particles. median times to cessation of viral shedding were 91·3 h (95% ci 67·5-96·0) for the placebo group, 77·0 h (48·0-95·5) for the nitazoxanide 300 mg treatment group, and 71·8 h (55·0-96·0) for the nitazoxanide 600 mg treatment group. at day 7, only one participant (placebo group) was positive for infl uenza infection by viral culture. infl uenza virus was detected by rt-pcr in samples from day 7 for 23 (39%) of 59 patients in the placebo group, 17 (19%) of 73 patients in the nitazoxanide 300 mg treatment group, and 15 (24%) of 63 patients in the 600 mg treatment group. we cultured infl uenza viruses from nasopharyngeal swabs taken on day 5 from 13 participants treated with nitazoxanide. viruses present in all 13 samples were inhibited by tizoxanide, with eff ective concentrations ec50 and ec90 similar to those inhibiting viruses present in the corresponding baseline samples (data not shown). to assess potential eff ect of treatment on humoral immune response, we measured antibody titres at baseline and day 28 for participants with laboratory-confi rmed infl uenza by rt-pcr or viral culture at baseline. we noted no signifi cant diff erences in antibody titre change from baseline to day 28 or in the proportions of participants with laboratory-confi rmed infl uenza seroprotected (antibody titre ≥40) or seroconverted (four-fold increase in antibody titre) at day 28 between the treatment groups. seroconversion rates were 34 (49%) of 70 participants for the placebo group, 36 (58%) of 62 for the nitazoxanide 300 mg treatment group, and 36 (55%) of 65 for the nitazoxanide 600 mg treatment groups (p=0·52). we analysed plasma concentrations of tizoxanide during the fi rst 12 h after dosing on the morning of day 2 for six participants treated with nitazoxanide 300 mg and ten participants treated with nitazoxanide 600 mg. mean maximum plasma concentrations of tizoxanide were 2·46 μg/ml (sd 1·36) for the nitazoxanide 300 mg group and 4·60 μg/ml (3·61) for the nitazoxanide 600 mg, and mean trough concentrations were 0·121 μg/ml (0·09) for the nitazoxanide 300 mg treatment group and 0·795 μg/ml (1·97) for the nitazoxanide 600 mg group. mean area under the curve (auc 0-12 h ) for tizoxanide plasma concentrations were 12·6 μg × h/ml (sd 9·2) for the nitazoxanide 300 mg group and 29·1 μg × h/ml (33·8) for the nitazoxanide 600 mg group. analysis of change in tcid 50 viral titre for participants with confi rmed infl uenza that we took daily nasopharyngeal swabs from. statistical comparison with mixed model for repeated measures including baseline viral titre, treatment group, and geographic location: p=0·0006 for the diff erence between nitazoxanide 600 mg and placebo, p=0·1553 for the diff erence between nitazoxanide 300 mg and placebo. group compared with none in the placebo group. this event was probably attributed to the colour of nitazoxanide metabolites. because of the mild nature and relative infrequency of chromaturia (14 [2%] of 624 participants enrolled), the blinding of the study was not aff ected. treatment with nitazoxanide given orally 600 mg twice daily for 5 days beginning less than 48 h after symptom onset signifi cantly reduced the time from fi rst dose to alleviation of symptoms in participants aged 12-65 years with acute uncomplicated infl uenza. the 300 mg dose produced an intermediate response that did not signifi cantly diff er to that in the placebo group. the trial was designed according to regulatory guidance and was consistent with previous trials of the neuraminidase inhibitors, oseltamivir and zanamivir (panel). [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] reductions in time to alleviation of symptoms (median diff erence 21·2 h for the 600 mg group) were in the range of those recorded in trials of neuraminadase inhibitors. [8] [9] [10] [11] [12] [17] [18] [19] diff erences in the size of treatment eff ect from one trial to another could arise as a result of diff erences in study design (eg, duration of symptoms at time of enrolment, populations selected for analysis, use of paracetamol) or characteristics of circulating infl uenza strains (eg, pathogenicity, drug susceptibility). in this study, we enrolled participants within 48 h of symptom onset, whereas most clinical trials of neuraminidase inhibitors enrolled participants within 36 h of symptom onset. time to symptom alleviation was fastest in the subset enrolled within 36 h of symptom onset into the nitazoxanide 600 mg group. by contrast with studies of neuraminidase inhibitors, the results from this study show a potential treatment benefi t in participants without infl uenza or other documented viral infection (17·3 h reduction in time to alleviation of symptoms for 600 mg vs placebo). comparisons of treatment eff ect of nitazoxanide and neuraminidase inhibitors in participants with confi rmed infl uenza infection or with or without other documented viral infections will be an interesting goal for future trials. the adverse event profi le of nitazoxanide is well known because it has been commercially available since 1996 in global markets for treatment of intestinal infections. this study used a new controlled-release formulation of nitazoxanide designed specifi cally to deliver drug into the blood and to the respiratory tract and to maintain blood concentrations for 12 h. in the 600 mg dose group, the drug was given at a dose that is 20% higher and for a duration of therapy that is 67% longer than the drug's approved dose for treatment of intestinal infections. 6 this increase in dose and duration did not result in any signifi cant change in the known side-eff ect profi le. frequency or severity of adverse events did not signifi cantly diff er between participants in any group of the study. the fi ndings of this clinical trial need to be confi rmed by other studies. limitations of this study include the number of participants and the fact that they were all enrolled during a single infl uenza season. the absence of statistical signifi cance for the 300 mg treatment group might refl ect an absence of statistical power because only about 60% of the planned number of participants were recruited. although this trial enrolled participants with infl uenza ah3n2, aph1n1, and b, examination of the effi cacy and safety of the drug against infl uenza strains from another season would be benefi cial. the drug does not seem to be associated with resistance, as might be expected because of its mechanism of action, but further studies to assess the potential for resistance would be valuable. furthermore, although this clinical trial has shown a treatment eff ect in otherwise healthy participants aged 12-65 years, the eff ect of the drug still needs to be studied in children and patients with serious illness or those at risk of infl uenza complications. infl uenza (seasonal) infl uenza-associated hospitalizations in the united states estimates of deaths associated with seasonal infl uenza-united states thiazolides, a new class of anti-infl uenza molecules targeting viral hemagglutinin at the post-translational level nitazoxanide, a novel potential anti-infl uenza drug, acting in synergism with neuraminidase inhibitors romark laboratories, lc. alinia prescribing information guidance for industry, infl uenza: developing drugs for treatment and/or prophylaxis effi cacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute infl uenza effi cacy and safety of oseltamivir in treatment of acute infl uenza: a randomized controlled trial. neuraminidase inhibitor flu treatment investigator group effi cacy and safety of the neuraminidase inhibitor zanamivir in the treatment of infl uenzavirus infections. gg167 infl uenza study group the mist (management of infl uenza in the southern hemisphere trialists) study group. randomised trial of effi cacy and safety of inhaled zanamivir in treatment of infl uenza a and b virus infections clinical effi cacy and safety of the orally inhaled neuraminidase inhibitor zanamivir in the treatment of infl uenza: a randomized, double-blind, placebocontrolled european study nitazoxanide pharmacokinetics and tolerability in man during 7 days of 0.5 g and 1 g bid dosing with food seasonal infl uenza in adults and children-diagnosis, treatment, chemoprophylaxis and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america centers for disease control and prevention. prevention and control of infl uenza with vaccines: recommendations of the advisory committee on immunization practices (acip) cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine infl uenza eff ectiveness of oseltamivir in adults: a meta-analysis of published and unpublished clinical trials effi cacy and safety of intravenous peramivir for treatment of seasonal infl uenza virus infection the value of neuraminidase inhibitors for the prevention and treatment of seasonal infl uenza: a systematic review of systematic reviews neuraminidase inhibitors for preventing and treating infl uenza in children (published trials only) antivirals for treatment of infl uenza: a systematic review and meta-analysis of observational studies neuraminidase inhibitors for preventing and treating infl uenza in healthy adults and children neuraminidase inhibitors for preventing and treating infl uenza in children neuraminidase inhibitors for preventing and treating infl uenza in healthy adults: systematic review and meta-analysis prescription of anti-infl uenza drugs for healthy adults: a systematic review and meta-analysis neuraminidase inhibitors for treatment and prophylaxis of infl uenza in children: systematic review and meta-analysis of randomised controlled trials neuraminidase inhibitors for infl uenza the clinical study was done in the usa under the us food and drug administration ind #107 316. we thank romark laboratories lc, tampa, fl, usa, for sponsoring the study; barc usa, lake success, ny, usa, for serving as central laboratory for the study and doing laboratory safety and virology studies; sgs life science services, gaithersburg, md, usa, for serving as contract research organisation for the study; and the laboratory of gabriella santoro, university of rome tor vergata, rome, italy, for doing resistance testing. the international phase 3 clinical trial (nct01610245) referred to in the discussion section is funded in whole or in part with federal funds from the us department of health and human services, offi ce of the assistant secretary for preparedness and response, biomedical advanced research and development authority under contract hhso100201300004c. an international phase 3 clinical trial in participants with acute uncomplicated infl uenza is ongoing (nct01610245), which is designed to compare nitazoxanide given orally 600 mg twice daily for 5 days with placebo and to compare the combination of nitazoxanide and oseltamivir with oseltamivir or nitazoxanide given as monotherapy. another trial in participants admitted to hospital with severe acute respiratory illness (nct02057757) is in progress. these studies and others should provide additional information related to the value of the drug in the treatment of infl uenza and other viral respiratory infections. j-fr designed the study. jh, ah, mh, hr, and ss recruited participants and collected data. cg supervised the clinical virology work. all authors reviewed and interpreted the data. mb and j-fr drafted the report. all authors approved the fi nal report. we searched pubmed for systematic reviews and meta-analyses published in english language between jan 1, 2009, and jan 29, 2014, with the search terms "infl uenza treatment", "infl uenza clinical trial", "oseltamivir", "zanamivir", and "neuraminidase inhibitor". with the same search terms, we also searched for articles published in english between jan 1, 1990, and jan 29, 2014, reporting placebo-controlled clinical trials of drugs for treatment of infl uenza. we identifi ed nine systematic reviews or meta-analyses within the past 5 years related to the use of the neuraminidase inhibitors, oseltamivir and zanamivir, and we identifi ed several articles reporting randomised placebo-controlled clinical trials reporting a treatment eff ect of oral oseltamivir, inhaled zanamivir, or intravenous peramivir 8-12,17-26 (another neuraminidase inhibitor) in reducing the time from fi rst dose to alleviation of symptoms in participants with acute uncomplicated infl uenza. articles have also reported benefi cial treatment eff ect of oseltamivir and zanamivir in otherwise healthy children infected with infl uenza. evidence to support treatment benefi ts of neuraminidase inhibitors in elderly and at-risk groups or their eff ects on rates of admission to hospital and mortality was insuffi cient. in our trial, oral administration of nitazoxanide 600 mg twice daily for 5 days reduced the time to alleviation of symptoms of infl uenza in otherwise healthy adults and adolescents by roughly 1 day. although many factors can aff ect the size of treatment eff ect observed in diff erent clinical trials, the eff ect recorded in this trial is similar to that previously reported for the neuraminidase inhibitors. [8] [9] [10] [11] [12] 17, 18, 27 data from this trial suggest a treatment benefi t of nitazoxanide in participants without infl uenza or other documented viral infection-a fi nding that has not been reported for the neuraminidase inhibitors. importantly, nitazoxanide possesses a novel mechanism of action against infl uenza viruses. a new drug with a diff erent mechanism of action could ultimately prove benefi cial in overcoming resistance to neuraminidase inhibitors or in providing more potent therapy when used in combination with existing drugs. as a fi rst step, it has been important to show eff ectiveness of nitazoxanide in a randomised placebo controlled trial by use of a method established for licensure of existing drugs. further studies are warranted to confi rm our fi ndings and to assess effi cacy of nitazoxanide alone or in combination with existing drugs in seriously ill patients and those at risk of infl uenza complications. key: cord-332237-8oykgp0h authors: omrani, ali s; saad, mustafa m; baig, kamran; bahloul, abdelkarim; abdul-matin, mohammed; alaidaroos, amal y; almakhlafi, ghaleb a; albarrak, mohammed m; memish, ziad a; albarrak, ali m title: ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study date: 2014-09-29 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70920-x sha: doc_id: 332237 cord_uid: 8oykgp0h background: middle east respiratory syndrome coronavirus (mers-cov) infection is associated with high mortality and has no approved antiviral therapy. we aimed to compare ribavirin and interferon alfa-2a treatment for patients with severe mers-cov infection with a supportive therapy only. methods: in this retrospective cohort study, we included adults (aged ≥16 years) with laboratory-confirmed mers-cov infection and pneumonia needing ventilation support, diagnosed between oct 23, 2012, and may 1, 2014, at the prince sultan military medical city (riyadh, saudi arabia). all patients received appropriate supportive care and regular clinical and laboratory monitoring, but patients diagnosed after sept 16, 2013, were also given oral ribavirin (dose based on calculated creatinine clearance, for 8–10 days) and subcutaneous pegylated interferon alfa-2a (180 μg per week for 2 weeks). the primary endpoint was 14-day and 28-day survival from the date of mers-cov infection diagnosis. we used χ(2) and fischer's exact test to analyse categorical variables and the t test to analyse continuous variables. findings: we analysed 20 patients who received ribavirin and interferon (treatment group; initiated a median of 3 days [range 0–8] after diagnosis) and 24 who did not (comparator group). baseline clinical and laboratory characteristics were similar between groups, apart from baseline absolute neutrophil count, which was significantly lower in the comparator group (5·88 × 10(9)/l [sd 3·95] vs 9·88 × 10(9)/l [6·63]; p=0·023). 14 (70%) of 20 patients in the treatment group had survived after 14 days, compared with seven (29%) of 24 in the comparator group (p=0·004). after 28 days, six (30%) of 20 and four (17%) of 24, respectively, had survived (p=0·054). adverse effects were similar between groups, apart from reduction in haemoglobin, which was significantly greater in the treatment group than in the comparator group (4·32 g/l [sd 2·47] vs 2·14 g/l [1·90]; p=0·002). interpretation: in patients with severe mers-cov infection, ribavirin and interferon alfa-2a therapy is associated with significantly improved survival at 14 days, but not at 28 days. further assessment in appropriately designed randomised trials is recommended. funding: none. since it was fi rst described in september, 2012, 855 cases of middle east respiratory syndrome coronavirus (mers-cov) infection have been confi rmed, 333 of which were fatal. 1,2 cases occur sporadically, as community clusters or as hospital outbreaks, and range in severity from asymptomatic or mild illness to rapidly progressive and fatal disease. [2] [3] [4] [5] [6] the management of patients with mers-cov infection consists of a combination of supportive measures, antimicrobial therapy for any associated bacterial or viral infections, and strict implementation of appropriate infection control precautions. 7 so far, no antiviral therapy has been approved for the treatment of patients with mers-cov infection. 8 several therapeutic interventions for coronavirus were investigated during the large multinational outbreak of severe acute respiratory syndrome (sars) in 2003. 9, 10 reviews of the available scientifi c literature suggest that a combination of ribavirin and interferon might be of benefi t in patients with severe mers-cov infection. 8, 11, 12 furthermore, this combination was shown to inhibit mers-cov in cell culture and seemed to improve outcomes in an animal study. 13, 14 both agents are associated with substantial potential adverse eff ects and hence their clinical use should be carefully balanced against any potential harm. 8 we aimed to assess outcomes of a treatment programme for patients with severe mers-cov infection that consisted of oral ribavirin and subcutaneous pegylated interferon alfa-2a. we report the results and outcomes in patients given treatment in accordance with this protocol by comparison with a historical group who received supportive therapy only. this single-centre, retrospective cohort study included individuals who were diagnosed with laboratoryconfi rmed mers-cov infection between oct 23, 2012, and may 1, 2014, at the prince sultan military medical city (riyadh, saudi arabia). eligible patients were those aged 16 years or older with severe pneumonia needing invasive or non-invasive ventilation. no exclusion criteria were applied at this stage. mers-cov infection was diagnosed by rt-pcr testing of respiratory tract samples for mers-cov upe, orf 1b, and n genes. 15 all rt-pcr tests for mers-cov were done at the saudi ministry of health regional laboratory in jeddah and riyadh, saudi arabia. pneumonia was defi ned as new, otherwise unexplained, lower respiratory tract symptoms such as cough or shortness of breath with at least one systemic feature such as fever or chills, and new focal chest signs on examination, in addition to new or progressive pulmonary infi ltrates on chest radiograph. 16 from sept 16, 2013, all eligible patients were off ered treatment with oral ribavirin and subcutaneous pegylated interferon alfa-2a after informed written consent had been obtained from the patients themselves or their next of kin. the treatment protocol was approved by pharmacy and therapeutics committee. the study was approved by the research ethics committee at the prince sultan military medical city to allow retrospective access to patients' records and fi les. pegylated interferon alfa-2a (pegasys; roche pharmaceuticals, basel, switzerland) was given by subcutaneous injection at a dose of 180 μg per week for 2 weeks. the dose of oral ribavirin (copegus; roche pharmaceuticals) was adjusted according to calculated creatinine clearance and continued for 8-10 days. 12 patients with a creatinine clearance of greater than 0·833 ml/sec/m 2 received a 2000 mg loading dose, followed by 1200 mg every 8 h for 4 days then 600 mg every 8 h for 4-6 days; those with a creatinine clearance of 0·333-0·833 ml/sec/m² received a 2000 mg loading dose, followed by 600 mg every 8 h for 4 days then 200 mg every 6 h for 4-6 days; and those with a creatinine clearance of <0·333 ml/sec/m² or on dialysis received a 2000 mg loading dose, followed by 200 mg every 6 h for 4 days then 200 mg every 12 h for 4-6 days. patients did not receive ribavirin and interferon alfa-2a therapy if they were diagnosed before sept 16, 2013, or if they declined consent. all patients received appropriate supportive care such as supplementary oxygen, vasopressor therapy, and renal replacement as needed. hydrocortisone 200 mg daily was given to patients with refractory septic shock and continued until vasopressor therapy was no longer needed. 17 in addition to regular clinical monitoring, renal function, liver enzymes, and blood count were assessed at baseline and daily throughout the treatment course. conscious patients were monitored for any clinical signs of depression or acute confusion. patients who received ribavirin and interferon alfa-2a therapy were classifi ed as being in the treatment group and those who did not made up the comparator group. two investigators, aso and kb, both of whom were masked to group allocation and the patients' clinical outcomes, compared baseline characteristics of the two groups. immunosuppressive therapy 1 (5%) 4 ‡ (18%) 0·66 data are number (%) or mean (sd). apache ii=acute physiology and chronic health evaluation ii. sofa=sequential organ failure assessment. *only 19 patients were assessed. †only 13 patients were assessed. ‡only 22 patients were assessed. the primary endpoints for the study were 14-day and 28-day survival from the date of mers-cov infection was diagnosis. we used χ² and fischer's exact tests for categorical variables, whereas we used the student's t test for continuous variables to assess the diff erences in means of the two groups. the log-rank test was used for assessing survival diff erences between the two groups. our cutoff for statistical signifi cance was 0·05. the graphical and statistical tests suggested that the pro portionalhazard assumption was not violated. we did statistical analyses using microsoft excel 2011 and stata statistical software, release 12. no external funding was received for this study. zm had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. 70 individuals were diagnosed with mers-cov infection between oct 23, 2012, and may 1, 2014. baseline characteristics were generally similar between patients who received ribavirin and interferon alfa-2a therapy and those who did not (table 1) , with the exception that end-stage renal failure was present in three patients who did not receive study treatment and in none who did. after excluding ineligible patients, 44 patients were included in the study: 20 in the treatment group and 24 in the comparator group (fi gure 1). the mean age of all 44 patients was 65·5 years (sd 18·2), and 32 (73%) were men (table 1) mean absolute neutrophil count was signifi cantly lower in the treatment group than in the comparator group (table 1) ; however, no other statistically signifi cant differences in baseline characteristics or support measures were noted between the two groups (tables 1, 2). of all 44 patients, 21 (48%) were still alive 14 days after diagnosis of mers-cov infection, whereas at 28 days only ten (23%) had survived. 14 (70%) of 20 patients in the treatment group were alive 14 days after diagnosis, compared with seven (29%) of 24 in the comparator group (p=0·004). however, six (30%) of 20 patients in the treatment group survived up to 28 days from diagnosis of mers-cov infection, whereas four (17%) of 24 did in the comparator group (p=0·054; fi gure 2). ribavirin and pegylated interferon therapy was well tolerated by the treatment group with no premature discontinuation secondary to adverse eff ects. however, the mean drop in haemoglobin over the treatment course was signifi cantly greater in the treatment group (4·32 g/l [sd 2·47]) than in the comparator group eff ective treatment interventions for patients with severe mers-cov infection are still urgently needed. in critically ill patients with severe mers-cov infection, our study shows that ribavirin and pegylated interferon alfa-2a therapy is associated with a signifi cant 14-day survival benefi t compared with standard treatment. 28-day survival also seemed to improve with ribavirin and pegylated interferon alfa-2a therapy, but the diff erence between groups was not signifi cant (panel). the loss of a signifi cant survival diff erence over time might be partly explained by most patients in our cohort having several comorbidities with high apache ii and sofa scores. mortality is known to be very high in patients with severe mers-cov infection who need critical-care support. 24 therefore, long-term survival benefi t, if present, might be diffi cult to show in smaller studies. treatment with ribavirin and interferon was well tolerated in our study. the only adverse event that was signifi cantly worse in the treatment group was mean decrease in haemoglobin (4.32 g/l in the treatment group compared with 2·14 g/l in the comparator group). anaemia is a well recognised complication of ribavirin therapy and was noted previously in studies investigating the role of ribavirin in the treatment of sars coronavirus infection. 25, 26 of note, receipt of packed red blood cells was not signifi cantly diff erent between the treatment and comparator groups in our study. furthermore, no treatment discontinuations occurred as a result of anaemia. therefore, the risk of ribavirin-associated anaemia-although substantial and in need of careful monitoring-might not hinder the use of ribavirin for patients with severe mers-cov infection, especially if a survival benefi t can be confi rmed. baseline absolute neutrophil count was signifi cantly lower in the treatment group and therefore a signifi cantly lower minimal absolute neutrophil count during the course of the illness is not surprising. several investigators showed that interferon α has useful in-vitro activity against mers-cov. 13, 20, 26 however, when compared with interferon α and interferon γ, interferon β seems to have the most potent inhibitory in-vitro activity against mers-cov. 21 ribavirin has slight anti-mers-cov activity in vitro when used alone or in combination with interferon α. 13, 22 mycophenolic acid is another compound that exhibits signifi cant in-vitro activity against mers-cov. 21 of 290 compounds screened, 60 were active in cell culture against mers-cov. 23 although only the combination of interferon α plus ribavirin has so far undergone in-vivo assess ment against mers-cov, 14 many others are potential candidates for further clinical assessment. one of the limitations of our study is its small size. however, only two previous reports of clinical use of ribavirin and interferon for mers-cov infection have been published. 18, 19 in a retrospective report, fi ve patients with severe mers-cov infection, all of whom had signifi cant comorbidities and needed mechanical ventilation, received a com bination of ribavirin and pegylated interferon alfa-2b a median of 19 days after admission. none of the patients survived and the investigators concluded that late commencement of therapy might not be benefi cial. 18 in another report, 19 a patient with severe mers-cov infection received ribavirin and interferon therapy with good clinical response and no signifi cant adverse eff ects. our study, albeit small, is the largest clinical investigation so far to assess the use of this combination in the treatment of patients with severe mers-cov infection. although baseline characteristics of our treatment and comparator groups seem to be reasonably balanced, substantial diff erences might not be apparent because of the small number of patients in the study. our study is also limited by its retrospective, nonrandomised nature. inevitably, selection and unmeasured confounding bias cannot be completely excluded. undoubtedly, new interventions should ideally be assessed in randomised, controlled clinical trials. however, such an approach is generally accepted to not always be practically feasible in the context of an emerging and relatively uncommon disease such as mers-cov infection. 8 we carefully selected our comparator group, ensuring that the two cohorts were matched as closely as possible in their clinical characteristics and treatment interventions other than the receipt of ribavirin and interferon. we removed three individuals who had outlying baseline serum creatinine from the comparator group to minimise the risk of any spurious conclusions driven by clinical characteristics that might be potentially detrimental to clinical outcome. clinical outcomes for each individual were masked from investigators who selected patients and did matching assessments. the absence of serial viral load measurement in lower respiratory tract samples in our study makes it impossible to show any association between temporal viral load changes and antiviral therapy. such measurements should be included in any future clinical studies exploring the therapeutic benefi t of any antiviral intervention for patients with mers-cov infection. severe mers-cov is associated with poor overall survival. treatment with oral ribavirin and subcutaneous pegylated interferon alfa-2a is associated with signifi cantly improved survival at 14 days, but not at 28 days. the combination is associated with signifi cant falls in haemoglobin, but no other signifi cant adverse eff ects were noted. treatment with ribavirin and pegylated interferon might be considered in patients with severe mers-cov infection, provided that adequate monitoring and assessment can be ensured. further assessment, including in patients with less severe mers-cov infection, in appropriately designed randomised trials, is recommended. this study was initiated and designed by aso, mms, zam, and ama. aso, mms, ab, ma-m, aya, gaa, mma, zam, and ama obtained and collated patient data. kb undertook all statistical analyses for the study. aso and kb prepared all tables and fi gures. aso, mms, zam, and ama wrote the fi rst draft of the manuscript and all authors reviewed and contributed to subsequent drafts and the fi nal report. aso has received consultancy fees from gilead, pfi zer, msd, and viiv; payment for lectures from pfi zer, msd, glaxosmithkline, and sanofi -aventis; and sponsorship to attend international meetings and conferences from msd, pfi zer, biopharma, bristol-myers squibb, and janssen-cilag. ab has received travel funding to attend an international meeting from pfi zer. gaa has received travel funding to attend an international meeting from edwards lifesciences. all other authors declare no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov)-11th update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case hospital outbreak of middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical management of severe acute respiratory infections when novel coronavirus is suspected: what to do and what not to do international severe acute respiratory and emerging infection consortium (isaric) the severe acute respiratory syndrome sars: systematic review of treatment eff ects applying lessons from sars to a newly identifi ed coronavirus therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)--possible lessons from a systematic review of sars-cov therapy inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections surveillance defi nitions for specifi c types of infections surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon (ifn)-alpha-2b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada severe acute respiratory syndrome: report of treatment and outcome after a major outbreak we thank staff of prince sultan military medical city, riyadh, saudi arabia, for the clinical care given to the patients and for facilitating access to the relevant medical records. we searched pubmed for reports published in english any time before june 24, 2014, with the search term "[(mers-cov or hcov-emc or novel coronavirus) and (therapy or interferon or ribavirin]". we found one animal study, 14 two small case series in human beings, 18, 19 and several in-vitro studies. 13, [20] [21] [22] [23] the data suggested that combination therapy with ribavirin and interferon alfa could have potential benefi ts for patients with severe mers-cov infection. this is, to our knowledge, the largest clinical study done so far assessing the potential benefi t and safety of combination therapy with pegylated interferon alfa-2a plus ribavirin in patients with severe mers-cov infection. because we noted a signifi cant 14-day survival benefi t in patients who received the combination compared with those who received supportive therapy only, but no survival benefi t at 28 days, we recommend further assessment in appropriately designed randomised clinical trials to provide further information about the role of this combination in the treatment of patients with severe mers-cov infection. key: cord-270602-599vweqe authors: donia, marwa; hamann, mark t title: marine natural products and their potential applications as anti-infective agents date: 2003-05-22 journal: lancet infect dis doi: 10.1016/s1473-3099(03)00655-8 sha: doc_id: 270602 cord_uid: 599vweqe the oceans are a unique resource that provide a diverse array of natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria. as infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases. many marine natural products have successfully advanced to the late stages of clinical trials, including dolastatin 10, ecteinascidin-743, kahalalide f, and aplidine, and a growing number of candidates have been selected as promising leads for extended preclinical assessment. although many marine-product clinical trials are for cancer chemotherapy, drug resistance, emerging infectious diseases, and the threat of bioterrorism have all contributed to the interest in assessing natural ocean products in the treatment of infectious organisms. in this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent in-vitro activity against infectious and parasitic diseases. the oceans are a unique resource that provide a diverse array of natural products, primarily from invertebrates such as sponges, tunicates, bryozoans, and molluscs, and from marine bacteria and cyanobacteria. as infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases. many marine natural products have successfully advanced to the late stages of clinical trials, including dolastatin 10, ecteinascidin-743, kahalalide f, and aplidine, and a growing number of candidates have been selected as promising leads for extended preclinical assessment. although many marineproduct clinical trials are for cancer chemotherapy, drug resistance, emerging infectious diseases, and the threat of bioterrorism have all contributed to the interest in assessing natural ocean products in the treatment of infectious organisms. in this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent in-vitro activity against infectious and parasitic diseases. although the diversity of life in the terrestrial environment is extraordinary, the greatest biodiversity is in the world's oceans, with 34 of the 36 phyla of life represented. the oceans cover more than 70% of the earth's surface and contains more than 300 000 described species of plants and animals. 1, 2 macroscopic plants and animals have adapted to all regions of the oceans, including polar, temperate, and tropical areas. the diversity in species is extraordinarily rich on coral reefs, where there are around 1000 species per m 2 in some areas, and the indo-pacific ocean has the world's greatest tropical marine biodiversity. 2 the marine environment represents a treasure of useful products awaiting discovery for the treatment of infectious diseases (figure 1). ecological pressures, including competition for space, the fouling of the surface, predation, and successfully reproducing have led to the evolution of unique secondary metabolites with various biological activities. 3 the importance that these secondary metabolites play in the control of infectious and parasitic organisms was for many years largely overlooked. in the past 30-40 years, marine plants and animals have been the focus of a worldwide effort to define the natural products of the marine environment. a small number of marine plants, animals, and microbes have already yielded more than 12 000 novel chemicals, with hundreds of new compounds still being discovered every year. these discovery efforts have yielded several bioactive metabolites that have been successfully developed by the pharmaceutical industry. 4, 5 several clinically useful drugs, investigational drug candidates, and pharmacological tools have already resulted from these marine-product discovery programmes. 4, 5 one of the marine natural products that is currently under clinical investigation as a potential new anticancer agents is the marine alkaloid ecteinascidin-743, isolated from the tunicate ecteinascidia turbinate. 6 ecteinascidin is a broad-spectrum antitumour agent and is among the most advanced compounds with clinical applications. 6 in this review, we focus on the pharmacologically tested marine leads that have shown in-vivo efficacy or potent invitro activity against infectious and parasitic diseases, including malaria, toxoplasmosis, trypanosomiasis, and viral, bacterial, and fungal infections. our objective was to highlight the most promising compounds that have the greatest potential to lead to clinically useful treatments. there was not room to review treatments for specific infectious diseases, but there are several helpful reviews for antiparasitic drugs, 7 antinematodal drugs, 8 antituberculosis agents, 9 antiviral leads, 10 and antifungal agents. 11 furthermore, there are some excellent general and extensive reviews focusing on marine natural products, such as those by capon, 12 newman and colleagues, 13 and faulkner. 14 the antifungal screening of marine samples has led to the characterisation of many unprecedented natural products in regard to antifungal activity and chemical structures. the frequency of invasive fungal infection has risen substantially with the increasing numbers of immunocompromised patients, such as those infected with hiv, receiving cancer chemotherapy, immunosuppressive therapy, or treatment with broad-spectrum antibiotics. 11 evidence is mounting that fungi display highly specific adaptations in the marine environment that include the production of unique secondary metabolites. 11 the fact that marine organisms contain secondary metabolites different from their terrestrial counterparts in structure and biological activity has led to the hypothesis that marine organisms may contain efficient antifungal compounds with different modes of action and selective antifungal activity compared with human cells. 11 jasplakinolide is the first example of a cyclodepsipeptide isolated from a sponge (figure 1) and was identified from a jaspis sp collected in fiji. 15 jasplakinolide, also named jaspamide, is a 19-membered macrocyclic depsipeptide with selective in-vitro antimicrobial activity and a minimum inhibitory concentration (mic) of 25 g/ml against candida albicans. the in-vivo topical activity of a 2% solution of jasplakinolide against a candida vaginal infection in mice is similar to that of miconazole nitrate. 15 gambieric acids are extremely potent antifungal metabolites isolated from a strain of the epiphytic marine dinoflagellate gambierdiscus toxicus. these metabolites are the first antifungal representatives of the brevetoxin-type (fused polyether rings) structures, consisting of nine contiguous ether rings and one isolated tetrahydrofuran (figure 2). gambieric acid a inhibits the growth of aspergillus niger at a concentration of 10 ng/disk. the potency of gambieric acids exceeds that of amphotericin b by 2000-fold. 16 toxic effects in mice or cultured mammalian cells are moderate for a dose of 1 mg/kg given by intraperitoneal injection. additional marine natural products with antifungal activity are listed in table 1. most antifungal compounds from marine origin are cytotoxic. consequently, they have not generally been considered promising antifungal agents for clinical applications. we have reviewed marine natural products that show potent antifungal activity, but evidence of cytotoxicity was not available for all. 11 in many cases, the assessment of whether antifungal activity outweighs cytotoxic effects will be required, followed by rational modifications to improve the therapeutic index for these molecules. the search for new antituberculosis agents has become increasingly important with the emergence of multidrugresistant strains of mycobacterium tuberculosis. despite the small number of investigators looking at marine products as potential leads against m tuberculosis, the research in this area has been productive (figure 3). anti-infectives from the sea the alkaloid (+)-8-hydroxymanzamine a is characterised by a complex heterocyclic ring system attached to a ␤-carboline moiety. (+)-8-hydroxymanzamine a was first isolated from a sponge pachypellina sp and later from an undescribed petrosiidae genus. 26 this alkaloid exhibits potent inhibitory activity against m tuberculosis h37rv, with an mic of 0·91 g/ml. 27 the likely biogenetic precursor ircinol a does not have the ␤-carboline moiety but still has an mic of 1·93 g/ml. ircinol a represents a useful candidate for assessment in vivo against m tuberculosis, since it shows low cytotoxicity and structural complexity compared with the other manzamine-type alkaloids. 27 in addition, manzamine a inhibits m tuberculosis h37rv, with an mic of 1·56 g/ml. 27 axisonitrile-3 is a cyanosesquiterpene isolated from the sponge acanthella klethra and shows potent inhibitory activity against m tuberculosis, with an mic of 2·0 g/ml. 28 pseudopteroxazole, a benzoxazole diterpene alkaloid isolated from the west indian gorgonian pseudopterogorgia elisabethae, induces 97% growth inhibition for m tuberculosis h37rv at a concentration of 12·5 g/ml without substantial toxic effects. 29 ergorgiaene, a serrulatane-based diterpene (also known as biflorane) was isolated from the hexane extract of the same west indian gorgonian, and induced 96% growth inhibition for m tuberculosis h37rv at a concentration of 12·5 g/ml. 30 litosterol is a c-19 hydroxysteroid isolated from an okinawan soft coral litophyton viridis. it inhibited 90% of the growth of m tuberculosis with an mic of 3·13 g/ml. the poor solubility of litosterol in the aqueous tissue culture media obscured assessment of cytotoxic effects. 9, 31 puupehenone induced 99% inhibition of m tuberculosis h37rv growth, with an mic of 12·5 g/ml and a 50% inhibitory concentration (ic 50 ) of 2·0 g/ml. the puupehenones are shikimate-sesquiterpene derived metabolites isolated from sponges of the order verongida and dictyoceratida, collected from the hawaiian islands. 9, 32 anthelmintic activity anthelmintics are drugs used to rid host organisms of helminth parasites. parasitism by nematodes (unsegmented worms that constitute the phylum nematoda) represents a major issue in the commercial livestock industry, and contributes substantially to malnutrition and disease in human beings. particularly difficult to eradicate is ascaris lumbricoides, the large gut worm, which causes malnutrition and obstructive bowel disease, and the soiltransmitted blood-sucking hookworms ancyclostoma duodenale and necator americanus, which lead to severe blood loss and iron-deficient anaemia, decreased food intake, impaired digestion, malabsorption, and poor growth rate. 33 despite the availability of excellent commercial anthelmintics, growing resistance to key structural classes (benzimidazoles and macrolides) necessitates the search for new bioactive agents. 33 jasplakinolide represents a potent antiparasitic as well as antifungal agent. it exhibited an in-vitro 50% effective dose of less than 1 g/ml against the nematode nippostrongylus braziliensis. 15 respectively. 34 this tetrahydrofuran inhibits the development of eggs for the infective free-living third stage of these two species. this degree of nematocidal activity is similar to that of the commercially available nematocides levamisole and closantel. unfortunately, attempts to translate this in-vitro nematocidal activity to in vivo proved unsuccessful, but is not surprising given the hydrophobic properties of this drug. 34 however, understanding of the in-vitro mode of action of these compounds may yet contribute to the discovery of new and improved anthelmintics. the amphilactams, isolated from a sponge of amphimedon sp collected in the great australian bight, feature an unusual carbon skeleton and an enamino lactone or lactam moiety (figure 4). amphilactam d has an in-vitro nematocidal activity against the free-living stages of the parasitic nematode h contortus, with an ld 99 of 0·39 g/ml. this substance inhibits larval development at the l1 stage, but has little or no activity against nematode eggs. this degree of in-vitro activity is similar to that of existing commercial anthelmintics, such as levamisole and closantel, and is thought to merit in-vivo assessment. 35 a sponge, geodia sp, collected from southern australia has yielded a potent nematocidal agent, geodin a magnesium salt, which is a macrocyclic polyketide lactam tetramic acid showing an ld 99 value of 1·0 g/ml. 36 diseases caused by protozoan parasites lead to high rates of mortality and morbidity worldwide. we have focused on the marine-derived natural products showing promising efficacy against the protozoal infections caused by leishmania sp, trypanosoma sp, toxoplasma sp, and plasmodium sp ( figure 5 ). leishmaniasis is a disease caused by an obligate intracellular parasite of the genus leishmania. types of disease range from self-healing ulcers (cutaneous leishmaniasis) to progressive nasopharyngeal infections (mucocutaneous leishmaniasis) to disseminating visceral leishmaniasis, which is generally fatal if left untreated. 37 in mediterranean countries adult visceral leishmaniasis is recognised as an aids-related opportunistic disease, largely due to reactivation of latent infections by immunosuppression. the most common drugs for the treatment of leishmaniasis contain pentavalent antimonials, such as sodium stibogluconate and meglumine antimonite, that have cardiotoxic effects at the recommended doses and consequently signify the urgent need for alternative treatments. 38 sponges of the genus plakortis are well known for their production of cyclic peroxides. two peroxides produced by the palauan sponge plakortis aff angulospiculatus are active against leishmania mexicana. the most active cyclic peroxide (ld 50 0·29 g/ml) causes lysis of the cell membrane after 24 h at a concentration of 1·0 g/ml, and a striking decrease in motility after 30 min. this peroxide is, however, less effective than ketoconazole (ld 50 0·06 g/ml). 39 toxoplasmosis can be transmitted to human beings from cats infected with the parasite toxoplasma gondii. the severity ranges from a self-limiting adenopathy to fatal encephalitis. this infection is particularly dangerous for pregnant women and immunocompromised individuals; congenital infection of infants is fatal in most cases. 40 new drugs or drug combinations that can eradicate the tissue cysts that cause relapses are urgently needed, particularly for patients intolerant to folate inhibitors (pyrimethamine, trimethoprim), which frequently cause undesirable side-effects. the remaining group of drugs used for the treatment of toxoplasmosis include the macrolides, which act only against the tachyzoite, leaving the cysts unaffected. 41 the alkaloid manzamine a displays 70% inhibition of the t gondii parasite, at 0·054 g/ml concentration, without cell toxic effects. the activity increases notably at concentrations of 0·54 g/ml and 5·40 g/ml, even though it is accompanied by an increase in the cytotoxic effects for the host cells, and was therefore selected for in-vivo assessment. a daily intraperitoneal dose of 8 mg/kg of manzamine a for 8 consecutive days, started on day 1 after infection, prolonged the survival of swiss webster mice to 20 days, compared with 16 days for untreated controls. exploration of new manzamines, structure-activity relation studies, and studies of optimum dose will be important to improve the in-vivo efficacy of the manzamines against t gondii. 42 sigmosceptrellin-b isolated from the red sea sponge diacarnus erythraeanus, exhibits potent in-vitro activity against t gondii at a concentration of 0·039 g/ml without severe toxic effects. sigmosceptrellin-b is active (84-99% inhibition) against the parasite in human diploid fibroblast cells. 43 a cyclic peroxylactone, plakortide, was isolated from the jamaican sponge plakinastrella onkodes and has potent in-vitro activity against t gondii, with an ic 50 of 0·023 g/ml and no toxic effects for the host cells up to a concentration 0·12 g/ml. 44 the flagellated protozoa trypanosoma cruzi and trypanosoma brucei are, respectively, the causal agents of south american trypanosomiasis (chagas disease) and human african trypanosomiasis (sleeping sickness). the drugs used to treat these diseases, including pentamidine and suramin, require parenteral administration and are effective only against the early haemolymphatic stage of the disease. the arsenical drug melarsoprol is available for late-stage central nervous system infection, but it causes reactive encephalopathy and is difficult to administer. the nitroheterocyclic drug benznidazole, used to treat acute stages of chagas disease, is not effective against the chronic phases of the disease and is poorly tolerated, which clearly indicates the need for new drugs with structures and mechanisms of action different from those that are presently used. 45 from the green alga ulva sp the endophytic and obligate marine fungus ascochyta salicorniae was isolated and cultivated on a preparative scale, producing a structurally unusual tetramic acid metabolite ascosalipyrrolidinone a, which shows activity against t cruzi with an mic of 1·1 g/ml, whereas the control (benznidazole) has an mic of 30·0 g/ml. the main limitation for further development of this compound is its cytotoxic effects at 3·7 g/ml tested against rat skeletal muscle myoblast cells. 46 malaria is a particularly serious disease in sub-sahran africa, 47 but it is also a serious public-health issue in certain regions of southeast asia and south america. most malaria cases and deaths are caused by the parasite plasmodium falciparum. 47 since removal of the vector of transmission (the anopheles mosquito) is almost impossible, new antimalarial agents providing novel mechanisms of action will always be needed to combat resistance to drugs such as chloroquine, mefloquine, quinine, and sulfadoxine-pyrimethamine. manzamine a exhibits potent in-vitro activity against p falciparum (d6 clone), with an mic of 0·0045 g/ml, compared with control-drug (chloroquine and artemisinin) mics of 0·0155 g/ml and 0·010 g/ml, respectively. 48 manzamine a inhibits the growth of the rodent malaria parasite plasmodium berghei in vivo, with more than 90% of the asexual erythrocytic stages of p berghei inhibited after one intraperitoneal injection of 50 or 100 mol/kg manzamine a into infected mice. this treatment prolongs the survival of highly parasitaemic mice to more than 10 days compared with 2-3 days among untreated controls, 2 days among mice treated with artemisinin, and 6 days among mice treated with chloroquine; it has a 40% recovery rate 60 days after one injection. oral administration of an oil suspension of manzamine a also substantially reduces parasitaemia. interestingly, the manzamine a hydroxyl derivative (-)-8-hydroxymanzamine a, as well as the dimer neo-kauluamine, extend the lives of p berghei-infected mice far more than the two most important human therapeutic antimalarial drugs when administered as one intraperitoneal dose of 100 mol/kg. manzamine a and selected derivatives have a rapid onset of action because of high bioavailability and sustained antiparasitic activity with no apparent toxic effects. 48 manzamine a has a similar therapeutic index to chloroquine, whose toxic dose at 500 mol/kg is ten times higher than the dose required (50 mol/kg) to clear the parasite if administered three times with 2-day intervals. 48 the effectiveness of manzamine a and selected analogues against malaria makes them one of the most promising antiinfective leads to be discovered from the oceans, and understanding the structure-activity relation of this unique class of alkaloids is certain to lead to more effective and safer manzamine-related antimalarial drug leads. the previously mentioned antifungal macrolide halichondramide also has notable activity against p falciparum (d6 clone), with an ic 50 of 0·002 g/ml, which is approaching the value for the most active clinically used compounds (mefloquine 0·0003 g/ml). although halichondramide has a much lower selectivity index than currently used drugs, it represents a potential new class of compounds in the development of alternative chemotherapy for the treatment of malaria after further structure-activity relation investigations. 49 an example of a marine secondary metabolite containing the isonitrile group and eliciting significant antimalarial activity is di-isocyanoadociane, a tetracyclic diterpene with an isocycloamphilectane skeleton. di-isocyanoadociane has been isolated from the sponge cymbastela hooperi (axinellidae, halichondrida) and displayed antiplasmodial potency, with ic 50 values of 0·005 g/ml, and selectivity that rivals the invitro results obtained from clinically used antimalarial drugs (chloroquine and artemisinin). 50 the kalihinane diterpenoids exhibit several types of biological activities, of which the antimalarial activity of kalihinol a is the most noteworthy. kalihinol a was isolated from an okinawan marine sponge, acanthella sp, and it possesses notable in-vitro antimalarial activity (ec 50 0·0005 g/ml) and selectivity index (si 317) similar to those for mefloquine. 51 the development of resistance to current antibacterials continues to be a serious difficulty in the treatment of infectious diseases, and therefore the discovery and development of new antibiotics has become a high priority review anti-infectives from the sea in biomedical research. in the continuing effort by the marine natural products community, many antibacterial agents have been identified; we have focused only on those that seem to possess the greatest potential ( figure 6) . squalamine is the first aminosterol isolated from the dogfish shark squalus acanthias (squalidae). it has potent antimicrobial activity, with an mic of 1·0 g/ml against staphylococcus aureus, and antiangiogenic and antitumour properties. it is currently being tested in clinical trials for the treatment of advanced non-small-cell lung cancer. 52 cribrostatins were isolated from a blue sponge cribrochalina sp, and showed potent antineoplastic and antimicrobial activities. cribrostatin 3 has potent inhibitory activity against neisseria gonorrheae, with an mic of 0·09 g/ml. cribrostatin 3 is also active against penicillinresistant n gonorrheae (clinical isolate), with an mic of 0·39 g/ml. 53 sphaerococcus coronopifolius, a cosmopolitan red alga collected along the atlantic coast of morocco, contains the potent antibacterial diterpene, bromosphaerone. it shows antibacterial activity against s aureus, with an mic of 0·047 g/ml. 54 a marine fungus isolated from the surface of the brown alga rosenvingea sp, of the genus pestalotia, collected in the bahamas, was cocultured with a unicellular marine bacterium to yield pestalone. pestalone has potent antibiotic activity against meticillin-resistant s aureus, with an mic of 0·037 g/ml, and vancomycin-resistant enterococcus faecium, with an mic of 0·078 g/ml. these results represent an important achievement in showing that mixed fermentation can induce the biosynthesis of novel antibiotics, suggesting that this method may have use for drug discovery in the future. in addition, the potency of this agent toward drug-resistant pathogens suggests that pestalone should be assessed in more advanced animal models against infectious diseases. 55 jorumycin is a dimeric isoquinoline alkaloid that was isolated from the mantle and mucus of the pacific nudibranch jorunna funebris. it inhibits the growth of various grampositive bacteria-eg, bacillus subtilis, s aureus-at a concentration of 0·050 g/ml, with an inhibition zone of 16 mm. despite this high potency, there are several limitations for further investigation, which include its cytotoxic effects at an ic 50 of 0·012 g/ml and the difficulty in obtaining a pure stable preparation. 56 viruses have remained resistant to treatment or prophylaxis longer than any other infectious organism. the search for viral chemotherapeutic agents from marine sources has yielded several promising therapeutic leads reported to display notable antiviral activity (figure 7). perhaps the most important antiviral lead of marine origin reported thus far is the nucleoside ara-a. ara-a is a semisynthetic compound based on the arabinosyl nucleosides isolated from the sponge cryptotethia crypta. 57 once it was realised that biological systems would recognise the nucleoside base after modifications of the sugar moiety, chemists began to substitute the typical pentoses with acyclic entities or with substituted sugars, leading to the drug azidothymidine (zidovudine). ara-a (vidarabine), ara-c (1-␤-d-arabinosylcytosine; cytarabine), acyclovir, and azidothymidine are in clinical use and are all examples of products of semisynthetic modifications of the arabinosyl nucleosides. 58 the didemnins are a family of closely related cyclic depsipeptides obtained from trididemnum solidum, a caribbean tunicate, or sea squirt, of the family didemnidae. 59 in addition to their antitumour activity, they exhibit significant in-vitro and in-vivo antiviral properties. in 1982, canonico and colleagues 60 reported that didemnin b has in-vitro inhibitory effects against the viruses causing rift valley fever (ld 50 0·04 g/ml), venezuelan equine encephalomyelitis (ld 50 0·08 g/ml), and yellow fever (ld 50 0·08 g/ml). in-vivo didemnin b at a dose of 0·25 mg/kg daily in mice infected with rift valley fever virus gave a 90% survival rate. despite its important antiviral activity, didemnin b is cytotoxic and inhibits cellular dna, rna, and protein synthesis at concentrations close to those at which viral growth was inhibited, and hence it has low antiviral selectivity and therapeutic index. the didemnins might, however, be modifiable or useful in combination with other antiviral agents to combat such difficult diseases. 61 didemnin b interferes with mitogenic signal transmission such as inhibiting kinases, phosphatases, and elongation factors. since 1986, didemnin b has been subjected to phase 1 and 2 clinical trials for cancer by different groups, and the consensus is that with the dose schedules used, the drugs provided little efficacy and substantial toxic effects. 61 eudistomins represent a large family of ␤-carboline alkaloids that have been isolated from shallowwater tunicates from the genus eudistoma. there are four types of eudistomins, including: unsubstituted, pyrrolyl-substituted, pyrrolinyl-substituted, and tetrahydro-␤-carbolines containing a uniquely condensed oxathiazepine ring system. the in-vitro antiviral potency of the eudistomins against herpes simplex viruses 1 and 2 ranges from 5 ng/disk to 500 ng/disk and follows a trend in which the oxathiazepino-tetrahydro-␤-carbolines show greater potency than 1-pyrrolinyl-substituted, which in turn have better antiviral activity than the 1-pyrrolyl-substituted compounds. 62 perry and colleagues 63 reported isolating and investigating the structure of mycalamide a from a new zealand sponge mycale sp. they found that a material consisting of 2% mycalamide a was effective against a59 coronavirus in vivo in mice at 0·2 g/kg daily, with 100% survival after 14 days. when pure mycalamide a has been obtained, it inhibited herpes simplex virus 1 and poliovirus type 1 at 0·005 g/disk. 63 when mycalamide b was isolated, it showed greater antiviral activity and cytotoxicity than mycalamide a. in-vitro antiviral testing showed a minimum dose of inhibition of 0·001-0·002 g/disk for mycalamide b. mycalamide a and b are protein synthesis inhibitors. 64 further antiviral marine natural products are presented in table 2. the unprecedented diversity of marine natural products combined with an improved global awareness about the need for new anti-infective treatments is certain to result in an increased effort to move the product of the marine environment into clinical applications. although marineproduct leads have historically been confronted with many obstacles, including a sustainable supply-ie, the compounds may account for less than 10 -6 % of the wet weight 73 -there have been substantial advances, which suggest that sustainable sourcing will be achievable. we have learned from terrestrial drug leads that continuous and exhaustive harvesting from nature is not reliable and puts the respective species under risk of extinction. consequently, the large-scale production of metabolites from marine natural products for clinical applications is a real challenge and alternative strategies for environmentally sound and economically feasible supplies are clearly needed. traditionally, among the first options to be explored for the supply of marine-derived small molecules is chemical synthesis. unfortunately, the structural complexity of marine molecules, which suggests novel mechanisms of action and high selectivity, has also resulted in few economically feasible strategies for total chemical synthesis. one successful example of the synthetic production of a marine-product drug in unlimited quantities is the conus toxin ziconotide, because of its peptide nature. 74 a second but also fairly labour-intensive strategy is to do studies of the biological roles of marine natural product pharmacophores, with a clearly defined structural moiety, and attempt to define whether the critical pharmacophore via synthesis, chemical degradation, modification, or a combination of these, can result in more practical drugs based on a marine prototype. the aquaculture of the source organisms, including sponges, tunicates, and bryozoans, with the aim of securing a steady supply of drug product, has progressed notably in cancer applications, although in most cases the biomass currently generated is still far short of those required should a marine-based drug finally enter the market. 75 furthermore, the cultivation of invertebrates in their natural environment is subject to several uncertainties, such as destruction by storms or disease. an intriguing strategy has been to identify the true producers of bioactive compounds to find out whether or not they are of microbial origin, based on the fact that most if not all of the marine invertebrates harbour large communities of microorganisms, including bacteria, cyanobacteria, and fungi within their tissues. 76 many studies have successfully provided data to support the involvement of microorganisms in the biosynthesis of natural products isolated from invertebrates. the major breakthrough in the characterisation of the microbial communities associated with sponges involve the application of molecular methods such as fluorescence in-situ hybridisation, with the use of group-specific 16 s rrna-targeted oligonucleotide probes. this approach has led to the identification of the bacterial communities associated with many marine invertebrates. 77 review anti-infectives from the sea if bacteria or other associated microorganisms do produce these compounds of interest, the careful design of special media for culture would be useful for large-scale fermentation. currently, only an estimated 5% or less of the bacteria seen in marine samples by microscopic methods can be cultivated under standard conditions. 78 as a result, molecular approaches offer particularly promising alternatives through the transfer of biosynthetic gene clusters to a vector suitable for large-scale fermentation, thereby avoiding the difficulties in culturing symbiotic bacteria. clearly, the world's oceans will play an important part in the future control of the global infectious-disease burden. although substantial progress has been made in identifying novel drug leads from the ocean's resources, great efforts are still needed to advance to clinical applications. none declared. a 56-year-old man presented to the emergency department with a 4-week history of a painful left-sided preparotid swelling. computed tomography suggested that the lesion was most likely a tuberculous abscess (figure). samples for a clinical armamentarium of marinederived anti-cancer compounds the bioprocess-technological potential of the sea marine and freshwater products handbook biological activities of selected marine natural products marine natural products et-743 natural products as potential antiparasitic drugs secondary metabolites with antinematodal activity marine natural products as antituberculosis agents natural products as antiviral agents antifungal metabolites from marine sponges marine bioprospecting: trawling for treasure and pleasure the influence of natural products upon drug discovery marine natural products jasplakinolide a cyclodepsipeptide from the marine sponge jaspis sp gambieric acids: new potent antifungal substances with unprecedented polyether structures from a marine dinoflagellate gambierdiscus toxicus aurantosides d e and f: new antifungal tetramic acid glycosides from the marine sponge siliquariaspongia japonica phorboxazoles a and b: potent cytostatic macrolides from marine sponge phorbas sp halishigamides a-d new cytotoxic oxazolecontaining metabolites from okinawan sponge halichondria sp macrocyclic antifungal metabolites from the spanish dancer nudibranch hexabranchus sanguineus and sponges of the genus halichondria fascaplysin: an unusual antimicrobial pigment from the marine sponge fascaplysinopsis sp antifungal activity of meridine a natural product from the marine sponge corticium sp bengazoles c-g from the sponge jaspis sp synthesis of the side chain and determination of absolute configuration ptilomycalin a: a novel polycyclic guanidine alkaloid of marine origin haliclonadiamine: an antimicrobial alkaloid from the sponge haliclona sp 8-hydroxymanzamine a a ␤-carboline alkaloid from a sponge pachypellina sp 1234-oxamanzamines novel biocatalytic and natural products from manzamine producing indo-pacific sponges assessment of antimycobacterial activity of a series of mainly marine derived natural products novel antimycobacterial benzoxazole alkaloids from the west indian sea whip pseudopterogorgia elisabethae serrulatane diterpenes with antimycobacterial activity isolated from the west indian sea whip pseudopterogorgia elisabethae novel 19-oxygenated sterols from the okinawan soft coral litophyton viridis puupehenone-related metabolites from two hawaiian sponges hyrtios spp nutritional impact of intestinal helminthiasis during the human life cycle marine nematodes: tetrahydrofurans from a southern australian brown alga notheia anomala amphilactams a-d: novel nematocides from southern australian marine sponges of the genus amphimedon geodin a magnesium salt: a novel nematocide from a southern australian marine sponge chemotherapy of leishmaniasis: recent advances in the treatment of visceral disease leishmania and human immunodeficiency virus coinfection: the first 10 years antileishmanial cyclic peroxides from the palauan sponge plakortis aff angulospiculatus toxoplasmosis in aids patients anti-toxoplasmosis drugs new manzamine alkaloids with potent activity against infectious diseases antimalarial antiviral and antitoxoplasmosis norsesterterpene peroxide acids from the red sea sponge diacarnus erythraeanus new peroxylactones from the jamaican sponge plakinastrella onkodes with inhibitory activity against the aids opportunistic parasitic infection toxoplasma gondii recent advances in identifying and validating drug targets in trypanosomes and leishmanias ascosalipyrrolidinone a: an antimicrobial alkaloid from the obligate marine fungus ascochyta salicorniae survey of malaria treatment and deaths in vivo antimalarial activity of the betacarboline alkaloid manzamine a the marine environment: a resource for prototype antimalarial agents novel potent antimalarial diterpene isocyanates isothiocyanates and isonitriles from the tropical marine sponge cymbastela hooperi antimalarial activity of kalihinol a and new relative diterpenoids from the okinawan sponge acanthella sp aminosterols from the dogfish shark squalus acanthias antineoplastic agents 430 isolation and structure of cribrostatins 3 4 and 5 from the republic of maldives cribrochalina sp marine lit, and references from relevant articles. search terms were "antifungal marine new bromoditerpenes from the red alga sphaerococcus coronopifolius pestalone a new antibiotic produced by a marine fungus in response to bacterial challenge a new antitumor isoquinoline alkaloid from the marine nudibranch jorunna funebris contributions to the study of marine products: the nucleosides of sponges new anti-hiv agents and targets structures of the didemnins antiviral and cytotoxic depsipeptides from a caribbean tunicate inhibition of rna viruses in vitro and in rift valley fever-infected mice by didemnins a and b natural products as probes of cell biology: 20 years of didemnin research eudistomins c e k and l potent antiviral compounds containing a novel oxathiazepine ring from the caribbean tunicate eudistoma olivaceum mycalamide a an antiviral compound from a new zealand sponge of the genus mycale antiviral and antitumor agents from a new zealand sponge mycale sp 2: structures and solution conformations of mycalamides a and b papuamides a-d hiv-inhibitory and cytotoxic depsipeptides from the sponges theonella mirabilis and theonella swinhoei collected in papua new guinea inhibition of replication of the etiologic agent of acquired immune deficiency syndrome (human t-lymphotropic retrovirus/ lymphadenopathy-associated virus) by avarol and avarone debitus c: in vitro antiviral activity on dengue virus of marine natural products microspinosamide a new hiv-inhibitory cyclic depsipeptide from the marine sponge sidonops microspinosa solenolides new antiinflammatory and antiviral diterpenoids from a marine octocoral of the genus solenopodium hennoxazoles bioactive bisoxazoles from a marine sponge venustatriol a new anti-viral triterpenes tetracyclic ether from laurencia venusta antitumor and antiviral furanoditerpenoids from a marine sponge drugs from the seas: current status and microbiological implications mviia: from marine snail venom to analgesic drug aquacultural production of bryostatin 1 and ecteinascidin 743 electron microscope study of the association between some sponges and bacteria microbial diversity in the marine sponge aplysina cavernicola analyzed by fluorescence in situ hybridization (fish) chemical studies of marine bacteria: developing a new resource review anti-infectives from the sea the preparation of this review was supported by nih grants r01ai 36596 and ko2ai01502 from the national institute of allergy and infectious diseases, and the egyptian government (predoctoral fellowship for md). we thank charles stanley for his assistance in the preparation of this review. anti-infectives from the sea rabbit's revenge analysis were obtained from incision and drainage of the abscess; however, no acid-fast bacilli could be detected and culture media for bacteria, fungi, and mycobacteria remained without growth. a few days after his admission, the patient's wife joined her husband with a painful cervical lymph node. on further investigation it emerged that the couple had eaten a medium roasted wild rabbit in a berlin restaurant 2 months earlier. 1 week after the meal they both developed an exudative pharyngitis. oropharyngeal tularaemia was suspected and confirmed for both patients by positive serology. they both received a 2-week course of streptomycin and recovered uneventfully.tularaemia is caused by the gram-negative rod francisella tularensis. natural reservoirs and vectors include several small mammals, as well as insects like ticks and mosquitoes. transmission to people is through direct contact with infected animals, inhalation of contaminated fomites, insect bites, and ingestion of contaminated food. the primary clinical presentation of tularaemia depends on the site of entry of the bacterium into the body. oropharyngeal tularaemia results from oral exposure from contaminated fluids or foods. key: cord-316201-08pyx98r authors: grout, andrea; howard, natasha; coker, richard; speakman, elizabeth m title: guidelines, law, and governance: disconnects in the global control of airline-associated infectious diseases date: 2017-02-01 journal: lancet infect dis doi: 10.1016/s1473-3099(16)30476-5 sha: doc_id: 316201 cord_uid: 08pyx98r international air travel is increasingly affecting the epidemiology of infectious diseases. a particular public health, economic, and political concern is the role of air travel in bringing infectious passengers or vectors to previously non-endemic areas. yet, little research has been done to investigate either the infection risks associated with air travel or the empirical evidence for the effectiveness of infection control measures on aircraft and at borders. we briefly review the interface between international and national legislation, policy, and guidelines in the context of existing infection risks and possible scenarios. we have found that public health guidance and legislation, which airlines are required to follow, are often contradictory and confusing. infection control measures for air travel need to be underpinned by coherent and enforceable national and international legislation that is based on solid epidemiological evidence. we recommend further research investment into more effective on-board vector control, health screening, and risk communications strategies, and the development of enforceable and harmonised international legislation. low air fares and a multitude of social and economic factors have resulted in increased air travel. the number of journeys taken by passengers each year has grown from approximately 642 million in 1980 to more than 3·4 billion in 2015. 1 the epidemiology of infectious diseases associated with air travel and the challenges of infection control are important public health concerns, yet they are scarcely discussed in the literature. 2 aircraft can now travel to almost any part of the world within 24 h, and can enable spread of infection either by inflight infection transmission or by transporting infectious passengers or vectors-eg, malaria-infected mosquitoes-from endemic to non-endemic regions, thus putting populations in destination countries at risk. the combination of rising passenger numbers, new travel destinations, and on-board transmission events can influence transmission patterns of several imported diseases, including severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebola virus disease. 3 for example, the ongoing transmission of zika virus disease is believed to have been introduced to the americas by air travel. 4 management of these risks requires knowledge of the dynamics of infectious disease transmission and the potential effectiveness of control measures, suggesting that frontline employees (eg, airline staff) would need appropriate training to handle suspected disease cases. as a result of experiences with sars, the international air transport association (iata) issued the emergency response plan and action checklist, which consists of guidelines and best practices for aircrews during public health emergencies. 5 to reduce the risk of on-board disease transmission, the us centers for disease control and prevention (cdc) provides cabin crews with information on general infection control measures and guidelines to identify ill and potentially infectious passengers. 6 passengers with certain conditions-for example, someone recovering from measles-might require medical clearance; however, guidelines for these conditions vary between countries and can be subject to individual airline policy. 7 the effectiveness of infectious disease response strategies largely depends on prompt identification of cases. 8 current measures, such as entry and exit screening, isolation, quarantine, and travel health information might not be feasible or sufficient to control infectious disease transmission. for example, the value of entry screening measures has been questioned, 9, 10 while an evaluation of border entry screening measures in several countries concluded that a combination of communication methods (eg, in the form of pre-flight health information, in-flight videos, and clinical guidance) for passengers and clinicians might be a more effective strategy for global infectious disease control. 11 collectively, the unique dynamics and interactions at play in an aircraft environment require a distinct response to infectious disease control. in this personal view, we consider the disconnects between global health law, national jurisdictions, organisational guidelines, and aircrew compliance by discussing existing risks and presenting two infection scenarios based on current airline practice. 12 although the risk of disease transmission exists whenever people congregate in confined spaces, aircraft are unique in having individuals from often diverse geographical regions, with differing population immunity and exposure risks, interacting with aircrews and each other. 6 infection can occur via direct transmission through contact with skin, blood, or other bodily fluids (eg, ebola virus), or via indirect transmission without person-to-person contact. indirect transmission on an airplane can occur through infectious droplets (eg, influenza virus), through contaminated surfaces or objects (eg, meticillin-resistant staphylococcus aureus), or long-distance air travel in particular exposes passengers to several factors that could affect disease transmission. the transmission characteristics of a pathogen, ambient climatic conditions, time spent on board, and aircraft type can affect quantification of the general transmission risk. 13 absolute figures for the risk of in-flight disease transmission are therefore not readily available and the evidence base is inadequate. 14 mangili and gendreau 2 reported in-flight transmission of influenza, sars, tuberculosis, measles, smallpox, and other pathogens. on a 3 h flight from hong kong to beijing in 2003, 16 of 120 passengers were infected with the sars virus by a single ill passenger, 15 while modelling has demonstrated the possibility of in-flight transmission of mers coronavirus. 16 protective measures are in place in modern aircraft, but these measures are not necessarily as robust as assumed. for example, commercial aircraft use highefficiency particulate air (hepa) filters to restrict exposure to small airborne particles. however, there are no regulations requiring that hepa filters be in place, and little testing has taken place to assess the effectiveness of these filters. 17 in 2014, ebola virus disease was brought to the usa, 18 the uk, 19 and nigeria 20 by undiagnosed infected people aboard aircraft. brownstein and colleagues 21 demonstrated the effect of air travel on the global spread of seasonal influenza, noting that decreased air traffic following the terrorist attacks of sept 11, 2001 , was associated with a delayed influenza season. maloney and cetron 22 documented the air-travel-associated transmission of meningococcal disease. global air travel could spur the spread of epidemics by bringing viruses and parasites to new areas. 23 infected mosquitoes on intercontinental flights are believed to have contributed to the global spread of malaria. 23, 24 west nile virus is widely suspected to have been spread to the usa by an infected mosquito carried by plane. 24 similarly, the introduction of zika virus to the americas coincided with an upsurge of air travel to brazil from endemic countries in 2013. 4 management of the risk of transporting infected passengers requires knowledge of transmission dynamics and the potential effectiveness of airport entry and exit screening measures, the ability to appropriately isolate or quarantine individual passengers on an aircraft, and adequately trained aircrew who are able to identify signs of infection and take appropriate measures. for example, who maintains that the risk of vector-borne diseases being transmitted aboard aircraft is low, 25 but recommends aircraft disinsection (a public health measure involving insecticide treatment of aircraft interiors and holds), 25 stating that "there have been frequent instances of insects of public health importance being introduced from one country to another, with occasional dire consequences". 23 however, the effectiveness of disinsection is unclear. 26 minimisation of the risk of inadvertently carrying insect vectors requires consistent use of effective control measures, including insecticides that are safe for frequent aircrew and passenger exposure. public health measures for international air travel include a range of national and international legislative tools, policies, and guidelines. globally, 196 countries signed the legally binding international health regulations (ihrs), with the aim of controlling global disease spread. 27 however, the only ihr provision relating to air travel is the requirement that all chief pilots provide a brief aircraft general declaration on passenger health to ground staff before disembarkation. the international civil aviation organization (icao) and iata coordinate with who and provide recommendations, but specific controls are left to the discretion of individual countries. national guidance and legislation are uncoordinated across countries, and-with no strong evidence underpinning control measures-they are often inconsistent. following the sars epidemic, iata recommended that all air carriers create an emergency response plan for public health emergencies, but these are only guidelines and legislative powers lie with national authorities. 5 airlines face conflicting obligations, since they must comply with infectious disease controls in both origin and destination countries. 28 airlines owe a duty of care to three different groupspassengers, aircrew, and destination country populations-and these duties sometimes conflict. for example, the us environmental protection agency prohibits usage of some insecticides because of potential risks to aircrew, whereas national laws in australia and new zealand require usage of these insecticides. us airlines flying to these countries must purchase insecticides at stopovers, and airline unions have raised serious concerns about the "inconsistent and inappropriate application", toxicity, and potential adverse health effects of these agents. 29 other airlines have reported difficulties in aircraft storage of aerosol insecticides that were either banned or prohibited from import in some destination countries. 30 additionally, doubt exists as to the effectiveness of disinsection, with research identifying increasing resistance of mosquitos to insecticides. 26 although the icao encouraged more research into non-chemical disinsection procedures in 2013, 31 procedures have not changed and airplane disinsection policies and implementation remain inconsistent worldwide. airlines and national authorities can refuse to transport passengers they consider to be a health risk. the us air carrier access act states that carriage can be refused www.thelancet.com/infection vol 17 april 2017 e120 personal view where a passenger presents with a disease that "is both readily transmitted in the course of a flight and which has serious health consequences (eg, sars, but not aids or a cold)". 32 this rule applies to all flights of us carriers and flights to or from the usa, but it clearly requires any disease to be diagnosed before the flight. considerable debate continues about the effectiveness and practicality of screening passengers at entry, exit, or both. further research must be prioritised before national and international legislation can take a consistent, evidence-informed approach to screening, because flight duration and pathogen transmission dynamics are just two important factors that challenge one-size-fits-all recommendations. 33 enforcement of national laws is highly variable, with non-compliance carrying financial penalties and criminal sanctions in some countries, but little evidence of enforcement in others. 191 countries are signatories to the montreal convention, which imposes obligations to protect passengers. 34 however, although this convention enables compensation claims to be made, proving an airline's liability for a passenger contracting an infectious disease during the flight can be challenging evidentially. even if transmission time can be proven, airlines can defend the extent to which they should have been expected to identify the risk. they can argue that liability should lie with the infectious passenger who took the flight without notifying the airline or health authorities. 35 although industrial injury claims have been brought on behalf of aircrew for alleged adverse reactions to constant insecticide exposure in aircraft, these claims have been defended on the basis that airlines were following who guidelines. 36, 37 the montreal convention does not apply to individuals in a destination country who could become infected by a passenger or imported vector. although regulatory liability might still exist and personal litigation against an airline could be undertaken, proving causal transmission will again be very difficult, particularly if the disease in question did not become symptomatic until some time after the flight had landed. two hypothetical scenarios are provided to show the potential occurrence and wider implications of disease transmission on aircraft. ebola is an infectious and often fatal disease marked by fever, nausea, vomiting, and-less frequentlyhaemor rhaging, and spread through infected bodily fluids. on a flight from frankfurt to washington, dc, a 40-year-old passenger started complaining of a severe headache, abdominal pain, nausea, and sweating. he recalled no specific symptoms before boarding, but claimed he had been feeling generally unwell since his arrival from abuja, an interim stopover on his itinerary that had originated in kampala 2 days earlier. about 3 h into the flight, his symptoms worsened and the cabin supervisor requested medical assistance. as there was no doctor on board, a nurse examined the passenger and, suspecting he might be infectious, advised the crew to "isolate him as a precautionary measure". the passenger was taken to a seat near the galley and looked after by two crew members for the remainder of the flight. meanwhile, he had violent bouts of vomiting and became increasingly disoriented. the cabin supervisor notified the chief pilot of a sick passenger, but did not communicate the severity of his condition. the pilot assumed the situation was controlled and did not contact us health authorities. upon landing, the passenger's condition had deteriorated and an ambulance was requested. after 24 h, the passenger was determined to be positive for ebola virus. this scenario demonstrates an absence of communication between crew members and between aircrew and ground staff in the destination country. this miscommunication delayed notification of a potentially severe health risk from infected bodily fluids, such as vomit; moreover, an ambulance with infection control facilities should have been requested while the plane was airborne. this represents non-compliance with iata guidance and a potential criminal breach of us health and quarantine laws. us laws are enforceable against both individuals and organisations, with penalties including fines and imprisonment. 38,39 vector-borne diseases (eg, malaria, yellow fever, and zika virus) are transmitted by mosquitoes or other vectors to human beings, and contribute to a substantial proportion of the global infectious disease burden. 40 mosquito ecology suggests that aircraft are associated with a higher risk of introducing a live infected mosquito than are sea or road transport. 41 following national requirements, disinsection was carried out by aircrew during descent into mumbai airport. the flight had originated in london. a passenger who regularly travelled on this route objected to being sprayed with insecticide, pointing to potentially dangerous adverse health effects. he added that, having travelled with different airlines, he had not witnessed any in-flight spraying for years. on the return flight, several passengers complained about the presence of mosquitoes in the cabin before take-off. the aircraft had been parked on the apron of mumbai airport, with cabin and cargo doors open during baggage loading and passenger embarkation. passengers demanded protection from mosquitoes and wondered why spraying was done upon entering india, but not upon departure. this scenario reflects inconsistencies in, and in adequate monitoring of, disinsection policy. indian national law requires disinsection on inbound flights, but india is itself personal view a reservoir of vector-borne diseases. guidance from who and iata uses permissive rather than mandatory language on disinsection, and thus national policies determine whether countries choose to implement disinsection consistently for all arriving aircraft or only require the process on selected aircraft. policies are not always clear, and it is necessary to balance the fears of health risks from both insecticides and mosquitoes. to be effective, infection control measures for air travel need to be underpinned by coherent and enforceable national and international legislation that is based on solid epidemiological evidence. since aircrew are not infectious disease specialists and would not normally have medical training, recognition of potential disease cases and adequate communication of an in-flight illness remains challenging and ad hoc. the dynamics of existing, emerging, and re-emerging infectious pathogens mean that infectious diseases will always challenge control efforts as pathogens exploit novel evolutionary niches. incoherent guidelines and inconsistently applied laws unnecessarily hinder disease control efforts, and the evidence base underpinning control measures for airlineassociated infectious diseases needs to be strengthened considerably. public health involves balancing the rights of the majority against those of the individual, and issues related to air travel require particular review and improvement by the global health community. first, a systematic review should be done to appraise the evidence supporting control measures for transmission of infectious diseases via air travel. second, airlines and the global health community need to invest in research to identify better, non-toxic insecticides, or non-chemical means to control insect vectors. third, additional research and investment into airport health screening measures is required to better identify infectious passengers. disease transmission can be minimised if passengers take appropriate precautions before or during a flight, or refrain from flying altogether when ill. current education and communication strategies (and refund policies for missed flights) therefore warrant improvement. fourth, these measures cannot be implemented in the absence of enforceable and harmonised international legislation and governance. achieving this goal will be a major challenge, but a starting point could be for international or regional bodies-such as who or the european union-to produce model legislation or standards for the guidance of member states. close consultation with iata and icao would be required to develop such legislation or guidance. enforceability might be encouraged by treating this as a security issue, similar to ensuring the mechanical safety of aircraft. in the context of regular global air travel and evidence of dangerous non-endemic diseases appearing in new, vulnerable populations, the risks of airline-associated infection are growing. potential costs or inconvenience to passengers and aircrews might arguably be a lesser evil than transmission of potentially fatal infections to vulnerable populations. however, without concerted efforts from the global health community, the threat can be expected to worsen. ag developed the scenarios and drafted the manuscript with ems, who wrote on legal aspects. nh contributed to writing and interpretation. rc provided interpretation and critical review. all authors approved the version for submission. we declare no competing interests. air transport, passengers carried: international civil aviation organization, civil aviation statistics of the world and icao staff estimates transmission of infectious diseases during commercial air travel infectious risks of air travel zika virus in the americas: early epidemiological and genetic findings emergency response plan: a template for air carriers infection control guidelines for cabin crew members on commercial aircraft mode of travel: health considerations entry and exit screening of airline travellers during the a (h1n1) 2009 pandemic: a retrospective evaluation world health organization writing group. non-pharmaceutical interventions for pandemic influenza, international measures screening for influenza a (h1n1) pdm09, auckland international airport entry screening for infectious diseases in humans planning and response to communicable disease on us domestic air flights germs on a plane: aircraft, international travel, and the global spread of disease ebola: how easily do germs spread on planes transmission of the severe acute respiratory syndrome on aircraft predicting the potential for within-flight transmission and global dissemination of mers ticket to ride: spreading germs a mile high us to "rethink" ebola infection control after nurse falls ill second ebola patient is treated in uk assessment of the potential for international dissemination of ebola virus via commercial air travel during the 2014 west african outbreak empirical evidence for the effect of airline travel on inter-regional influenza spread in the united states investigation and management of infectious diseases on international conveyances (airplanes and cruise ships) why aircraft disinsection? assessing the risks of west nile virus-infected mosquitoes from transatlantic aircraft: implications for disease emergence in the united kingdom transmission of communicable diseases on aircraft pyrethroid resistance in african anopheline mosquitoes: what are the implications for malaria control? geneva: world health organization to spray or not to spray': developing a tourism-linked research agenda for aircraft disinsection disinsection and pesticides in aircraft cabins. twelfth session of the facilitation (fal/12) division of the international civil aviation organization disinfection of aircraft: appropriate disinfectants and standard operating procedures for highly infectious diseases agenda item 38. summary of work undertaken since the 37th assembly in the area of non-chemical disinsection of the aircraft cabin and flight deck for international flights 14 cfr part 382: nondiscrimination on the basis of disability in air travel; final rule entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation convention for the unification of certain rules for international carriage by air. montreal: international civil aviation organization preventing airline liability for spread of communicable diseases air quality in airplane cabins and similar enclosed spaces qantas steward with parkinson's to sue over pesticide link us code § 270: quarantine regulations governing civil air navigation and civil aircraft us code § 271: penalties for violation of quarantine laws vector-borne diseases: overview evidence in australia for a case of airport dengue key: cord-320970-ru2iw0py authors: peeling, rosanna w; wedderburn, catherine j; garcia, patricia j; boeras, debrah; fongwen, noah; nkengasong, john; sall, amadou; tanuri, amilcar; heymann, david l title: serology testing in the covid-19 pandemic response date: 2020-07-17 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30517-x sha: doc_id: 320970 cord_uid: ru2iw0py the collapse of global cooperation and a failure of international solidarity have led to many low-income and middle-income countries being denied access to molecular diagnostics in the covid-19 pandemic response. yet the scarcity of knowledge on the dynamics of the immune response to infection has led to hesitation on recommending the use of rapid immunodiagnostic tests, even though rapid serology tests are commercially available and scalable. on the basis of our knowledge and understanding of viral infectivity and host response, we urge countries without the capacity to do molecular testing at scale to research the use of serology tests to triage symptomatic patients in community settings, to test contacts of confirmed cases, and in situational analysis and surveillance. the who r&d blue print expert group identified eight priorities for research and development, of which the highest is to mobilise research on rapid point-of-care diagnostics for use at the community level. this research should inform control programmes of the required performance and utility of rapid serology tests, which, when applied specifically for appropriate public health measures to then be put in place, can make a huge difference. the covid-19 pandemic, now only a few months old, 1,2 has brought into sharp focus inequalities within and among countries. john nkengasong, director of the africa centres for disease control and prevention, reported that "the collapse of global cooperation and a failure of international solidarity have shoved africa out of the diagnostics market". 3 sadly, the same is true of many other low-income and middle-income countries (lmics) outside africa. why are diagnostics important? in any epidemic response, diagnostic testing plays a crucial role and this pandemic is no exception. because early clinical presentations of infected patients are non-specific, testing is needed to confirm the diagnosis of covid-19 in symptomatic patients, as soon as possible, so that these patients can be appropriately isolated and clinically managed. 1,4,5 diagnostic testing is also needed for individuals who have come into contact with someone with confirmed covid-19. some testing strategies examine only contacts who have symptoms or develop illness of any kind during the 14-day period after contact. other strategies examine all contacts when identified, regardless of whether they have any symptoms. studies have shown that a large number of infected individuals might have no symptoms at all, and there is concern that these individuals are still able to shed the virus and transmit infection through saliva droplets as they speak. [4] [5] [6] [7] [8] [9] tracking all contacts of confirmed cases and testing them for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is key to successful pandemic control. diagnostics are also needed to support rapid serosurveys that establish whether and to what extent sars-cov-2 has circulated in a community, and sur veillance systems, such as that for influenza-like illness, that monitor disease trends over time. diagnostics can also be used to identify atrisk populations and assess the effectiveness of control strategies. tedros adhanom ghebreyesus, director-general of who, urged countries to implement a comprehensive package of measures to find, isolate, test, and treat every case, and trace every contact. goodwill between countries has already been shown through the publishing of the sars-cov-2 genetic sequence and shared laboratory protocols to detect the virus. 10 however, as these molecular assays require sophisticated labora tory facilities, countries with insufficient infrastructure quickly accumulate a backlog of testing. the rapid spread of covid-19 around the world has led to a global shortage of reagents and supplies needed for testing. point-of-care molecular assays for sars-cov-2 detection are now available to enable community-based testing for covid-19 in lmics. unfortunately, the production of these test cartridges takes time and, again, global demand has outstripped supply, leaving lmics struggling for access. in march, 2020, who urged member states to "test, test, test". 11 widespread testing can help countries to map the true extent of the outbreak, including identifying hot spots and at-risk populations, and monitor the rate at which the epidemic is spreading. however, most lmics find that molecular testing, including point-of-care testing, is neither scalable nor affordable on a large scale. relying solely on centralised testing puts countries at risk of having nothing to use. what diagnostic alternatives are available to support decentralised testing that would allow countries to mount an adequate response to the pandemic? rapid antigen detection tests that are simple to do at point of care and can give results in less than 30 min would be viable alternatives to molecular testing for confirming covid-19 cases, enabling appropriate case management, and guiding public health measures, such as quarantine or self-isolation. however, although scaling up rapid antigen testing offers an effective means of triaging symptomatic individuals in community settings, early evaluations of rapid antigen detection tests show personal view suboptimal sensitivity for these tests to be recommended for clinical diagnosis or triage. 12 rapid antibody detection lateral flow tests are also simple to use, generally requiring a few drops of whole blood from a finger prick placed onto the test strip with no processing needed. these tests take 15-20 min to do with minimal training and can be done at the point of care as most do not require any equipment. rapid antibody testing is an attractive option for scaling up testing but only if these tests show satisfactory performance for a clearly specified use. the detection of sars-cov-2 infection and immune response has been described in relation to different diagnostic tests. 13 in this section, we summarise the evidence from studies to date. studies have shown that sars-cov-2 rna can be detected 2-3 days before onset of symptoms and can remain detectable up to 25-50 days after the onset of symptoms, particularly in patients who remain symptomatic for an extended period. 7,14,15 sars-cov-2 rna can be detected for longer in respiratory samples from patients with severe disease than in samples from patients with mild illness. 16 viral rna concentrations peak within the first 5 days after onset of symptoms and decrease slowly with rising antibody concentrations. 7, 17, 18 however, rna clearance is not always associated with rising antibody concentrations, particularly in patients who were critically ill. 6,18 an important question for the potential for spread of covid-19 is whether individuals who are rna-positive are shedding infectious virus. a small study in nine patients found that viral replication stopped 5-7 days after onset of symptoms but patients remained rna-positive for 1-2 weeks after this point. 6 hence, there remains some uncertainty as to whether a patient who is rna-positive is shedding live virus or not. maturation of the immune response typically takes 40 days with variations in the dynamics of the antibody response depending on disease severity and other factors still to be discovered. in most studies of laboratoryconfirmed covid-19 cases, igm antibodies start to be detectable around 5-10 days after onset of symptoms and rise rapidly. 14,18-21 igg antibody concentrations follow the igm response closely. seroconversion is typically within the first 3 weeks with the mean time for seroconversion being 9-11 days after onset of symptoms for total antibody, 10-12 days for igm, and 12-14 days for igg. 14, 18, 19, 22 antibodies against the receptor-binding domain of the spike protein and the nucleocapsid protein have been associated with neutralising activity. 14,23,24 neutralising anti bodies to these domains can be detected approximately 7 days after onset of symptoms and rise steeply over the next 2 weeks. 23, 24 several studies showed that patients can remain rna-positive despite high concentrations of igm and igg antibodies against the nucleocapsid protein and the receptor-binding domain of the spike protein. 18 whether the presence of neutralising antibodies translates into protective immunity in patients with covid-19 is unclear. some researchers speculate that antibodies can enhance infectivity as higher antibody concentrations have been observed in patients with severe disease than in those with mild disease. 18, 25 in one study (n=222), a greater proportion of patients with high igg concentrations had severe disease than did those with low igg concentrations (52% vs 32%, p=0·008). 26 the role of antibody response in the pathogenesis of covid-19 remains unclear pending further studies. who and the pan american health organization have stated that they do not currently recommend the use of immunodiagnostic tests except in research settings, 27, 28 because of scarce information on test performance and appropriate use when immunity to covid-19 is not well under stood. however, many countries are struggling to scale up testing to implement the key strategies of diagnosing all symptomatic patients and tracing all contacts. delays in confirming covid-19 cases allow continued transmission within communities and can result in failure to contain the pandemic despite other mea sures such as physical distancing and travel restrictions. countries are assessing all available testing options to address their range of needs. in settings where challenges with molecular testing exist or access to laboratories is scarce, rapid serology tests offer a needed additional option. a rapid serology test with good performance character istics is extremely important to avoid missing true cases of covid-19 and imposing unnecessary quaran tine for people with false-positive results due to cross-reactivity with seasonal coronaviruses. studies have shown more anti body cross-reactivity between the nucleo capsid proteins of sars-cov-2 and common corona viruses than between their spike proteins. 20, 22 tests that use the spike protein or fragments of the spike protein as targets might have the least amount of cross-reactivity with common coronaviruses, on the basis of sequence analysis. 22 clear articulation of the benefits and limitations of serology tests will hopefully incentivise manufacturers to improve performance. 29 when is serology testing recommended? where there is little or no access to molecular testing, rapid serology tests provide a means to quickly triage personal view suspected cases of covid-19, provided the test is highly specific for the disease. a positive result for igm in symptomatic patients fulfilling the covid-19 case definition is strongly suggestive of sars-cov-2 infection. this approach is probably most effective in individuals 5-10 days after symptom onset. in peru, public health facilities for molecular testing are sparse and only 500 beds in intensive care units exist for a population of 32 million. the ministry of health has set up a hotline and website for individuals who have symptoms to be interviewed by a health professional for possible follow-up, prioritising the visits according to age, risk factors, and severity of symptoms. a testing team visits the individual at home to do the rapid anti body test. individuals who are igm and igg positive and have mild symptoms are quarantined, whereas people who need critical care are referred to hospital. all contacts are also tested with the rapid serology test. anyone who tests negative in the antibody test has a swab collected for molecular testing. as of may 2, 2020, 355 604 people had been triaged in peru with 42 534 testing positive, 26 362 of whom were found to be positive by use of a rapid test. 30 this approach has allowed a large number of sympto matic individuals and contacts to be rapidly tested in the community, relieving the backlog, reducing waiting time for molecular testing, and preventing the health-care system from being overwhelmed. experience in china has also shown that, in symptomatic patients, the use of igm tests or total antibody tests can increase the sensitivity of covid-19 case detection. 18 further research should explore the performance and utility of rapid antigen-igm and antigen-igg combo tests and the timing of testing. in individuals who test negative for igg, research should also explore, if resources allow, the value of doing a follow-up antibody test 10-14 days later to document a definitive diagnosis through seroconversion. studies have shown that a large number of infected individuals could have only mild symptoms or no symptoms at all, but they can still transmit infection, with as much as 44% of infections being transmitted by presymptomatic individuals. 6,7,9 experience from singapore shows that tracking down all contacts of people with confirmed covid-19, testing them for evidence of infection, regardless of symptoms, and putting those contacts who test positive into isolation is an urgent priority for interrupting the chain of transmission and containing the epidemic. [31] [32] [33] this approach is particularly important in the early stages when there are only sporadic or clusters of cases, or for countries coming down from the peak to continue to reduce the extent of infection in the community. only individuals who test negative should have a throat swab collected for molecular testing, which will reduce the strain on laboratories doing these tests. in countries that have set up syndromic surveillance, such as surveillance for influenza-like illness or severe acute respiratory infections, and where blood or throat swabs are routinely collected at these sentinel sites, collected samples can be tested for covid-19 with molecular, antigen, or serology tests, either alone or in combination. if any of these samples are positive, it means covid-19 has been circulating in the community. where serial samples are available, it might be possible to date when covid-19 established itself in a community or country. in general, antibody tests can be used to establish the true extent of an outbreak, map its geographical distribution, and identify hotspots and populations that are particularly at risk. this information can in turn be used to inform public health measures and control strategies. in this case, researchers need to stick to the same serology test and test sentinel populations repeatedly, avoiding the variation in sensitivity between different rapid tests. the use of serology tests for population surveys is not recommended in low prevalence settings as this approach will probably result in more false-positive than truepositive results, even if a test with high specificity is used. for example, if the prevalence of infection is 1% in the general population, a test with 98% specificity will identify two false-positive results for every true positive result. these results could lead to a false sense of security regarding the extent of immunity in the population and premature easing of public health measures on the basis of misleading disease estimates. patients at an early stage in the disease course, or asympto matic or paucisymptomatic patients, might have low antibody concentrations that could give falsenegative results. patients' disease stage and severity are important points to consider, along with the population being tested. the estimated level of risk can be considered before using a serology test, because of the changing falsepositive rate or low positive predictive value across different populations. among the groups with the highest risk of the disease are symptomatic patients with clinical presentation of covid-19, patients with other respiratory symptoms, contacts of confirmed cases, and health-care workers in settings with little personal protective equipment. we suggest countries consider risk levels before using serology tests and creating public health guidance. scaling up testing, particularly at the community level, allows for better estimates of risks, which in turn allows more effective public health measures to be put into place than would be otherwise. in a pandemic, countries must strive to maintain a robust health-care workforce. key workers who develop personal view symptoms should be prioritised for molecular testing and receive care if infected. on recovery, should a serology test be used to decide when they can safely return to work? this strategy is based on the assumption that antibodies confer protective immunity. although antibodies against the receptor-binding domain of the spike protein and the nucleocapsid protein have been correlated with neutralising activity, 14, 23, 24 the development and duration of immunity has not yet been established. 34, 35 although it is tempting to speculate that serology tests based on the detection of neutralising antibodies can be used as markers of protective immunity, and people who test positive can get a so-called immunity passport to return to work, studies have shown that a significant proportion of patients remain rna-positive despite high concen trations of antibodies against the receptor-binding domain of the spike protein and the nucleocapsid protein. 6, 15, 18, 20, 21 wang and colleagues 36 found that elevated serum igm concentrations are correlated with poor outcomes in patients with covid-19 pneumonia, and tan and colleagues 21 found that high concentrations of igg antibodies were correlated with severe disease outcomes. hence a substantial igm or igg response is not necessarily a surrogate marker of protective immunity. to date, insufficient evidence exists to recommend the use of serology testing for health-care workers to return to work. a negative molecular test remains the safest option to establish whether health-care workers can work again safely. a policy brief by the world bank suggested that serology testing could potentially have a high net benefit if it can allow dilution of restrictions for essential workers to return to work and revive essential segments of the economy. 37 the type of tests that can be used for immunity passports remains unclear. a better understanding of the interaction between infection and immune response dynamics is needed before these passports can be considered. hospital beds are often in short supply. the recommended criteria for hospital discharge are two negative molecular tests over several days. however, molecular testing is often scarce or unavailable. can serology tests be used for discharging recovered patients when molecular testing is not available? as patients can remain positive for viral rna despite rising concentrations of antibodies against the nucleocapsid protein and receptor-binding domain of the spike protein, which are correlated with neutralising activities, antibody tests cannot be used in the place of molecular tests to confirm that the patient is virus-free or at least no longer shedding live virus. the events over the past few months have taught us that this pandemic is caused by an extraordinary pathogen that requires extraordinary measures to combat its spread and end the pandemic. the latest finding that as much as 44% of covid-19 transmission happens before index cases become symptomatic 7 means that a great deal still needs to be learnt about this novel pathogen and its spread through a population. the paucity of knowledge on the dynamics of the immune response to infection has led to much hesitation on recommending the use of rapid immuno diagnostic tests, particularly serology tests. on the basis of our current knowledge and understanding of viral infectivity and host response, we urge countries with restricted capacity for molecular testing to embark on research into the use of serology tests in triaging symptomatic patients in community settings, testing contacts of confirmed cases, and in situational analysis and surveillance. rapid and scalable tests are needed to deal with this pandemic. rapid serology tests, applied in the right situation for appropriate public health measures to be put into place, can make a huge difference. on feb 10, 2020, leading health experts from around the world identified eight research and development priorities at the who r&d blue print meeting in geneva, switzerland, of which the top priority was to "mobilize research on rapid point of care diagnostics for use at the community level". 38 in line with this decision, research on the use of rapid serology tests to inform control programmes of their required performance and utility is an urgent priority in the covid-19 pandemic response. rwp wrote the first draft of the manuscript. all authors contributed to the manuscript conception and supported manuscript revisions. we declare no competing interests. who. molecular assays to diagnose covid-19: summary table of available protocols director-general's opening remarks at the media briefing on covid-19 find evaluation update: sars-cov-2 immunoassays 2020 interpreting diagnostic tests for sars-cov-2 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china evidence summary for covid-19 viral load over course of infection antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 serology characteristics of sars-cov-2 infection since exposure and post symptoms onset profiling early humoral response to diagnose novel coronavirus disease (covid-19) viral kinetics and antibody responses in patients with covid-19 antibody responses to sars-cov-2 in patients with covid-19 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov-2 profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19 advice on the use of point-of-care immunodiagnostic tests for covid-19 advice on the use of point-ofcare immunodiagnostic tests for covid-19 developing antibody tests for sars-cov-2 covid-19 in peru investigation of three clusters of covid-19 in singapore: implications for surveillance and response measures connecting clusters of covid-19: an epidemiological and serological investigation interrupting transmission of covid-19: lessons from containment efforts in singapore covid-19 immunity passports and vaccination certificates: scientific, equitable, and legal challenges what policy makers need to know about covid-19 protective immunity elevated serum igm levels indicate poor outcome in patients with coronavirus disease 2019 pneumonia: a retrospective case-control study how-two-tests-can-help-contain-covid-19-and-revive-the-economy.pdf?sequence=1&isallowed=y world experts and funders set priorities for covid-19 research we thank sergio carmona and jilian sacks of the foundation for innovative new diagnostics for helpful discussions. key: cord-291821-ovfqfurf authors: memish, ziad a; stephens, gwen m; steffen, robert; ahmed, qanta a title: emergence of medicine for mass gatherings: lessons from the hajj date: 2011-12-19 journal: lancet infect dis doi: 10.1016/s1473-3099(11)70337-1 sha: doc_id: 291821 cord_uid: ovfqfurf although definitions of mass gatherings (mg) vary greatly, they consist of large numbers of people attending an event at a specific site for a finite time. examples of mgs include world youth day, the summer and winter olympics, rock concerts, and political rallies. some of the largest mgs are spiritual in nature. among all mgs, the public health issues, associated with the hajj (an annual pilgrimage to mecca, saudi arabia) is clearly the best reported—probably because of its international or even intercontinental implications in terms of the spread of infectious disease. hajj routinely attracts 2·5 million muslims for worship. who's global health initiatives have converged with saudi arabia's efforts to ensure the wellbeing of pilgrims, contain infectious diseases, and reinforce global health security through the management of the hajj. both initiatives emphasise the importance of mg health policies guided by sound evidence and based on experience and the timeliness of calls for a new academic science-based specialty of mg medicine. defi nitions of mass gatherings (mgs) vary greatly, with some sources specifying any gathering to be an mg when more than 1000 individuals attend, whereas others require the attendance of as many as 25 000 people to qualify. 1, 2 irrespective of the defi nition, mgs represent large numbers of people attending an event that is focused at specifi c sites for a fi nite time. these gatherings might be planned or unplanned and recurrent or sporadic. examples of mgs include world youth day, the summer and winter olympics, rock concerts, and political rallies. mgs pose many challenges, such as crowd management, security, and emergency preparedness. stampedes and crush injuries are common, the result of inevitable crowding. outdoor events are associated with complications of exposure, dehydration, sunburn, and heat exhaustion. other health hazards arise from lack of food hygiene, inadequate waste management, and poor sanitation. violence is unpredictable and diffi cult to mitigate whether the mg is a political rally or a sporting competition. with few exceptions, however, the rates of morbidity and mortality resulting from these hazards are rarely increased outside the event. global mgs, however, can lead to global hazards. mitigation of risks requires expertise outside the specialty of acute care medicine, event planning, and venue engineering. for centuries, muslim pilgrims have converged in mecca, saudi arabia, for the hajj (fi gure 1) to participate in a series of sacred rituals that defi ne islam. with about 1·6 billion muslims and the obligation on believers to attend hajj at least once in their lifetimes, this event has become the largest annually recurring mg in the world, with attendance reaching more than 2·5 million in 2009 despite warnings about pandemic infl uenza. pilgrims come from more than 183 countries, leading to enormous diversity in terms of ethnic origin and socioeconomic status. men, women, and children of all ages attend hajj together; however, a disproportionate number of people will be middle aged or older before they can aff ord the journey. comorbidities are common. the public health implications of the hajj are huge-nearly 200 000 pilgrims arrive from low-income countries, many will have had little, if any, pre-hajj health care, added to which are the saudi arabia's safety and security policies for hajj attendees are well developed after decades of planning the annual event. lessons learned have led to comprehensive programmes that are continually revised and coordinated by government sectors. public health has involved global partners for decades. far from being the only mg that aff ects global health, the hajj is a useful model to understand the nature of risk management and the benefi ts of international collaboration and cooperation. pilgrimage is central to many belief systems and also appeals to mankind's recurring desire to be homo viator-a universal fi gure common to many cultures and civilisations, who wanders in search of spiritual enlightenment. in hellenic civilisation, delphi-home to pythia the oracle-was long a focus for pilgrimage. 3 ancient tribal populations such as the huichol of western mexico, the lunda of central africa, and the shona people of southwest africa all included pilgrimage in their cultures. 4 institutionalised pilgrimage came to prominence with the advent of world religions. buddhism invites pilgrimage to nepal, the birthplace of siddharta. hindus journey to benares in india, and followers of judaism to jerusalem. christendom has a complex history of pilgrimages through the ages including the modern era. until the advent of modern air travel, the journey was associated with the greatest risks. a review of the historical data for the hajj shows these dangers: "…the oscillatory movement of the camel produces miscarriages, followed frequently by haemorrhage and death of the infant and mother. the caravan however cannot stop, and it is impossible to nurse effi ciently while the (journey) continues. if any portion of the caravan stopped it would certainly be attacked…" 5 kumbh mela is a huge hindu pilgrimage held at various locations along the river ganges according to the zodiac positions of the sun, moon, and jupiter. purifi cation rites involve bathing in the ganges and are believed to interrupt the cycle of reincarnation. the highest holy days arise every 144 years, but the normal kumbh mela is celebrated every 3 years, and often attract thousands of non-hindu enthusiasts. this is the largest human gathering, so large that in 2001 movements of the amassed individuals could be seen from space. 6, 7 the ardh kumbh mela in 2007 attracted 70 million pilgrims over 45 days in allahabad; on the most auspicious day of the festival, more than 5 million participated. 8 celebrations are accompanied by singing, religious readings, and ritual feeding of holy men and the poor. managing rival sects is a recurring challenge. administrators overseeing the event have to negotiate bathing schedules. clashes have resulted in deaths-eg, in 2010, a vehicle carrying members of the juna sect struck several people, setting off a stampede. 9 in 1954, a stampede killed 500 people. 10 the festival probably contributed to the 1817-24 asiatic cholera pandemic. pilgrims are believed to have carried the bacteria from an endemic area in the lower ganges to populations in the upper ganges, from there to kolkata and mumbai, and across the subcontinent. british soldiers and sailors took it home to europe and then to the far east. 11 the epidemic ended abruptly in 1824 after a very cold winter. although cholera returned to the kumbh mela in 1892, authorities of the hardiwar improvement society reacted to contain the outbreak. 12 diarrhoeal diseases, including cholera, continue to be a risk at the gathering despite rapid monitoring and prompt public health interventions. 13 another pilgrimage with a focus on water and religious rites is to lourdes, france. this village in the pyrenees attracts more than 5 million catholics and other enthusiasts every year. their destination is a shrine and nearby spring where a young village girl witnessed apparitions of the virgin mary in the mid 1800s. drinking and bathing in lourdes' water is believed to ensure health and cure disease, and is featured at the water walk where religious stations are situated and water is available for drinking or bottling. spring water is also routed to a series of bathing stalls used by more than 350 000 pilgrims every year. 14 although health issues have not been associated with lourdes' waters, the french writer emile zola visited the spring in 1891 and provided a graphic description of the baths at the time: "and the water was not exactly inviting. the grotto fathers were afraid that the output of the spring would be insuffi cient, so in those days they had the water in the pools changed just twice a day. as some hundred patients passed through the same water, you can imagine what a horrible slop it was at the end. there was everything in it: threads of blood, sloughed-off skin, scabs, bits of cloth and bandage, an abominable soup of ills...the miracle was that anyone emerged alive from this human slime." 15 stampedes and fi res continue to be major causes of death and injury at mgs-eg, the sabarimala in kerala, india, and the feast of the black nazarene in manila, philippines. inaccessible for 300 years after their construction, hindu temples of sabarimala in kerala's western ghat mountains have become increasingly popular despite the location and winter openings. with the increasing crowd sizes, tragedies have occurred. in 1952, 66 pilgrims burned to death when sheds containing fi reworks caught fi re, and more than 52 perished in 1999 when a hillside collapsed under the weight of 200 000 assembled worshipers triggering a stampede. 16 more than 50 million attended the most recent rites in series january, 2011, uneventful until the last day when a motor vehicle accident caused a panic that triggered a stampede, killing 104 people. 17, 18 although authorities off ered compensation packages, they could not quell unprecedented public criticism of kerala authorities and the national government. 17 manila's feast of the black nazarene has fared a little better after religious leaders and municipal authorities joined forces to change the route of the annual jan 9 procession after two deaths in 2008, and many stampedes and injuries caused by fi reworks and trauma over the years. the authorities responsible for the mg also recruited thousands of volunteers to manage the crowds. these changes and the addition of an information campaign have helped calm crowds and reduce injuries. despite an estimated attendance of 7-8 million in 2011, no deaths or serious injuries were reported. 19 protests during the arab spring in 2011 drew millions of largely peaceful protesters to central locations of tunis, tunisia, and then cairo, egypt. more than 5 million were present when the departure of egypt's president hosni mubarak was announced in february, 2011. other mgs include political protests of the antiwar movement during the vietnam war. 1968 was marked by massive student marches in major european, asian, and latin american capitals. chicago, il, usa, had a particularly violent succession of mgs that became riots after the assassination of the civil rights leader martin luther king and again a few months later during antiwar protests at the democratic national convention. by contrast, european marches in protest of the us-led invasion of iraq were larger and more peaceful. more than 3 million attended the largest march in rome in 2003 (fi gure 2). in 1999, antiglobalisation protesters assembled in seattle, wa, usa, ahead of a scheduled world trade organization meeting. along with international anticorporate interests and assorted domestic supporters, they successfully occupied seattle's downtown core and the convention centre. violence increased during the 5 days, culminating in a full-scale riot after anarchists joined in and police responded with tear gas and rubber bullets. the battle in seattle as it came to be known, caused damages that were estimated at more than us$3 billion. despite the violence and very large crowds, estimated to be hundreds of 23, 24 violent sports fans are as old as history. in 532, the nika riots in constantinople pitted rival charioteer factions and athletes against each other and emperor justinian. during the 1 month insurrection that ensued, half the city was destroyed and more than 30 000 people died. 25 although sports violence continues to be a risk during matches between rival teams, the massive crowds, crowds in motion, and immovable barriers cause the greatest loss of lives. the worst sports riot in history occurred in south america during a 1964 football playoff game between peru and argentina when fans responded in protest after a controversial decision to annul a goal by peru. police responded by throwing teargas canisters into the grandstand. more than 500 fans were injured and another 318 died. most were crushed trying to escape the locked stadium, others died from teargas asphyxiation. the disaster in hillsborough, uk, in 1989 was the worst stadium tragedy in british history. 96 fans died and another 766 were injured as crowds surged into the stadium crushing others in front who were pinned against fences. many of the deaths resulted from compressive asphyxia while standing. ineff ective crowd control and poorly designed venues have also resulted in deaths at music festivals, most recently in 2010 during the love parade in duisburg, germany, in which 21 people were crushed to death and 500 were injured as a result of a stampede in a narrow tunnel. occasionally, mgs cause structural stresses that threaten safety and security. in 1987, the 50th anniversary of the golden gate bridge, san francisco, ca, usa, was celebrated by closing it to vehicular traffi c. though not catastrophic, the suspension cables had the greatest load factor ever when 500 000 pedestrians crowded onto the deck, fl attening its centre span. 26 although the hajj was undertaken in the middle east before the arrival of islam, the movements and rituals of pilgrims today have not changed since the prophet mohammad inaugurated the islamic hajj in his lifetime. 27 it has been recorded in arabic literature known as adab al rihla. persian literature records hajj in the safarnameh (travel letter). at the core of islamic belief is trust and this trust has been best exemplifi ed by the risks muslims take when travelling. the muslim individual must trust in his maker and, in ancient times, in the benevolence of strangers who would host him on his perilous journey to mecca. nowadays, as a result of the dissemination of islam across the world, hajj removes national, cultural, and social boundaries between diverse people like no other event. hajj has been the focus of public health initiatives for centuries, as shown in contemporary medical reports. [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] during the 19th century, the hajj attracted the interest of european powers, particularly the maritime travel to the hajj, which dominated until the arrival of air travel. colonial powers at the time were suspicious of political islam, which was referred to as wahabism. direct engagement in hajj-related aff airs was seen as too intrusive by politically savvy imperialists who recognised the sanctity of this little understood religious pilgrimage. instead, supervision, albeit series displaced, and management of hajj were gradual processes, including surveillance, regulation, secure passage through the red sea and protection of british littoral interests, and eventually formal organisational processes, which would quickly become central to these hidden concerns. imperial organisations linked cholera morbus, a non-epidemic diarrhoea, to hajj, allowing a public health industry to develop that used health concerns to control immigration, pilgrim passports, proof of suffi cient funds to allow return travel, maritime regulation, and vessel quarantine procedures. by the mid 19th century, most of the muslim populations using maritime travel for hajj were from the malay peninsula and indian subcontinent. about 2000 pilgrims travelled from the malay peninsula and between 5000 and 7000 arrived from the indian subcontinent. although there are few reliable data, the total number of pilgrims was estimated to be 10 000. 38 "according to the turko-egyptian sanitary commissioners at mecca, the number of mohammedan pilgrims collected in and about the holy city…amounted to two hundred thousand persons; composed of natives of turkey, india, egypt, morocco, arabia, syria, persia, java etc." 29 most travellers came in small vessels of 100-300 tons under diff erent international aegis. departures were concentrated around singapore, calcutta and madras in india, aceh in indonesia, and other regional cities. most pilgrims then, like today, disembarked in jeddah, though some would land on southern arabian coastal ports and then make a land journey through yemen to hijaz. well into the 20th century, the conditions of passage were often appallingly cramped and unsanitary. 34 many people died along the route from infection and dehydration. 83 pilgrims died on board a maritime vessel, which had embarked from jeddah with 520 pilgrims en route home to singapore. 34 "when she drew abreast of the watcher she proved to be a pilgrim ship; the afternoon being hot, the travellers had all crowded to the port side to catch what little wind was stirring. their numbers were so great that they appeared to cover all the deck space, while the ship was unable to right herself from the list…" 34 eff orts to manage hajj were initiated by dutch-indonesian authorities, not for wholly altruistic reasons. the dutch had established an association between returning pilgrims and societal unrest, so they introduced heavily surcharged passports as a way of restricting the number of travellers to mecca. the ruling empires focused on health issues and justifi ed inspections of hajj sites for compliance with contemporary public health directives, often focusing on quarantine as a means of protection at a time when many international arrivals, including maritime travellers, were reaching mecca. their inspections were disappointing-the annual sanitary commission visited the sites of hajj and noted that the focus was not on prevention, but rather on the easy option of quarantine. 37 when cholera was reported at hagar's well within the holy mosque in mecca, the british consul at jeddah requested a scientifi c assessment. samples were analysed at the royal college of chemistry in the south kensington museum, london, uk, and compared with those of london sewage, which was a source of cholera at that time. recommendations after their alarming fi ndings were sent to the secretary of state for india who reported the well to be infected with the bacterium. 35, 36 similarly, entrepôt cholérique (cholera reservoir) was noted when authorities visited pilgrims from india intending to do the hajj. these pilgrims were routinely detained on the island of camaran as a quarantine station in the red sea to restrict the ingress of cholera into the holy sites. 37, 38 pilgrims were detained for 5-10 days without adequate provisions or clean water. the long exposure to sun, however, was thought to be benefi cial for elimination of infection. after quarantine, pilgrims were often permitted into the site. results of later studies showed a link between the pilgrims quarantined on camaran with a series of eight subsequent outbreaks. the conclusions drawn from a review of these events at an international public health meeting at the international sanitary conference of paris, france, 1895, were that the "turkish possession of camaran remains the greatest hindrance to the abolition of cholera at mecca". 37 infection was a frequent feature of the hajj in the 19th and 20th centuries, not unexpected since infectious disease medicine became better elucidated and the fascination with the developing specialty increased. epidemics of smallpox occurred in iraq and sudan between october, 1928, and april, 1929. a small epidemic of plague occurred pamela das www.thelancet.com/infection vol 12 january 2012 series in upper egypt and a larger one in morocco (161 cases). 34 653 cases of typhus were reported in egypt and 32 in palestine during the same period. 34 these fi ndings led to some strong recommendations that are still relevant: "the yearly pilgrimage will remain a danger to all the countries from which pilgrims are drawn as long as the conditions of transport and accommodation remain…as at present. effi cient reorganization of the pilgrimage in every direction is needed and should be facilitated by the governments of the large number of the countries involved." 34 by the early 20th century, non-muslim european powers were heavily engaged in the management of the hajj and would remain so until modern saudi arabia came into existence and acquired fi nancial independence through petrochemical wealth. the comparison of hajj in the imperial era with the modern hajj shows the absence of muslim public health experts or authorities in managing this pilgrimage. 39, 40 this absence would gradually change and with the arrival of ibn saud's modern kingdom and its investments in hajj. from this point, muslims would solely administer the modern hajj in its entirety. 41, 42 the islamic calendar is a lunar calendar, so the date of the hajj moves forward by 10-11 days every year, presenting planners with additional challenges of health risks that are associated with seasonal variation. temperature fl uctuations in mecca might be extreme depending on the time of year; daytime highs can be 40°c and higher, and night-time temperatures occasionally fall to 10°c. hajj can coincide with the northern hemisphere's infl uenza season, as in 2009, increasing public health risks. [43] [44] [45] [46] [47] attendance in 2009 was not blunted despite offi cial recommendations encouraging pregnant women, and elderly and very young people to stay at home. 48 more than 2·5 million people attended, including 1·6 million foreign citizens, 753 000 of whom did not have valid hajj permits. 49 to put the event in its local context, the infl ux of pilgrims is so great that it trebles the resident population of mecca, which is normally 1·4 million. access to the hajj for pilgrims has changed greatly with air travel gradually replacing maritime and overland travel. in the past decade, the breakdown includes about 92% of pilgrims arriving by air, 1% making the maritime journey, and 7% travelling over land. 50 although a few pilgrims will arrive at medina's international airport, jeddah remains the major port of entry for all travellers as it has been for centuries. increasing numbers of people attending the modern hajj led to a 1980 decision by saudi aviation authorities to partition jeddah's king abdulaziz international airport and create a separate south terminal to serve all pilgrims. now two-thirds completed, the terminal's capacity is 80 000 travellers at any time. when completed, its fi nal capacity will be greater than 30 million passengers per year. important new features include health-screening systems, customs, and immigrations security. each of its 18 hubs receives pilgrim fl ights; all hubs have two examination rooms. the terminal also features large holding areas that allow effi cient reviews of selected arrivals in segregated parts of the terminal. this permits verifi cation of the immunisation status and administration of any prophylactic drugs and vaccines according to set protocols. the overall design of the terminal permits visitors arriving without required visas and health records to be managed outside the main fl ow of pilgrims who continue through the facility to join assigned groups or agents who are responsible for coordinating details of travel and housing. these regulated services will also escort their charges through the hajj site. in islam, umrah is a shorter pilgrimage to mecca. although not compulsory, umrah draws an additional 5 million pilgrims per year to the country; jeddah's airport plays a major part throughout the year, controlling access and enforcing health protocols. groups exiting the country and returning home are also monitored, allowing comparative studies between the two populations. at various times of the year, but most intensely during the hajj season, public health teams, both stationary and mobile, use mobile devices to monitor inbound and outbound populations. protocols are based on regularly reviewed case defi nitions. gathered data are sent to centralised databases for real-time analysis. many diseases are monitored during a hajj season. those given specifi c attention every year include both mild and severe respiratory diseases, food poisoning and gastroenteritis syndromes, haemorrhagic fevers, and meningococcal diseases. reports of all diseases, but particularly those with immediate eff ect worldwidesevere acute respiratory syndrome (sars), infl uenza, cholera, yellow fever, polio, plague, meningitis, and viral haemorrhagic syndromes-are expedited to who epidemiologists who work closely with saudi authorities reuters/jim young series to analyse information and coordinate a response. the airport is also equipped with clinics for management of medical problems. humility, faith, and unity are emphasised throughout the hajj. the pilgrims wear simple clothing, women and men comingle, women are enjoined not to cover their faces, children and adults of all ages are included, and families journey together. on arrival in mecca, hajj pilgrims do a series of synchronised acts based on events in the lives of ibrahim (abraham), his wife hajra (hagar), and their son ishmael. each pilgrim does an initial circumambulation (tawaf ) around the central ka'aba seven times. when completed, the pilgrim leaves for arafat, about 22 km east of mecca. hajj culminates in arafat on the day of standing, when all 2·5-3·0 million visitors stand and supplicate together on the mountain. mount arafat is believed to be the site of mohammad's last sermon to his followers. many people attempt to pray at the summit believing prayers there are the most blessed. on the way to arafat, the pilgrims make overnight stops for prayers and contemplation in mina. leaving arafat, the pilgrims return to muzdaliff ah, where stones are gathered; on the way to mina, they stop at jamarat bridge to throw stones at the pillars that are effi gies of satan. when the pilgrimage is complete, the new hajjee (pilgrim who has completed the hajj) makes an animal sacrifi ce thanking allah for accepting his hajj. this is often a proxy sacrifi ce because the saudi government has established modern abattoirs that are staff ed by professionals who will do this on behalf of the pilgrims. meat is then distributed to the poor, family, and friends. the fi nal farewell is undertaken with another seven circuits around the ka'aba. muslim men on completion of a successful hajj shave their heads. after completion of the hajj, most pilgrims exit the country at jeddah airport, which has congestion so great that the telecommunications infrastructure has to be constantly updated to allow suffi cient capacity. a smaller number of pilgrims will visit the holy mosque in medina. some will also visit tourist sites in the hijaz and the old city of jeddah. because all hajj pilgrims travel as part of small informal groups, there is order in what could otherwise be chaos. groups take their shepherding of individual pilgrims seriously, with easily identifi ed group leaders who carry placards and fl ags and lead the entire group through the rituals without losing stragglers, infi rm individuals, or temporarily distracted people. further, this fl exibility safeguards hajj at the most pressured points, which could otherwise become treacherous. despite this fl exibility, hajj stampedes have been recurring events, most notably at the jamarat site. 42 according to islam, only adults should undertake the hajj. the age at which hajj is undertaken varies according to culture. some nationalities seem to undertake hajj at a uniformly young age (eg, indonesian and malaysian), whereas other nationalities defer hajj until the late phase of life as a precursor to preparing for death. there might also be diff erences in sex distribution. malaysia for instance has had a female dominated hajj attendance for more than three decades. 42 in keeping with the islamic spirit of compassion, muslims are enjoined to undertake hajj only when adequately healthy. despite this strong scriptural admonition, many muslims insist on hajj even when wheelchair bound. special accommodations for wheelchairs are provided at the holy mosque despite the tremendous crowd densities. these channels are wide enough to admit wheelchairs and one person pushing the wheelchair and are divided into two lanes (one for each direction). pilgrims who are not well are provided transport by the ministry of health ambulance to hajj sites as needed so they can complete their pilgrimage. because of the islamic belief that death during the hajj has a benefi cial outcome in the afterlife, a few sick pilgrims attend, hoping for death during the hajj. public health and religious offi cials do much to dissuade this belief, which is often tenacious. this cultural belief system aff ects care providers at hajj, all of whom are muslims (non-muslims are not permitted to enter the holy sites). anecdotally, this belief aff ects resuscitation eff orts of those in cardiac arrest, which once initiated (if the patient reaches the emergency rescue services in time) are unlikely to be pursued if not immediately successful. a do-not-resuscitate status is often requested by pilgrims who can speak for themselves. 51 hajj itself has several qualities that aid public health security. 52 attendees must practise specifi c behaviours for their hajj to be considered valid, and these requirements are strict and closely adhered to by both clerical and community leaders. crime is strictly forbidden at hajj and the risk of violent altercation is reduced because of the weapon-free, drug-free, and alcohol-free environment. 42 tobacco intake is also banned, curtailing the risk of inadvertent fi re hazards. by contrast with some other mgs, sexual relations are not allowed during hajj and male and female pilgrims are accommodated separately even when travelling as families, eliminating the risk of sexually transmitted disease. this observant, penitent, and sober crowd engrossed in worship is thus likely to remain cooperative and coherent if sudden events demand rapid cooperation with authorities. insurrection, rioting, disinhibited behaviour, or hooliganism of any kind does not arise even in these extraordinarily massive crowds. pilgrims are urged to safeguard themselves or others at all times, aiding the infi rm and assisting the fallen, behaviours that symbolise peaceful islamic societies that enhance the public health security. the spirit of cooperation is central to a successful series acceptance of the hajj by allah in the islamic belief system and reduces the potential risk of disastrous events in such massive crowds. saudi arabia's responsibility for the hajj has aff ected the country's advanced health-care infrastructure and its multinational approach to public health. although other jurisdictions have administered the hajj, saudi arabia has invested in it. within the immediate vicinity of the hajj, there are 141 primary health-care centres and 24 hospitals with a total capacity of 4964 beds including 547 beds for critical care. the latest emergency management medical systems were installed in 136 healthcare centres and staff ed with 17 609 specialised personnel. more than 15 000 doctors and nurses provide services, all at no charge. this event requires the planning and coordination of all government sectors; as one hajj ends, planning for the next begins. infection and prevention strategies are reviewed, assessed, and revised every year. coordination and planning requires the eff orts of 24 supervising committees, all reporting to the minister of health. the preventive medicine committee oversees all key public health and preventive matters during the hajj and supervises staff working at all ports of entry. public health teams distributed throughout the hajj site are the operational eyes and ears of the policy planners. in hosting the modern hajj, saudi arabia has weathered a 20th century world war, global outbreaks due to newly emerging disease (including sars and meningococcal meningitis w135), and regional confl icts. in this time, the country has acquired a unique, resilient expertise concerning hajj-related public health. important observations that are relevant to public health planners everywhere are part of this experience. one of the best examples of such cross-cultural translation has been in the preparation for barack obama's presidential inauguration and crowd management informed by the hajj experience. yet the process of exchanging expertise is possibly even more instructive. collaborative work on this scale shows the increasingly important global health diplomacy in which the muslim world has an enormous part to play. first articulated by the us health and human services secretary tommy thompson, global health diplomacy usually includes the provision of a service by one nation to another. 6 the usa's rebuilding of maternity hospitals in afghanistan or the deployment of the ship uss comfort to serve as a site for temporary clinics in vietnamese coastal waters are two recent examples. 53 as they struggled with the best responses to the global threat of pandemic infl uenza a h1n1, which coincided with the hajj in 2009, colleagues at the us centers for disease control and prevention and the saudi ministry of health worked together to deploy one of the largest real-time mobile databasing systems, which was designed to detect disease in real time at any mg. senator john kerry discussed precisely this joint eff ort in a speech in doha at the 2010 us-islamic world forum. 54, 55 this international collaboration was realised only through both intense personal dedication and the confi dence the agencies had in their people. such collaboration strongly resonates with president obama's renewed hopes for us engagement with the muslim world, as articulated in his speech in cairo, egypt, in june, 2009. 54 people who collaborate, write, and disseminate information internationally have long been aware of the latent value of such informal, positive exchange. in the fl at world of medical academia, individuals have immediate and palpable eff ects. fostering such professional dialogues are everyday (albeit unseen) acts of global health diplomacy. when investigators and physicians work in a shared space, unfettered by the global geopolitics, global health diplomacy becomes alive and vibrant. hajj medicine, as part of the emerging specialty of mg medicine, provides an extraordinary platform. saudi arabia's experience in international service through public health is substantial and is promoting the emergence of the formalised specialty of mg medicine. hajj continues to provide insights into advanced and complex public health challenges, which are unlocked through collaborative exchange. 56 disease and suff ering remain universal, even in the 21st century. solving these challenges is relevant to humanity everywhere. islamic scholars have long referred to hajj as a metaphor for ideal societal behaviour. 42 at the centre of these ideals is a unifying theme: collaboration. saudi arabia's experience of hajj medicine contains rapidly developing public health solutions to several global challenges. multiagency and multinational approaches to public health challenges are likely to become major factors in the specialty of global health diplomacy, engaging societies globally, and drawing the west a little closer to the east. in view of the global public health threats that might originate from mgs, medicine relevant to mgs has become an essential specialised, interdisciplinary branch of public health, particularly hybridised with global health response, travel medicine, and emergency or disaster planning. 52 agencies outside the realm of public health should be closely involved in mg medicine. in the operation and management of an mg, several sectorshealth care, security, and public communications-need to know how to interface with public health services and resources quickly and eff ectively. involving public health experts with the broader civic planning for any mg helps with parallel transparency in needs and expectations, ensuring that public health considerations are factored into the entire planning process instead of intruding too late in development, relegating public health security series concerns to little more than ineff ective afterthought. delayed entry of these actors into the planning process can debilitate or completely disable adequate responses to potential diseases during mgs. experts must educate civic planners about the values of early collaborative approaches to mgs for these reasons. conventional concepts of disease and crowd control do not adequately address the complexity of mgs. the need for mg health policies that are guided by sound evidence but anchored in experience shows the importance of calls for a new academic medical and science-based discipline. mgs have been associated with death and destructioncatastrophic stampedes, collapse of venues, crowd violence, and damage to political and commercial infrastructure, but little is known about the threats from mgs to the global health security. who has worked closely with international agencies to address such risks. [57] [58] [59] mgs pose complex challenges that require a broad expertise and saudi arabia has the experience and infrastructure to provide unique expertise with respect to mgs. zam and gms co-wrote the text. imperial powers and 19th century hajj, hajj culture, and most of the global health diplomacy sections were contributed by qaa. rs compiled the table. we declare that we have no confl icts of interests. we identifi ed references for this review by searching medline and the national health service hospital search service for articles published in english from 1880 to august, 2011. additional articles were identifi ed through searches of extensive fi les belonging to the authors. search terms used were "mass gathering", "disease", "pilgrimage", "hajj", "outbreak", "public health", "prevention", "travel", or "modeling". we reviewed the articles found during these searches and relevant references cited in the articles. mass gathering medicine: a predictive model for patient presentation and transport rates mass gathering medical care: a twenty-fi ve year review from medieval pilgrimage to religious tourism: the social and cultural economics of piety the lancet. a mohamedan doctor on the mecca pilgrimage kumbh mela pictured from space millions of hindus wash away their sins five die in stampede at hindu bathing festival what is hinduism?: modern adventures into a profound global faith epidemics and pandemics: their impacts on human history use of telemedicine in evading cholera outbreak in mahakumbh mela the baths lourdes: body and spirit in the secular age another black friday for sabarimala pilgrims sabarimala stampede death toll crosses 100 sabarimala stampede, 50 injured list of largest peaceful gatherings in history promed mail. varicella, asian games-qatar ex maldives centennial olympic park bombing summer olympics procopius: justinian suppresses the nika revolt muhammad: a biography of the prophet return pilgrims from mecca. egyptian quarantine at torr. (from a correspondent) the pilgrimage to mecca: medical care of pilgrims from the sudan the lancet. the origin of cholera in mecca cholera at mecca and quarantine in egypt the lancet. the risks of the mecca pilgrimage the lancet. the mecca pilgrimage the cholera and hagar's well at mecca hagar's well at mecca camaran: the cause of cholera to mecca pilgrims sanitation and security: the imperial powers and the nineteenth century hajj mecca pilgrimage quarantine and the mecca pilgrimage-the growth of an idea the lancet. a medico-sanitary pilgrimage to mecca the lancet. the pilgrimage to mecca guests of god pilgrimage and politics in the islamic world pandemic h1n1 infl uenza at the 2009 hajj: understanding the unexpectedly low h1n1 burden global public health implications of a mass gathering in mecca, saudi arabia during the midst of an infl uenza pandemic infl uenza a (h1n1) in the kingdom of saudi arabia: description of the fi rst one hundred cases and the jeddah hajj consultancy group. establishment of public health security in saudi arabia for the 2009 hajj in response to pandemic infl uenza a h1n1 pandemic h1n1 and the 2009 hajj health conditions for travellers to saudi arabia for the pilgrimage to mecca (hajj) royal embassy of saudi arabia. 2,521,000 million pilgrims participated in hajj 1430 global public health implications of a mass gathering in mecca, saudi arabia during the midst of an infl uenza pandemic emergency room to the intensive care unit in hajj. the chain of life the quest for public health security at hajj: the who guidelines on communicable disease alert and response during mass gatherings citation?related-urls=yes&-legid=healthaff remarks by the president on a new beginning. www.whitehouse. gov/the_press_offi ce/remarks-by-the-president-at-cairo-university-6-04-09 chairman kerry addresses the us-islamic world forum jeddah declaration on mass gatherings health international health regulations who. communicable disease alert and response for mass gatherings: key considerations we thank abdullah a al rabeeah, the saudi minister of health, for his leadership and support for hosting the lancet conference on mg medicine: implications and opportunities for global health security, jeddah, saudi arabia, oct 23-25, 2010, which generated the series of reviews. key: cord-278256-dmrtsxik authors: qiu, haiyan; wu, junhua; hong, liang; luo, yunling; song, qifa; chen, dong title: clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study date: 2020-03-25 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30198-5 sha: doc_id: 278256 cord_uid: dmrtsxik background: since december, 2019, an outbreak of coronavirus disease 2019 (covid-19) has spread globally. little is known about the epidemiological and clinical features of paediatric patients with covid-19. methods: we retrospectively retrieved data for paediatric patients (aged 0–16 years) with confirmed covid-19 from electronic medical records in three hospitals in zhejiang, china. we recorded patients' epidemiological and clinical features. findings: from jan 17 to march 1, 2020, 36 children (mean age 8·3 [sd 3·5] years) were identified to be infected with severe acute respiratory syndrome coronavirus 2. the route of transmission was by close contact with family members (32 [89%]) or a history of exposure to the epidemic area (12 [33%]); eight (22%) patients had both exposures. 19 (53%) patients had moderate clinical type with pneumonia; 17 (47%) had mild clinical type and either were asymptomatic (ten [28%]) or had acute upper respiratory symptoms (seven [19%]). common symptoms on admission were fever (13 [36%]) and dry cough (seven [19%]). of those with fever, four (11%) had a body temperature of 38·5°c or higher, and nine (25%) had a body temperature of 37·5–38·5°c. typical abnormal laboratory findings were elevated creatine kinase mb (11 [31%]), decreased lymphocytes (11 [31%]), leucopenia (seven [19%]), and elevated procalcitonin (six [17%]). besides radiographic presentations, variables that were associated significantly with severity of covid-19 were decreased lymphocytes, elevated body temperature, and high levels of procalcitonin, d-dimer, and creatine kinase mb. all children received interferon alfa by aerosolisation twice a day, 14 (39%) received lopinavir–ritonavir syrup twice a day, and six (17%) needed oxygen inhalation. mean time in hospital was 14 (sd 3) days. by feb 28, 2020, all patients were cured. interpretation: although all paediatric patients in our cohort had mild or moderate type of covid-19, the large proportion of asymptomatic children indicates the difficulty in identifying paediatric patients who do not have clear epidemiological information, leading to a dangerous situation in community-acquired infections. funding: ningbo clinical research center for children's health and diseases, ningbo reproductive medicine centre, and key scientific and technological innovation projects of wenzhou. coronaviruses are non-segmented positive-stranded rna viruses with a roughly 30 kb genome surrounded by a protein envelope. most coronaviruses cause diseases in their particular host species; 1 those that can infect humans through cross-species transmission have become an important threat to public health. two serious coronavirus disease outbreaks have happened in the past two decades: severe acute respiratory syndrome (sars) in 2003, 2 and middle east respiratory syndrome (mers) in 2012. 3 since december, 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been recognised as the causal factor in a series of severe cases of pneumonia originating in wuhan in hubei province, china. 4 this disease has been named coronavirus disease 2019 (covid-19) by who. sars-cov-2 has been shown to cause disease via a mechanism analogous to the sars coronavirus, with potential damage to vital organs such as lung, heart, liver, and kidney, and infection poses a considerable risk to patients by the high prevalence of pneumonia. 5 by march 1, 2020, sars-cov-2 had led to 80 981 confirmed cases of covid-19 in china, and 44 067 cases in 117 countries. 6 a cohort study of 44 672 chinese cases reported that 2·1% of patients were younger than 20 years, and 1·2% were asymptomatic. 7 pneumonia is the leading cause of mortality in children. 8 distinct immunological responses to viral infections can exist in children and result in severe damage to vital organs. 9 although short-to-medium-term outcomes for children who recovered from sars seemed good, the disease caused exercise impairment after more than 6 months. 10 a comprehensive literature search indicated that mers was rare in children (31 cases identified between 2012 and 2016), 11 because transmission depends heavily on animal exposure and direct contact in the household or health-care facilities. collectively, these two coronavirus diseases did not widely affect children because of the short-term epidemic of sars and the strict transmission route of mers. in a study of 15 children with covid-19 (aged 4-14 years), six patients had small nodular ground-glass opacities in the lungs. 12 at the time of the covid-19 outbreak, all schools were on the spring festival holiday, which might have prevented children from exposure to transmission sources. however, the school community is a place that can enhance rapid spread of the highly infectious sars-cov-2. ningbo and wenzhou are two cities in zhejiang province, located 900 km east of wuhan. on jan 17, 2020, the first covid-19 case was reported in wenzhou. up to march 1, 2020, 661 cases of covid-19 had been reported in ningbo and wenzhou, of which 36 were in children. we aimed to describe the epidemiological and clinical features of these paediatric patients, information that is urgently needed for prevention and treatment of covid-19 in children. we did an observational cohort study at three hospitals in zhejiang province, china (ningbo women and children's hospital, the third affiliated hospital of wenzhou medical university, and wenzhou central hospital of wenzhou). we included all paediatric patients (aged 0-16 years) with laboratory confirmed covid-19. the diagnosis of covid-19 was based on guidelines issued by the national health commission of the people's republic of china. 13 we screened all patients who had cough, fever, and radiographic presentation at the initial assessment, or who underwent epidemiological investigation because of a history of exposure to epidemic areas or close contact with an infected individual. we confirmed sars-cov-2 infection by rt-pcr of samples taken from upper nasopharyngeal swabs. epide miological investigation was focused on the route of transmission, and we assessed travel history, residence in epidemic areas, and close contact with patients with confirmed covid-19 within 14 days. this study was approved by the ethics committee of ningbo women and children's hospital and followed the declaration of helsinki. written consent was obtained from the guardians of the patients. sample collection, rt-pcr, and interpretation of results was done as previously described. 14 15 on admission, patients were assessed for clinical type, according to guidelines for scoring paediatric patients with covid-19 (recommendations issued by the paediatrics branch of the chinese medical association; panel 2). 16 the therapeutic principles included general support therapy; monitoring of lung, liver, kidney, and myocardial functions; active control over high fever; oxygen uptake if necessary; and antiviral treatment with interferon alfa and lopinavir-ritonavir. treatment evidence before this study we searched pubmed from nov 1, 2019, to march 1, 2020, for studies published in any language using the terms "covid-19", "coronavirus disease 2019", "novel coronavirus", "pediatric patients", "children", "transmission", "clinical feature", and "epidemiological features". we found 13 studies about coronavirus disease 2019 (covid-19) in children. these studies were related to treatment recommendations, ct features in 15 children, case reports, and covid-19 in infants. to our knowledge, no study has been done to comprehensively investigate a cohort of paediatric patients with covid-19 and their distinctive clinical features. published work about adult patients has shown that manifestations vary strikingly by individual. since children are different from adults in many aspects, such as immunological development, we aimed to investigate the clinical and epidemiological features of paediatric patients with covid-19. between jan 17 and march 1, 2020, 661 cases of covid-19 were reported in ningbo and wenzhou (zhejiang province, china). of these cases, 36 were in children. all children with covid-19 had been infected either by close contact with adults infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) or by exposure to the epidemic area. although fever, cough, and pneumonia were the most common signs, about half the children had mild disease with no presenting symptoms. fewer children with covid-19 had obvious symptoms compared with adult patients with covid-19 and paediatric patients with h1n1 influenza described in previous studies. by contrast with findings in adults, children with covid-19 had milder clinical manifestations; nearly half of paediatric patients were asymptomatic (ie, no fever and no cough). this asymptomatic condition is relevant if community-acquired transmission becomes the primary mode; identification of paediatric patients without presenting symptoms will become a great challenge. fortunately, the number of children infected with sars-cov-2 accounted for a small proportion of total people infected, and paediatric patients also had clear epidemiological information. devising measures to protect children from infection with sars-cov-2 is very important. outcomes were defined as improved, cured, and failed. improved outcome referred to the end of fever and improve ment in pneumonia (via ct scan) and improvement in upper respiratory manifestations. cured outcome referred to normal body temperature for 3 days, plus improved outcomes and two negative results on rt-pcr for sars-cov-2. failed outcome referred to disease progression to critical illness or death. patients who were discharged from hospital had to be quarantined for 2 weeks. we obtained data retrospectively from electronic medical records, and these were reviewed by two trained doctors (lh and yl). information retrieved included demographic data, medical history, exposure history, underlying comorbidities, symptoms, laboratory examina tions, chest ct scans, and treatment. laboratory examinations included routine testing, analysis of immuno logical responses, and measurement of bio markers for monitoring lung, liver, myocardial, and renal functions. we compared the prevalence of abnormal clinical indices of the paediatric patients with previously reported data for 175 adults with covid-19 in wenzhou, 17 we presented continuous variables as mean (sd) and categorical variables as number (%). we compared means of continuous variables between groups using independent group t tests when values were normally distributed; otherwise, we used the mann-whitney u test. we compared proportions for categorical variables bet ween groups using fisher's exact test, because of limited numbers. we judged a two-sided α less than 0·05 statistically significant. all data analyses were done with spss version 20. 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 data in the study and had final responsibility for the decision to submit for publication. generally, two types of abnormal radiographic presentations were seen: multiple opacities and patchy opacities. 19 (53%) paediatric patients had pulmonary ground-glass opacities on ct scan, suggesting pneu monia (figure 1). pneumonia plus either fever or cough was seen in 11 (30%) paediatric patients and eight (22%) had pneumonia as the only symptom. ten (28%) children showed no symptoms of pneumonia, five (14%) reported fever only, and two (5%) only had a dry cough. with moderate clinical type also had a positive pcr result for longer (p=0·0050) and spent more days in hospital (p=0·017) than did those with mild clinical type. table 2 shows findings of laboratory examinations related to immunological responses and cardiac, liver, and renal damage, according to age group. ten patients were aged 5 years or younger and 26 patients were 16 years or younger but older than 5 years. the older children had decreased lymphocytes (p=0·029), elevated procalcitonin (p=0·032), and decreased creatine kinase (p=0·032) compared with the younger patients. treatments and primary manifestations were traced to assess disease progression and outcome, and four case types were identified: asymptomatic, upper respiratory symptoms or fever only, pneumonia only, and both pneumonia and upper respiratory symptoms or fever (figure 2). on admission, all children were treated with interferon alfa by aerosolisation twice a day, 14 (39%) received lopinavir-ritonavir syrup twice a day, and six (17%) needed oxygen inhalation (table 1). in the 13 patients with fever, mean duration of fever was 3 (sd 2) days. improve ment in pneumonia was seen 4-10 days after treatment initiation. sars-cov-2 rt-pcr results became negative after a mean of 10 (sd 2) days of treatment, regardless of the various initial manifestations of patients (figure 2). the mean number of days in hospital was 14 (sd 3) days. by feb 28, 2020, all patients were cured, according to the criteria for cured outcome, and quarantined for a further 2 weeks. follow-up is continuing every week, with samples taken to measure serum igg and igm and sars-cov-2 in blood, faeces, and nasopharyngeal swabs. the comparison of paediatric patients with covid-19 with adult patients with covid-19 in the same city (table 3) showed that paediatric patients had a significantly lower prevalence of abnormal values of several variables indicating disease severity, such as fever (36% for children and 86% for adults), cough (19% and 62%), pneumonia (53% and 95%), elevated c-reactive protein (3% and 49%), and severe disease type (0% and 23%; p<0·0001 for all features). nevertheless, no differences were noted between children and adults in prevalence of leucopenia, lymphopenia, and elevated myocardial enzymes. when compared with children with sars, paediatric patients with covid-19 had much milder disease in terms of the prevalence of fever, cough, pneumonia, and severe case type. compared with children with h1n1 influenza, paediatric patients with covid-19 had fewer upper respiratory symptoms (eg, cough and pharyngeal congestion) but pneumonia was more frequent. notably, when the three groups of patients with coronavirus infections were compared with the paediatric patients with h1n1 influenza, the most striking difference was that patients with h1n1 influenza had a much higher prevalence of pharyngeal congestion and a lower prevalence of pneumonia. history of exposure to epidemic area 2 (20%) 10 (38%) .. family members with covid-19 10 (100%) 22 (85%) 0·60 dry cough 1 (10%) 6 (23%) .. body temperature, °c 37·7 (0·3) 37·9 (0·4) 0·16 fever (body temperature >37°c) 3 (30%) 10 (38%) .. pulmonary ground-glass opacities 6 (60%) 13 (50%) 0·70 white blood cells ( unclear routes of transmission, were identified. this transmission feature means that identification of these paediatric patients was straightforward. however, previous work has shown that potential transmission routes of sars-cov-2 can be highly variable, 20 compared with mers coronavirus, which is less transmissible. 11 this situation could be altered if covid-19 persists for a long time in an area with increased risk for communityacquired infections. among the paediatric patients in our study, just under a third were asymptomatic. moreover, a fifth had pneumonia only and needed radiographic examination to be identified. symptoms seen on admission were variable, including cough and fever. all asymptomatic patients and children who did not present with pneu monia needed laboratory and radiographic examinations for diagnosis. the large proportion of children with asymptomatic sars-cov-2 infection contrasts with paediatric patients with h1n1 influenza 19 and sars, 10 and adult patients with 17 in terms of presenting symptoms such as cough and pharyngeal congestion (table 3) , indicating sars-cov-2 has little effect on the upper respiratory tract of children. covid-19 is highly transmissible, similar to sars and h1n1 influenza, but can have a covert presentation in children. pneumonia is the leading infectious cause of death in children younger than 5 years. 21 although covid-19 in children seemed to be mild in term of presenting symptoms, the prevalence of pneumonia with covid-19 (53%) was higher than with h1n1 influenza (11%), but very similar to the prevalence with sars (65%). 18 a striking characteristic of covid-19 is that it affects several vital organs (eg, lungs and heart), as shown by increased amounts of myocardial enzymes, even though all children with covid-19 had the mild or moderate clinical type (table 3) . with high mortality in adult patients with covid-19 in epidemic areas 6 (eg, >4% mortality in wuhan), paediatric patients should continue to be monitored after discharge from hospital. compared with adults, children rarely have comorbidities such as hypertension, cardiovascular disease, and diabetes. paediatric patients in our study had a lower prevalence of pneumonia and symptoms such as fever, cough, and dyspnoea compared with adult patients with covid-19 (table 3) . however, in our study, the prevalence of leucopenia, lymphopenia, and increased myocardial enzymes in children with covid-19 was similar to that in adults. notably, adults have a much higher prevalence of increased c-reactive protein than do children, suggesting a much milder immunological response in children and less immune damage. 22 except for antiviral drugs, no special treatments (eg, glucocorticoid therapy and invasive mechanical ventilation) were administered, and few children needed inhaled oxygen. taking all these findings into con sideration, the presentation of covid-19 in paediatric patients is much milder than in adults. it is noteworthy that few patients with coronavirus disease (ie, covid-19 and sars) have pharyngeal congestion or sore throat, which contrasts with patients with h1n1 influenza, despite the high prevalence of cough and fever in all patients. pharyngeal congestion is more like an upper respiratory symptom than is fever (which is a systemic symptom) and cough, which can also be induced by a lower respiratory infection. the findings of our study also showed that, although all paediatric cases were mild or moderate, the number of days spent in hospital and time needed for these patients to have negative pcr results was still considerable (table 1, figure 2) . the time to achieve a negative pcr result seemed to be unaffected by severity of disease in terms of symptoms and the presence of pneumonia and treatment choices. nevertheless, fever resolved faster in mild cases than in moderate cases (figure 2). considering that delayed clearance of viral rna in patients' stools is a potential risk for transmission, 23 particularly in patients in rehabilitation, the reliability of viral rna-negative criteria in asymptomatic patients, in the long run, needs further investigation. the major limitation of our study was the small sample size. however, most results were consistent within the study and, with several case reports on children with covid-19, we believe our conclusions are valid. since covid-19 is spreading in more countries and has become an urgent public health event, we present an early report on paediatric patients and expect additional data to improve our findings. in conclusion, our study shows that paediatric patients with covid-19 have a simple transmission mode, either by close contact with infected adults or by exposure to epidemic areas. although fever, dry cough, and mild pneumonia are common manifestations, nearly half of patients have neither obvious symptoms nor abnormal radiological findings. the proportion of asymptomatic cases indicates the difficulty in identifying paediatric patients without clear epidemiological information. this finding suggests a dangerous situation if communityacquired infections occur. hq and jw designed the study and did the literature search. lh and yl were responsible for disease diagnosis and treatment and data collection. dc collected and analysed data. qs analysed data and wrote the report. we declare no competing interests. a review of studies on animal reservoirs of the sars coronavirus epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china, as at 22 tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis who. coronavirus disease 2019 (covid-19): situation report-41 novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china the remaining challenge of pneumonia: the leading killer of children child pneumonia at a time of epidemiological transition severe acute respiratory syndrome among children middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia analysis of ct features of 15 children with 2019 novel coronavirus infection national health and health commission of the people's republic of china. diagnosis and treatment guidelines for 2019 novel coronavirus pneumonia (draft version 5) clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china national institute for viral disease control and prevention. novel primers and probes for detection of novel coronavirus 2019 diagnosis and treatment recommendation for pediatric coronavirus disease-19 hypokalemia and clinical implications in patients severe acute respiratory syndrome (sars): chest radiographic features in children clinical features of 167 children with the novel influenza a (h1n1) virus infection in xi'an, china an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) pneumonia: the leading killer of children biomarkers for pediatric sepsis and septic shock persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients this study was funded by ningbo clinical research center for children's health and diseases, project of ningbo reproductive medicine centre (ppxk2018-06), and key scientific and technological innovation projects of wenzhou (zy202004). key: cord-273910-fna7s9te authors: bochud, pierre-yves; bochud, murielle; telenti, amalio; calandra, thierry title: innate immunogenetics: a tool for exploring new frontiers of host defence date: 2007-07-20 journal: lancet infect dis doi: 10.1016/s1473-3099(07)70185-8 sha: doc_id: 273910 cord_uid: fna7s9te the discovery of innate immune genes, such as those encoding toll-like receptors (tlrs), nucleotide-binding oligomerisation domain-like receptors (nlrs), and related signal-transducing molecules, has led to a substantial improvement of our understanding of innate immunity. recent immunogenetic studies have associated polymorphisms of the genes encoding tlrs, nlrs, and key signal-transducing molecules, such as interleukin-1 receptor-associated kinase 4 (irak4), with increased susceptibility to, or outcome of, infectious diseases. with the availability of high-throughput genotyping techniques, it is becoming increasingly evident that analyses of genetic polymorphisms of innate immune genes will further improve our knowledge of the host antimicrobial defence response and help in identifying individuals who are at increased risk of life-threatening infections. this is likely to open new perspectives for the development of diagnostic, predictive, and preventive management strategies to combat infectious diseases. environmental and host factors are important determinants of susceptibility to infection. in recent years, a rapidly growing body of evidence has underscored the importance of host genetic factors. the eff ect of genetic and environmental factors on the risk of death was assessed in a study of 960 adoptees. 1 death of a biological parent (but not of an adoptive parent) from infection before the age of 50 years resulted in a six times increase in the relative risk of dying from infection in the adoptee, strongly suggesting that susceptibility to infection aggregates in families. individuals who are heterozygous for haemoglobin s are known to be protected against malaria, whereas homozygous individuals have sickle-cell anaemia. 2 the high frequency of sickle-cell anaemia and other red blood cell disorders in regions where malaria is highly prevalent suggests that infectious agents (eg, plasmodium falciparum) can exert quite substantial selective pressure on human populations. 3 although natural immunity ensures survival of the species as a whole, individuals themselves are not likely to be immunocompetent to all pathogens, and individual diff erences in susceptibility to specifi c pathogens are quite common. 4 the development of the human genome project in 1990 propelled the scientifi c community into a new era, allowing genetic mapping and the development of large-scale gene identifi cation that has greatly facilitated the study of gene polymorphisms. we review recent advances in the fi eld of innate immunogenetics of host defences and show how an interdisciplinary approach of combining genetic epidemiology, genetics, genomics, and molecular and cellular biology will improve our understanding of the pathogenetic basis of infectious diseases, and help the development of new preventive and therapeutic treatment strategies. little inter-individual variation exists within the human genome. in fact, all genetic diff erences between individuals are estimated to be caused by variability in 3 million bp, which represent about 0·01% of the human genome. 5 since the mutation rate in mammalian genomes is low (10 -⁹ per bp per year), most interindividual variations are inherited. the most frequent variation is the single nucleotide polymorphism (snp), which occurs on average every 1300 bp. another type of genetic mutation is the variable number of tandem repeat (vntr); vntrs consist of repeats of sequences ranging from a single basepair to thousands of basepairs. 6 the term microsatellite is used for repeats of one to six nucleotides, whereas repeats of longer units are called minisatellites (seven to 100 nucleotides) or, in the extreme case, satellite dna (more than 100 nucleotides). since the number of repeats varies among individuals, vntrs have been widely used as genetic markers. within a coding region of a gene, an snp can either induce an aminoacid change (non-synonymous snps) or not (synonymous snps). snps may be located in the promoter region of a gene and therefore infl uence gene expression or splicing. similarly, diff erent lengths of vntr regions have been associated with diff erential gene expression. 7 certain snps or vntr alleles, or both, may be linked together so that non-functional polymorphisms can be used as genetic markers of functionally important mutations. only 1·5% of snps are thought to be located in a coding region of a gene. the functions of nearly all snps that are located outside gene-coding or regulatory regions are unknown. in recent years, snp genotyping technologies with high throughput and aff ordable costs have become available. these technologies are based on a few basic biochemical reactions (hybridisation, pcr with diff erential primer extension, specifi c ligation, and diff erential cleavage), which are used on diff erent support media and can be detected by diff erent methods (fi gure 1). 8 recent highthroughput technologies allow genotyping at low cost (ie, a few cents per snp per sample). 9 once markers have been typed, two main approaches can be used to analyse them: single marker analysis or haplotype analysis. a haplotype refers to the arrangement of two or more alleles on the same chromosome. currently, there is much debate about which approach is the most appropriate. studies have proposed that the underlying structure of the human genome can be described by use of a relatively simple framework in which the data are parsed into a series of discrete haplotype blocks. 10, 11 this observation has led to the development of haplotype tagging methods that aim to capture the haplotype structure in a candidate region. 11 haplotype tagging refers to the concept that most of the haplotypic structure in a particular chromosomal region can be captured by genotyping a smaller number of markers than all of those that constitute the haplotypes. the crucial markers to type would be the minimum set of markers that unambiguously identify each possible haplotype. the detection and estimation of familial aggregation is usually the fi rst step in the genetic analysis of a trait. once familial aggregation has been documented, the traditional approach has been to narrow down the genetic region of interest by use of linkage analysis, followed by fi ne mapping and association studies (table 1) . linkage and association studies are based on the same underlying principle: once a mutation occurs on a particular chromosome, it is subsequently transmitted to off spring together with nearby loci. this association is broken down at each successive generation by recombination (ie, homologous chromosomes pair during the meiotic cell division and exchange genetic material). when two loci are close enough on the same chromosome that their alleles cosegregate when passed on to the next generation, we say that the two loci are linked. 12 linkage disequilibrium refers to allelic association that is caused by linkage, or in other words, that has not yet been broken up by recombination. 12 an association between two loci, such as the non-independence of alleles at these loci, may be caused not only by linkage, but also to factors such as population stratifi cation or chance. population stratifi cation refers to the situation in which study participants are selected from genetically diff erent subpopulations. population stratifi cation will only lead to a spurious association (and hence be a confounder) if both the allele and disease frequencies diff er across subpopulations. 13 some researchers have argued that too much emphasis has been put on this issue and surprisingly few examples can be found that unequivocally show that population stratifi cation has led to a spurious association. 14, 15 whereas linkage and association studies can be done in families, only association studies can be done in unrelated cases and controls (table 1). the main diff erence between related and unrelated cases is the number of meiotic events that separate them, so that unrelated cases share a much shorter chromosomal segment around a particular causative mutation than related cases. linkage and association can be obscured by incomplete penetrance (ie, there is no one-to-one correspondence between genotype and phenotype), misdiagnoses, genetic heterogeneity (several genes can produce a similar phenotype), phenocopies (ie, environmental factors mimicking the eff ect of certain genes), and disease an important advance toward enabling effi cient wholegenome-scan association studies is the determination of linkage disequilibrium patterns on a genome-wide scale through the hapmap project. 16 because most diseases are likely to be infl uenced by several genes and environmental factors, the analysis of gene-gene interactions (epistasis) and gene-environment interactions will represent an important task in the future, but this is, and will remain, a challenging issue for the years to come. the innate immune system assumes an essential role in the natural host defences against microbes. the recognition of microbial pathogens, either in tissue in contact with the host's environment or in the systemic circulation after invasion of the bloodstream, is done by macrophages, dendritic cells, natural killer cells, granulocytes, and monocytes, which act as sentinels of the innate immune system (fi gure 2). this process involves coordinated action of several families of proteins, such as toll-like receptors (tlrs), 17 nucleotide-binding oligomerisation domain (nod)-like receptors (nlrs), 18, 19 rna helicase-containing proteins, 20 and the c-type lectins. 21 tlrs are essential components of the innate immune system. 17, 22, 23 tlrs are type i transmembrane proteins that function as homodimers or heterodimers. the extracellular domain comprises multiple leucine-rich repeat structures that vary among diff erent tlrs and are implicated in the selective recognition of a vast range of microbial-associated molecular patterns (mamps). 24 so far, 12 members of the tlr family have been identifi ed in mammals. several molecules, including cd14, 25 cd36, 26 and md2, 25 have also been shown to participate in the sensing of microbial products and are therefore integral components of these receptor complexes. binding of microbial products to microbial-recognition molecules activates signal transduction pathways and the transcription of immune genes that code for costimulatory molecules expressed at the cell surface or for immunoregulatory eff ector molecules (including cytokines and chemokines) released in the extracellular milieu that orchestrate the host innate immune defence response. 23, 27 in addition to lipopolysaccharide of gram-negative bacteria, 28, 29 tlr4 detects other mamps that are structurally unrelated to lipopolysaccharide, such as mannan (candida albicans) or the fusion protein of respiratory syncytial virus (fi gure 3). other endogenous ligands, including fi brinogen, fi bronectin, hyaluronic acid, heparin sulphate, beta-defensins, or heat-shock proteins, have been reported to activate tlr4. 17 however, endotoxin contamination has been argued to account for tlr5 tl r2 tl r1 microbial-associated molecular patterns are recognised by transmembrane receptors (1: eg, toll-like receptors [tlrs]), which trigger the activation of several signal-transducing pathways, leading to the production of cytokines and expression of costimulatory molecules. cytokines induce and regulate the infl ammatory response and orchestrate the adaptive immune response. by contrast with other tlrs, tlr3, tlr7, tlr8, and tlr9 are expressed mainly in the endosomal compartment (2), where local acidifi cation is required for recognition of microbial products by their cognate receptors. intracellular pathogens or microbial products released intracellularly after lysis of ingested microorganisms may also interact with intracytoplasmic receptors, such as nucleotide-binding oligomerisation domain-like (nlr) proteins (3), or the rna helicase-containing molecules (4: rig-i or mda5). tcr=t-cell receptor. tlr4 detects lipopolysaccharide (lps), mannan (candida albicans), and the fusion protein of the respiratory syncytial virus. tlr2 forms a heterodimer with either tlr1 to detect triacyl lipopeptide or tlr6 to detect diacyl lipopeptide and zymosan. tlr2 is also involved in the recognition of lipoteichoic acid (lta), peptidoglycan (pg), lipoarabinomannan (lam), porins (neisseria spp), glycosylphosphatidylinositol mucin (trypanosoma spp; tgpi), and the haemaglutinnin protein (ha, measles virus). tlr3, tlr7, tlr8, and tlr9 are located in the endosomal compartment and detect nucleic acids and/or haemozoin (plasmodium spp, tlr9). through their intracellular domain, tlrs interact with specifi c adaptor proteins, including the myeloid diff erentiation primary response protein 88 (myd88), the tir domaincontaining adaptor protein (tirap), the tir domain-containing adapter inducing interferon (trif), and the trifrelated adapter molecule (tram). these adaptors lead to the activation of several transcription factors such the nuclear factor κb (nfκb), the activating-protein 1 (ap1), and/or the interferon regulatory factors 3 and 7 (irf3/7) that ultimately induce the production of pro-infl ammatory mediators. ss=single-stranded. ds=double-stranded. the tlr4 specifi city of some of these putative tlr ligands. 30 tlr2 and tlr6 heterodimers detect diacyl lipopeptides, whereas tlr2 and tlr1 heterodimers recognise triacyl lipopeptides. 17 tlr2 has also been proposed to sense lipoteichoic acid, peptidoglygan, lipoarabinomannan, phospholipomannan (c albicans), zymosan (saccharomyces cerevisiae), porins (neisseria spp), glycosylphosphatidylinositol mucin (trypanosoma spp), and the haemagluttinin protein of the measles virus. 17 tlr3, tlr7, tlr8, and tlr9, which are mainly expressed in endosomes, serve to detect viral or bacterial nucleic acids. tlr3 detects double-stranded rna and tlr8 detects single-stranded rna. 17 tlr9 senses dna containing the unmethylated cpg motifs found in bacteria and viruses and the malaria pigment haemozoin. 17 compartmentalisation of tlr3, tlr7, tlr8, and tlr9 thus allows the detection of pathogenic dna and rna within the endosomal compartment, while avoiding the detection of self-dna and mrna. 23 on binding of cognate ligands, the intracellular tollinterleukin-1 receptor (tir) domain of tlrs recruits and activates diff erent adaptor proteins, including myeloid diff erentiation primary response protein 88 (myd88), tir domain-containing adaptor protein, tir domaincontaining adapter-inducing interferon β (trif; also known as ticam), and trif-related adapter molecule, ultimately leading to the activation of several specifi c signal-transducing pathways and transcription factors such as nuclear factor κb (nfκb) and activating protein 1 (ap1; fi gure 3 and fi gure 4). 40 myd88-dependent signalling pathways (nfκb and ap1) are activated by all tlrs, whereas myd88-independent, trif-dependent signalling path ways (interferon regulatory factor [irf] 3) are activated only by some tlrs (such as tlr3 and tlr4). the observation that diff erent tlrs may activate diff erent signalling pathways with diff erent biological conse quences shows that the innate immune system can produce pathogen-specifi c defensive responses. in addition to the tlrs, the family of proteins comprising nod proteins and the nalps (neuronal apoptosis inhibitor [like] proteins), also known collectively as nlrs or nacht-leucine-rich-repeat-containing proteins, have been shown to have a crucial role in the sensing of microbial products, invasive pathogens, and endogenous host proteins. nlrs are cytosolic proteins composed of three diff erent structural domains, a carboxy-terminal ligand-binding domain consisting of leucine-rich repeats, a nucleotide oligomerisation domain, and an aminoterminal eff ector domain consisting of various caspaserecruitment domains (card), a pyrin domain, or a baculoviral inhibitor-of-apoptosis repeat. 17, 41 nod1 and nod2 have been shown to recognise specifi c bacterial peptidoglycan motifs, 42 and to interact with tlr signalling pathways. 19, 42 nod2 detects muramyldipeptide, a peptidoglycan fraction of gram-positive and gram-negative bacteria, 43 traf6=tumour necrosis factor-receptor-associated factor 6. snp=single nucleotide polymorphism. γ-d-glutamyl-meso-diaminopimelic acid, a peptidoglycan fraction found in gram-negative bacteria and in a few gram-positive bacteria (listeria and bacillus spp). 44, 45 on exposure to microbial products, nods activate transcription factors, including nfκb and the mitogenactivated protein kinase, and induce the cleavage of prointerleukin 1β into active interleukin 1β. [46] [47] [48] the nalp subfamily of nlr proteins interact with several adaptor molecules, including asc (apoptosis-associated specklike protein containing a card domain), caspase 1, and caspase 5, and are essential for the activation of interleukin 1β. 49 the nalp-related protein card12 (also known as ipaf) is involved in salmonella typhimuriuminduced activation of caspase 1. 50 nalp3 is implicated in the detection of atp, 51 bacterial rna, 52 and uric acid crystals. 53 however, most nalps are orphan recognition proteins with no known ligands. a series of fascinating articles have provided strong evidence implicating the innate immune system in the host defence against viruses. two intracytoplasmic molecules have been implicated in the detection of viral rna. retinoic-acid-inducible protein i (rig-i) 54 and melanoma diff erentiation-associated gene 5 (mda5) 55-57 possess a card domain and rna helicase domains that function as sensors of double-stranded rna. 58 rig-i and mda5 signal through the adaptor molecule mavs (mitochondrial antiviral signalling protein; also known as cardif or visa) 18, 59, 60 and interact with several other signal-transducing molecules, including fadd (tumour necrosis factor receptor superfamily member 6 precursor [tnfrsf6, also know as fas]-associated death domain protein), ripk1 (receptor-interacting serine/threonineprotein kinase 1; also known as rip or rip1), tbk1 (traf family member-associated nf-kappa-b activator [tank]-binding kinase-1), and ikke (inhibitor of nfκb kinase subunit epsilon; also known as ikk-i). 40 these molecules are involved in the production of type i interferons (interferons α and β) in response to infection by rna viruses. therefore, rig-i and mda5 are able to detect single-stranded rna present in the cytoplasmic compartment and thus not accessible to endosomal tlr3. interestingly, rig-i and mda5 can discriminate between diff erent types of viruses. rig-i is essential for the production of interferons in response to paramyxoviruses, infl uenza virus, and japanese encephalitis virus, whereas mda5 is crucial for detection of picornavirus. 20 a newly described form of innate immunity, termed intrinsic immunity, ensures protection by providing a constitutive, always-on line of defence, relying on intracellular obstacles to hinder the replication of pathogens. 61 this component of the immune system has gained much attention as a cornerstone of the resistance of mammals against several classes of retroelements and retroviruses. 61 among the best studied proteins are the family of apolipoprotein b mrna-editing enzyme catalytic polypeptide 3 (apobec3) proteins, which interfere with the viral lifecycle by incorporating themselves into viral particles, leading to viral dna hypermutation on the next round of infection. 62, 63 a series of studies involving infection of human cd4+ t cells and macrophages with wild-type hiv-1 and hiv-1 defi cient in the vif gene showed that the antiviral eff ect of abc3g (also known as cem15 or apobec3g) is counteracted by vif. 64 interestingly, in non-human primates, abc3g orthologues provide antiviral activity against wild-type hiv-1, 65 but not their cognate simian immunodefi ciency viruses, suggesting that virus permissiveness in diff erent primates results from species-specifi c diff erences within vif. 63 one human variant of abc3g has been associated with rapid hiv-1 disease progression. 66 the tripartite motif (trim) family is a well-conserved family of proteins characterised by a structure comprising a ring-fi nger domain, one or two b-box motifs, and a predicted coiled-coil region. 67 additionally, most trim proteins have additional carboxy-terminal domains. members of the trim protein family are involved in various cellular processes, including cell proliferation, diff erentiation, development, oncogenesis, and apoptosis. 68, 69 some trim proteins exert antiviral properties. trim5α is reported to restrict retroviral infection by specifi cally recognising the viral capsid and promoting its premature disassembly. 70 human trim5α has limited effi cacy against hiv-1, whereas some primate trim5α orthologues can potently restrict this particular lentivirus. 68, 69 substantial interspecies sequence diversity characterises trim5α and may underlie diff erences in the pattern and breadth of restriction of multiple lentiviruses. human trim5α variants do not modify susceptibility to hiv-1; however, they change susceptibility to other retroviruses, such as n-tropic murine leukaemia virus. 71 polymorphisms found in trim5α might conceivably have been selected in past epidemics by viruses unrelated to hiv-1. the increasing availability of genomic data allows comparative analyses of genetic sequences involved in innate and intrinsic immunity. this approach, also described as evolutionary genomics, identifi es the role of adaptive forces on protein-encoding genes by determining signs of positive (diversifying) or negative (purifying) selection. for example, positive selection in the human genome indicates shifts in living conditions experienced by modern human populations, such as diff erent habitats, food sources, population densities, and exposure to pathogens. 72 several families of innate immunity genes have been investigated by use of comparative genomics. vertebrate tlrs are an example of evolutionary conservation that review indicate the diffi culty for the microbes to mutate genes that encode mamps. 73, 74 the cd209 (dc-sign) proteins, a family of c-type lectins that participate in the recognition of various pathogens, display a complex pattern of evolution. whereas cd209 has been under a strong selective constraint that prevents accumulation of aminoacid changes, cd209l (also known as dc-sign2 or dc-signr) exhibits greater variation across human populations. 75 such variations may be tolerated because of the potentially redundant functional activities of the molecules encoded by these genes. 76 the killer-cell immunoglobulin-like receptor (kir) genes encode a family of receptors expressed by natural killer cells, which participate in early responses against infected or transformed cells through production of cytokines and direct cytotoxicity. 77, 78 by contrast with tlrs and cd209, only a small proportion of kir alleles are conserved among primates, showing a rapid species-specifi c diversifi cation of the kir gene family members and a plasticity of the genomic region that parallels that of the mhc loci. 79 thus, the evolutionary forces driving the genesis of natural killer receptors and their hla ligands represent a concerted response to pathogens. finally, a remarkable success of evolutionary genomics in infectious diseases is the identifi cation of protein regions relevant for host-pathogen interactions in hiv-1 infection. comparative analysis of the primate antiretroviral cellular defence genes encoding for abc3g and trim5α have revealed the powerful selective pressures emerging from a long-standing battle between retroviruses and their hosts. [80] [81] [82] singular aminoacids or regions (patches) contain key residues that confer primates the ability to combat hiv-1. given that the innate immune system is at the interface between the host and the pathogen, polymorphisms of innate immune genes are very likely to aff ect the host susceptibility to infections. since the innate immune system senses only a limited number of highly conserved microbial-associated molecular patterns 23 via a limited number of receptors and signalling molecules, as anticipated, several polymorphisms have been found to confer an increased susceptibility to specifi c pathogens (table 2, table 3 , and fi gure 4). a study from turkey revealed an association between susceptibility to tuberculosis and an snp (r753q) in the tlr2 gene. 94 14 (9·3%) of 151 tuberculosis patients were homozygous for the minor allele compared with two (1·7%) of 116 healthy controls (odds ratio 6·0, 95% ci 1·3-3·9, p=0·009). of note, r753q was associated with decreased responsiveness to bacterial lipopeptides. 95 the role of a microsatellite polymorphism (gt repeat) in the exon 2 of tlr2 has been studied in 176 korean patients with tuberculosis and 196 healthy controls. 7 a shorter gt repeat was found more frequently in tuberculosis patients than healthy individuals (49·4% vs 37·7%, p=0·02), and was associated with weaker promoter activities and lower tlr2 expression in cd14positive peripheral blood monocytes. two nonsynonymous snps in the extracellular domain of tlr4 found to be in linkage disequilibrium (d299g and t399i) have been associated with an increased susceptibility to infections caused by gram-negative bacteria, 103, 104 brucella spp, 98 respiratory syncytial virus, 88 and p falciparum. 99 individuals heterozygous for the d299g and t399i snps were hyporesponsive to lipopolysaccharide as measured by bronchospastic response after inhalation of endotoxin. 31 furthermore, airway epithelial cells isolated from heterozygous individuals had defi cient response to lipopolysaccharide, suggesting that d299g and t399i acted in a dominant fashion with respect to the wild-type allele. 31 however, monocytes and whole blood isolated from heterozygous individuals did not show abnormal responses to lipopolysaccharide, 127, 128 suggesting that the eff ects of these mutations may vary between cell types. although two studies had shown that d299g, 103 or d299g and t399i, 104 were associated with an increased risk of gram-negative infections or septic shock, three subsequent studies did not fi nd an association in patients with meningococcal sepsis. [120] [121] [122] however, in one study, rare heterozygous missense mutations of tlr4 were linked with the development of meningococcal disease. 120 unexpectedly, d299g and t399i were associated with decreased rather than increased susceptibility to legionella pneumophila infection. 32 a stop codon polymorphism in tlr5 (r392stop), shown to abolish the ability of tlr5 to detect bacterial fl agellin, has been associated with increased susceptibility to pneumonia caused by l pneumophila. 123 several studies have shown associations between mutations in genes encoding several proteins of the tlr signalling pathways (irak4, 33,34 ikbkg, [36] [37] [38] and iκbα 35, 129 ) and rare inherited immunodefi ciencies. complete recessive interleukin-1 receptor-associated kinase 4 (irak4) defi ciency is characterised by recurrent infections with pyogenic bacteria at an early age that tend to disappear over time. by contrast, mutations aff ecting the other genes result in x-linked (ikbkg) or autosomaldominant (iκbα) anhydrotic ectodermal dysplasia, which is characterised by increased susceptibility to a broader range of pathogens, such as atypical mycobacteria or pneumocystis jirovecii and a complex disorder involving impaired development of skin appendages, conical teeth, and hypotrichosis. [36] [37] [38] taken together, these data clearly show that mutations in the genes encoding tlrs and downstream signal-transducing molecules infl uence innate immune responses and increase susceptibility to many infectious diseases. similarly, polymorphism of cytokines and cytokine receptor genes, which are key eff ector molecules, have also been associated with altered susceptibility to invasive pathogens. 130 polymorphisms in genes encoding nlrs have been shown to infl uence susceptibility to infl ammatory diseases. polymorphisms in nod2 have been associated with susceptibility to crohn's disease, 131 common limitations of genetic association studies are shown in table 4. genetic studies done to date often fail on the following factors: (1) to properly account for confounding factors (such as lack of information on ethnicity), and selection and information biases (insuffi cient data on the source population of cases and controls or study endpoints); (2) to present appropriate statistical analyses (such as lack of sample size calculation and correction for multiple testing); and (3) to provide convincing information about biological plausibility. as an example, among fi ve studies that assessed the eff ect of tlr4 polymorphisms on susceptibility to, and outcome of, severe infections, only two included more than 100 patients, 105 two provided information about patient's ethnicity, 105, 106 and only one limited the analysis to a specifi c ethnic group. 106 comparison of data is often impaired by the fact that apparently similar studies used markedly diff erent controls groups and endpoints. [103] [104] [105] [106] [107] proving causality is never trivial. associations are likely to occur when noncausal markers are in linkage disequilibrium with the true disease locus. although the replication of a fi nding in an independent sample decreases the risk of a falsepositive result, the functional signifi cance of the genetic variant should ultimately be shown in biological studies. however, proving biological plausibility may be diffi cult in view of the limitations of in-vitro studies used as proxy of complex in-vivo biological processes. for example, use of gene-silencing techniques often reduces the biological observation to that of an on/off system, which does not allow the detection of quantitative variations (ie, a dose-response eff ect) of gene expression or discrete functional alterations. with the increasing use of high-throughput genotyping techniques, the number of genetic associations that will be reported in the years to come will most probably exceed our capacity to do proper functional studies and hence to provide convincing evidence for biological plausibility. 139 36 jain et al, 37 jain et al, 38 functional studies should therefore focus on genetic polymorphisms that exert a strong eff ect, have been replicated by independent investigators, and have potential diagnostic or therapeutic implications. to limit the importance of positive publication bias, it will be crucial for investigators and journal editors to become less reluctant to publish well-conducted negative studies. 139 in recent years, innate immunogenetic studies of inherited genetic disorders have provided researchers and clinical investigators with crucial information that has improved our understanding of the host defences against microbial pathogens. table 5 shows examples of the eff ect of recent discoveries in the fi eld of innate immunogenetics with foreseeable applications for the short, middle, and long term in areas such as vaccine development and predictive and preventive medicine. the persistence or emergence of potentially devastating infectious diseases, such as tuberculosis, malaria, hiv/ aids, and, most recently, severe acute respiratory syndrome or avian infl uenza, underscore the need to develop new vaccines and therapeutic treatment strategies. a better understanding of microbial genomics and genetics and host innate immunogenetics is likely to provide important information for the development of new vaccines. vaccine immunogenicity is determined not only by the chemical and physical nature of microbial antigens and adjuvants, but also by the genetic make-up of vaccine recipients. analyses of polymorphisms of innate immune genes may also help understand why some individuals exhibit suboptimum responses to vaccination. 140 immuno suppression as a result of myeloablative chemotherapy, solid organ or haematological stem-cell transplantation, or corticosteroid therapy for autoimmune diseases represent other clinical conditions for which immune gene polymorphisms may help to predict the risk of life-threatening infectious complications. the recent discoveries of genes encoding tlrs, nlrs, and the related signal-transducing molecules has markedly improved our understanding of innate immunity. the availability of high-throughput genotyping techniques opens new perspectives to further improve our understanding of the pathogenesis of infectious diseases and for the development of new diagnostic, predictive, and preventive treatment strategies. clinicians and researchers should be aware of the results and far-reaching implications of recent innate immunogenetic studies that have associated genetic polymorphisms with susceptibility to, or outcome of, infectious diseases. collecting dna should now be an integral part of epidemiological or clinical infectious disease studies. national and international consortia should be created to put together large cohort studies to promote and facilitate research in the fi eld. we declare that we have no confl icts of interest. relevant articles for this review were identifi ed by searching medline (1966 to november, 2006 ) by use of the terms "genetics", "single nucleotide polymorphisms", "toll-like receptors" or "tlrs", "nucleotide-binding oligomerization domain receptors" or "nods", "immunology", and "innate immunity", and by extracting references from these articles. the review was limited to articles published in the english language. genetic and environmental infl uences on premature death in adult adoptees protection aff orded by sickle-cell trait against subtertian malareal infection the immunogenetics of resistance to malaria genetic dissection of immunity to mycobacteria: the human model a map of human genome sequence variation containing 1·42 million single nucleotide polymorphisms microsatellites: simple sequences with complex evolution the association between microsatellite polymorphisms in intron ii of the human toll-like receptor 2 gene and tuberculosis among koreans accessing genetic variation: genotyping single nucleotide polymorphisms single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput the structure of haplotype blocks in the human genome haplotype blocks and linkage disequilibrium in the human genome introduction and overview. statistical methods in genetic epidemiology reporting, appraising, and integrating data on genotype prevalence and gene-disease associations population stratifi cation and spurious allelic association counterpoint: bias from population stratifi cation is not a major threat to the validity of conclusions from epidemiological studies of common polymorphisms and cancer the international hapmap consortium. the international hapmap project pathogen recognition and innate immunity cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus signalling pathways and molecular interactions of nod1 and nod2 diff erential roles of mda5 and rig-i helicases in the recognition of rna viruses myeloid c-type lectins in innate immunity toll-like receptors in the induction of the innate immune response inferences, questions and possibilities in toll-like receptor signalling innate immunity: an overview lps, tlr4 and infectious disease diversity cd36 is a sensor of diacylglycerides innate immune recognition defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene md-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4 endotoxin contamination in recombinant human heat shock protein 70 (hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages tlr4 mutations are associated with endotoxin hyporesponsiveness in humans tolllike receptor 4 polymorphisms are associated with resistance to legionnaires' disease distinct mutations in irak-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections pyogenic bacterial infections in humans with irak-4 defi ciency a hypermorphic ikappabalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and t cell immunodefi ciency a novel x-linked disorder of immune defi ciency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in ikkgamma (nemo) specifi c missense mutations in nemo result in hyper-igm syndrome with hypohydrotic ectodermal dysplasia specifi c nemo mutations impair cd40-mediated c-rel activation and b cell terminal diff erentiation x-linked anhidrotic ectodermal dysplasia with immunodefi ciency is caused by impaired nf-kappab signaling nod-lrr proteins: role in host-microbial interactions and infl ammatory disease the role of toll-like receptors and nod proteins in bacterial infection host recognition of bacterial muramyl dipeptide mediated through nod2. implications for crohn's disease nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan an essential role for nod1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid rick/rip2/ cardiak mediates signalling for receptors of the innate and adaptive immune systems role of nod2 in the response of macrophages to toll-like receptor agonists heterotypic interactions among nacht domains: implications for regulation of innate immune responses nlrs join tlrs as innate sensors of pathogens diff erential activation of the infl ammasome by caspase-1 adaptors asc and ipaf cryopyrin activates the infl ammasome in response to toxins and atp bacterial rna and small antiviral compounds activate caspase-1 through cryopyrin/nalp3 goutassociated uric acid crystals activate the nalp3 infl ammasome the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter expression analysis and genomic characterization of human melanoma diff erentiation associated gene-5, mda-5: a novel type i interferon-responsive apoptosis-inducing gene mda-5: an interferon-inducible putative rna helicase with double-stranded rna-dependent atpase activity and melanoma growth-suppressive properties shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity visa is an adapter protein required for virus-triggered ifn-beta signaling ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction lentiviral vectors and antiretroviral intrinsic immunity role and mechanism of action of the apobec3 family of antiretroviral resistance factors innate cellular defenses of apobec3 cytidine deaminases and viral counter-defenses isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein a single amino acid determinant governs the species-specifi c sensitivity of apobec3g to vif action apobec3g genetic variants and their infl uence on the progression to aids the tripartite motif family identifi es cell compartments trim family proteins: retroviral restriction and antiviral defence control of viral infectivity by tripartite motif proteins specifi c recognition and accelerated uncoating of retroviral capsids by the trim5alpha restriction factor highfrequency persistence of an impaired allele of the retroviral defense gene trim5alpha in humans a map of recent positive selection in the human genome toll-like receptor 5 recognizes a conserved site on fl agellin required for protofi lament formation and bacterial motility the evolution of vertebrate toll-like receptors novel member of the cd209 (dc-sign) gene family in primates the heritage of pathogen pressures and ancient demography in the human innate-immunity cd209/cd209l region mhc class i molecules and kirs in human history, health and survival the impact of variation at the kir gene cluster on human disease single haplotype analysis demonstrates rapid evolution of the killer immunoglobulin-like receptor (kir) loci in primates ancient adaptive evolution of the primate antiviral dna-editing enzyme apobec3g positive selection of primate trim5alpha identifi es a critical species-specifi c retroviral restriction domain patterns of evolution of host proteins involved in retroviral pathogenesis polymorphisms in cd14, mannose-binding lectin, and toll-like receptor-2 are associated with increased prevalence of infection in critically ill adults cd14 and tlr4 gene polymorphisms in adult periodontitis combined carriership of tlr9 −1237c and cd14 −260t alleles enhances the risk of developing chronic relapsing pouchitis cd14 promoter polymorphism −159c>t is associated with susceptibility to chronic chlamydia pneumoniae infection in peripheral blood monocytes the cd14 functional gene polymorphism −260 c>t is not involved in either the susceptibility to chlamydia trachomatis infection or the development of tubal pathology association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease genetic polymorphisms of cd14, toll-like receptor 4, and caspase-recruitment domain 15 are not associated with necrotizing enterocolitis in very low birth weight infants gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specifi c genetic predisposition to sepsis tlr1 and tlr6 polymorphisms are associated with susceptibility to invasive aspergillosis after allogeneic stem cell transplantation polymorphisms in toll-like receptor 2 are associated with increased viral shedding and lesional rate in patients with genital hsv-2 infection a microsatellite polymorphism in intron 2 of human toll-like receptor 2 gene: functional implications and racial diff erences the arg753gln polymorphism of the human toll-like receptor 2 gene in tuberculosis disease a novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection lack of association between toll-like receptor 2 polymorphisms and susceptibility to severe disease caused by staphylococcus aureus heterozygous arg753gln polymorphism of human tlr-2 impairs immune activation by borrelia burgdorferi and protects from late stage lyme disease tlr4 polymorphism in iranian patients with brucellosis toll-like receptor (tlr) polymorphisms in african children: common tlr-4 variants predispose to severe malaria common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy the toll-like receptor 4 (asp299gly) polymorphism is a risk factor for gram-negative and haematogenous osteomyelitis role of tlr4 receptor polymorphisms in boutonneuse fever human toll-like receptor 4 mutations but not cd14 polymorphisms are associated with an increased risk of gram-negative infections relevance of mutations in the tlr4 receptor in patients with gram-negative septic shock tlr4 and tnf-alpha polymorphisms are associated with an increased risk for severe sepsis following burn injury eff ects of functional toll-like receptor-4 mutations on the immune response to human and experimental sepsis polymorphisms in toll-like receptor 4 and the systemic infl ammatory response syndrome functional gene polymorphisms in aggressive and chronic periodontitis gingival epithelial cells heterozygous for toll-like receptor 4 polymorphisms asp299gly and thr399ile are hypo-responsive to porphyromonas gingivalis gene polymorphisms in pro-infl ammatory cytokines are associated with systemic infl ammation in patients with severe periodontal infections tolllike receptor (tlr) 2 and 4 mutations in periodontal disease toll-like receptor 4 thr399ile polymorphisms are a risk factor for candida bloodstream infection candida-specifi c interferon-gamma defi ciency and toll-like receptor polymorphisms in patients with chronic mucocutaneous candidiasis role of the toll-like receptor 4 asp299gly polymorphism in susceptibility to candida albicans infection relationship between a toll-like receptor-4 gene polymorphism, bacterial vaginosis-related fl ora and vaginal cytokine responses in pregnant women diff erences in infl ammatory cytokine and toll-like receptor genes and bacterial vaginosis in pregnancy the toll-like receptor 4 asp299gly variant: no infl uence on lps responsiveness or susceptibility to pulmonary tuberculosis in the gambia innate immunity genes infl uence the severity of acute appendicitis the role that the functional asp299gly polymorphism in the toll-like receptor-4 gene plays in susceptibility to chlamydia trachomatis-associated tubal infertility assay of locus-specifi c genetic load implicates rare toll-like receptor 4 mutations in meningococcal susceptibility variation in toll-like receptor 4 and susceptibility to group a meningococcal meningitis in gambian children a functional polymorphism of toll-like receptor 4 is not associated with likelihood or severity of meningococcal disease a common dominant tlr5 stop codon polymorphism abolishes fl agellin signaling and is associated with susceptibility to legionnaires' disease host susceptibility and clinical outcomes in toll-like receptor 5-defi cient patients with typhoid fever in vietnam polymorphisms in toll-like receptor 9 infl uence the clinical course of hiv-1 infection innate immune receptor genetic polymorphisms in pouchitis: is card15 a susceptibility factor? heterozygous tolllike receptor 4 polymorphism does not infl uence lipopolysaccharide-induced cytokine release in human whole blood monocytes heterozygous for the asp299gly and thr399ile mutations in the toll-like receptor 4 gene show no defi cit in lipopolysaccharide signalling inherited disorders of human toll-like receptor signaling: immunological implications cytokine promoter polymorphisms in severe sepsis host recognition of bacterial muramyl dipeptide mediated through nod2. implications for crohn's disease nod2 is a general sensor of peptidoglycan through muramyl dipeptide (mdp) detection card15 mutations in blau syndrome early-onset sarcoidosis and card15 mutations with constitutive nuclear factor-kappab activation: common genetic etiology with blau syndrome both donor and recipient nod2/card15 mutations associate with transplant-related mortality and gvhd following allogeneic stem cell transplantation mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinfl ammatory syndrome and muckle-wells syndrome chronic infantile neurological cutaneous and articular syndrome is caused by mutations in cias1, a gene highly expressed in polymorphonuclear cells and chondrocytes the bare lymphocyte syndrome and the regulation of mhc expression host genetic determinants in hepatitis c virus infection lipopolysaccharides from distinct pathogens induce diff erent classes of immune responses in vivo this work was supported by grants from the swiss foundation for medical and biological grants (1121 to pyb), the swiss national science foundation (81la-65462 to pyb, 32-49129.96 and 3100-066972.01 to tc), the bristol-myers squibb foundation (to tc), the leenaards foundation (to pyb and tc), and the santos-suarez foundation for medical research (to tc and at). key: cord-281529-2rec51xg authors: haagmans, bart l; al dhahiry, said h s; reusken, chantal b e m; raj, v stalin; galiano, monica; myers, richard; godeke, gert-jan; jonges, marcel; farag, elmoubasher; diab, ayman; ghobashy, hazem; alhajri, farhoud; al-thani, mohamed; al-marri, salih a; al romaihi, hamad e; al khal, abdullatif; bermingham, alison; osterhaus, albert d m e; alhajri, mohd m; koopmans, marion p g title: middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation date: 2013-12-17 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70690-x sha: doc_id: 281529 cord_uid: 2rec51xg background: middle east respiratory syndrome coronavirus (mers-cov) causes severe lower respiratory tract infection in people. previous studies suggested dromedary camels were a reservoir for this virus. we tested for the presence of mers-cov in dromedary camels from a farm in qatar linked to two human cases of the infection in october, 2013. methods: we took nose swabs, rectal swabs, and blood samples from all camels on the qatari farm. we tested swabs with rt-pcr, with amplification targeting the e gene (upe), nucleocapsid (n) gene, and open reading frame (orf) 1a. pcr positive samples were tested by different mers-cov specific pcrs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. we tested serum samples from the camels for igg immunofluorescence assay, protein microarray, and virus neutralisation assay. findings: we obtained samples from 14 camels on oct 17, 2013. we detected mers-cov in nose swabs from three camels by three independent rt-pcrs and sequencing. the nucleotide sequence of an orf1a fragment (940 nucleotides) and a 4·2 kb concatenated fragment were very similar to the mers-cov from two human cases on the same farm and a mers-cov isolate from hafr-al-batin. eight additional camel nose swabs were positive on one or more rt-pcrs, but could not be confirmed by sequencing. all camels had mers-cov spike-binding antibodies that correlated well with the presence of neutralising antibodies to mers-cov. interpretation: our study provides virological confirmation of mers-cov in camels and suggests a recent outbreak affecting both human beings and camels. we cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible. funding: european union projects emperie (contract number 223498), antigone (contract number 278976), and the virgo consortium. an emerging betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-causes illness characterised predominantly by mild-to-severe respiratory complaints, with most patients requiring admission to hospital because of pneumonitis or acute respiratory distress syndrome. old age and the presence of comorbidities or immunosuppression seem to increase the risk of infection and are associated with severe forms of the disease. however, some patients can remain asymptomatic or mildly symptomatic and atypical presentations such as gastroenteritis have occurred. 1 as of dec 2, 2013, 163 laboratory-confi rmed cases, including 71 deaths, have been reported to who. 2 all of these cases were directly or indirectly linked to the middle east region, including saudi arabia, qatar, united arab emirates, kuwait, oman, and jordan. coronaviruses reside in many animal hosts and can adapt to diff erent species, including people. mers-cov belongs to the lineage c betacoronaviruses, which are associated with bats 3 and the closest relatives of mers-cov have been identifi ed in vesper bats from europe, asia, and south africa. [4] [5] [6] notably, this coronovirus is able to replicate in various bat cell lines. 7 molecular clock dating of epidemiologically unlinked human mers-cov isolates estimated their divergence from a common ancestor in mid-2011, with a cluster of isolates from the eastern parts of the arabian peninsula diverging in late 2012. these fi ndings suggest that the reported mers-cov diversity in human beings is the result of several independent, geographically structured, zoonotic events from an unknown reservoir in the middle east. 8, 9 human-to-human transmission has been noted, especially in health-care settings and households, but is thought to be relatively ineffi cient. 1 to determine how circulation of mers-cov in human beings is maintained and to mitigate the chain of transmission, identifi cation of possible animal reservoirs and modes of transmission is needed. 10 limited available exposure history of patients suggest that contact with livestock, including dromedary camels, might have a role. 1, 9, 11, 12 in addition, our recent investigations have provided evidence for the circulation of mers-cov or a related virus in dromedary camels. 13 both mers-cov spike protein binding antibodies and virus neutralising antibodies were reported in dromedary camels from diff erent regions, including oman and egypt, but no virus shedding could be detected and, therefore, the signifi cance of these observations remained an issue of debate. 13, 14 in this study we aimed to assess presence of mers-cov in dromedary camels from a farm in qatar that was linked to two human cases of mers-cov. in october, 2013, the qatar supreme council of health recorded two cases of laboratory confi rmed mers-cov. case 1 involved a 61-year-old man who was the owner of a farm that he visited regularly. the patient had substantial contact with animals including camels, sheep, pigeons, and hens, and no history of travel outside of qatar in the 2 weeks before becoming ill. mers-cov infection was diagnosed by e gene (upe) pcr on a sputum sample collected on oct 13, 2013, in the national virology laboratory of qatar. case 2 involved a 23-year-old male employee of the farm owned by case 1 and was identifi ed in the close-contact investigation of that case. he was reported on oct 17, 2013, and had no underlying medical conditions and no recent travel history, but had regular contact with the animals on the farm. mers-cov infection was diagnosed in a throat swab taken on oct 17, 2013, by the national virology laboratory of qatar. diagnosis for both cases was confi rmed (orf1b and n gene pcr) by public health england. 15, 16 qatari authorities, with support from who, did an epidemiological investigation at the farm within 1 week of diagnosis of the fi rst case, which included the collection of various clinical samples from the farm's dromedary camels. full details of the outbreak investigation will be described elsewhere. we collected serum, rectal swabs, and nasal swabs from all camels present on the premises where the two men had been in contact with the animals. after an experienced animal handler fi xated the camel by nose pinching, samples were obtained through swabbing with fl ocked swabs (floqswabs, copan improve diagnostics, brescia, italy) that were inserted deep into one of the nostrils of the camel. after sampling, swabs were put into tubes containing viral transport medium-utm (copan diagnostics, brescia, italy). in addition, we obtained fi ve stool samples from three diff erent cages. the sample collection was done jointly by the communicable disease control outbreaks rapid response team of the public health department and the animal health team, which used full personal-protective equipment including n95 masks, goggles, disposable gowns, gloves, and head covers during the handling of the animals, and during the environmental and human sampling. serum samples were stored at -20°c and all other samples were stored at -80°c and were shipped to the netherlands on dry ice. we tested nose swabs with two independent rt-pcr targets specifi c for mers-cov (upe and nucleocapsid [n gene]) in one laboratory (department of viroscience, erasmus medical center, rotterdam, netherlands), and did rt-pcr testing with a third target (orf1a) in another laboratory (national institute of public health and the environment, bilthoven, netherlands). we inoculated vero and huh-7 cells for 1 h with cellculture medium containing samples from camel nose or rectal swabs, or with mers-cov (emc isolate) as a positive control. after washing, the cells were incubated with medium containing 1% fetal bovine serum at 37°c. we stained formaldehyde-fi xed huh-7 cells infected with mers-cov by use of heat-inactivated (at 56°c for 30 min) camel sera or a mers-cov patient's serum according to standard protocols with fl uorescein isothiocyanateconjugated antibodies as a second step. we isolated rna from 140 μl of swab medium or culture supernatant with the qiaamp viral rna minikit (qiagen, hilden, germany) and eluted it in 50 μl. camel mers-cov rna was quantifi ed on the abi prism 7700 with the taqman fast virus 1-step master mix (applied biosystems, bleiswijk, netherlands), with 10 μl isolated rna, one taqman mix, 0·5 u of uracil-n-glycosylase, primer, and probes targeting the n gene, upe, or orf1a as described elsewhere. 17, 18 we used rna dilutions isolated from a mers-cov isolate emc stock as a standard control. 10 μl of rna was reverse transcribed with the superscript iii fi rst strand synthesis system (invitrogen, bleiswijk, netherlands) with random hexamers. cdna was used to amplify six diff erent camel mers-cov fragments, including a partial spike gene by use of pfu ultra ii fusion hs dna polymerase (agilent technologies, santa clara, ca, usa). we did the pcr as follows: one initial denaturation step of 95°c for 5 min, followed by 39 cycles of 95°c for 20 s, 50°c for 20 s, 72°c for 2 min, and a fi nal extension of 72°c for 5 min. the amplifi ed camel mers-cov fragments were sequenced directly on both strands with the bigdye terminator version 3.1 cycle sequencing kit on an abi prism 3100 genetic analyser (applied biosystems). we generated phyml phylogenetic trees with seaview 4 software with the approximate likelihood-ratio test based on a shimodaira-hasegawa-like procedure, which used a general time reversible as substitution model. we used nearest neighbour interchange and subtree pruning and regrafting-based tree search algorithms to estimate the tree topologies. 19 rna from human samples (sputum for qatar 3 case and throat swab for qatar 4 case) was extracted with the nuclisens easymag system (biomérieux, basingstoke, uk). viral sequences from the two human cases were isolated (with the same techniques as for camel cases) in the reference department, public health england, london, uk. we used primers designed against sequences of mers-cov jx869059_emc/2012 and bat coronaviruses hku4 and hku5 to amplify the entire genomes from human cases for sequencing on an illumina miseq sequencer, subsequently indicated as qatar_3_2013 (case 1) and qatar_4_2013 (case 2). the sequences obtained in this study were deposited in genbank under the following accession numbers: camel_mers-cov/qatar_1_2013_orf1a_1_ partial, kf933380; camel_mers-cov/qatar_1_2013_ orf1a_3_partial, kf933381; camel_mers-cov/ qatar_1_2013_orf1b_partial, kf933382; camel_ mers-cov/qatar_1_2013_spike_partial, kf933383; camel_mers-cov/qatar_1_2013_orf4b_full, kf933384; camel_mers-cov/qatar_1_2013_orf1a_2_ partial, kf933385; qatar_3_2013, kf961221; and qatar_4_2013, kf961222. we tested all sera for the presence of igg antibodies reactive with mers-cov (residues 1-747), severe acute respiratory syndrome coronavirus (sars-cov; residues 1-676), and human coronavirus oc43 (residues 1-760) s1 antigens as described previously. 13, 20 we report results as the relative mean fl uorescent intensity (rfu) for each set of quadruplicate spots per antigen. we did igg antibody testing only because anti-camel igm conjugates were not available. for virus neutralisation, serum samples were heatinactivated by incubation for 30 min at 56°c. we prepared twofold serial dilutions with 96 well plates, starting dilution at 1/10. mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with penicillin, streptomycin, and 1% fetal bovine serum, to a dilution of 2000 tcid 50 per ml. we then added 50 μl of virus suspension to the plates and the plates were incubated at 37°c for 1 h. next, we incubated virus-serum mixtures on 96 well plates containing vero cells for 1 h, and then washed them with phosphate buff er saline and incubated them with imdm and 1% fetal bovine serum for 5 days. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. samples from 14 camels were obtained on oct 17, 2013 (table, fi gure 1). nose swab specimens from fi ve camels were positive for all three assays (upe, n gene, and orf1a), one sample reacted in two assays, and fi ve camels tested positive in only one rt-pcr assay. subsequently, all samples were subjected to a pcr assay targeting the spike gene of mers-cov (nucleotides 22449-22806) to obtain a fragment for sequencing as defi nitive confi rmation. such sequencing confi rmed the presence of sequences specifi c to mers-cov in three camels. alignment of these partial spike gene sequences with known human mers-cov sequences, including those determined from the two related human cases and some other human mers-cov isolates such as hafr-al-batin_1_2013 (kf600628) and riyadh_3_2013 (kf600613), showed 100% identity, but the sequences diff ered from emc mers-cov, which is used routinely in the erasmus mc laboratory, by one nucleotide. the three camel mers-cov sequences obtained by this approach were identical. virus culture attempts were not successful, although one sample tested for camel 7 yielded a positive rt-pcr of the supernatant (table) and staining of the fi xed virus-infected cells (data not shown). further passage of the virus on vero cells, however, was not successful. in addition, serum from camel 9 showed low levels of mers-cov as determined by rt-pcr, and all other camel sera tested negative (data not shown). rectal swabs and faecal materials were negative for mers-cov when tested for reactivity in the upe and n gene rt-pcrs. further sequencing of the virus from camel 5 with primers specifi c to mers-cov rendered six diff erent fragments covering 4·2 kb across the mers-cov genome (fi gure 1). fragments 1 (nucleotides 6708-7459), 2 (8095-9034), and 3 (10073-10658) were located in orf1a, fragment 4 (18792-19584) in orf1b, fragment 5 (22449-22806) in the spike gene, and fragment 6 (26074-26846) in orf4b. the sequences obtained from the human cases from the same farm diff ered by one nucleotide diff erence in orf1a and one in orf4b. the mers-cov emc isolate diff ers by eight nucleotides from the camel mers-cov in orf1a. alignment of the divergent 940 nucleotide orf1a fragment (data not shown) and a 4·2 kb concatenated sequence fragment was used for phyml phylogenetic analysis. the camel mers-cov clustered with viral sequences obtained from the two human cases related to the farm and with a sequence from hafr-al-batin as the next closest relative (fi gure 1). less stringent methods of analysis do show limited bootstrap support (data not shown), in line with relatively limited sequence variation within this fragment. all camel sera were positive in the immunofl uorescence assay when tested in a 1/200 dilution (fi gure 2). when serum samples from camels were tested at diff erent dilutions on the microarray, we noted reactivity with mers-cov antigen, but not the sars-cov antigen. reactivity to human coronavirus oc43 antigen, as a proxy to betacoronavirus, varied between negative (less than the cutoff value of 4000) and saturating signals (data not shown). consistent with the array data, all serum samples had mers-cov neutralising capacity, with titres varying between 160 and 5120 (fi gure 2). we also noted a strong correlation between microarray titres and virus neutralising antibody titres (fi gure 2). the two camels with the highest mers-cov load in the swabs as shown by taqman assay (camels 5 and 7) had a relatively low serum neutralisation titre of 640. we present, to our knowledge, the fi rst virological confi rmation of mers-cov in dromedary camels (panel). we and others previously reported mers-cov neutralising antibodies in dromedary camels from the canary islands, oman, and egypt, suggesting circulation of mers-cov or a mers-cov-like virus in camels. 13, 14 high prevalences and antibody titres were reported in the camels from oman and egypt suggesting widespread circulation. however, virological testing was unable to detect mers-cov viral sequences in camels, probably because only faecal and serum samples were analysed. in addition, shedding kinetics of mers-cov in camels (and human beings) are unknown, and therefore, inter pretation of negative rt-pcr results from screening is diffi cult because positive and negative predictive values remain to be determined. 22 in the outbreak reported, we showed evidence for presence of the virus in six camels according to internationally recommended criteria of two independent rt-pcr targets. furthermore, we used sequence confi rmation as an additional test to increase the level of certainty-during an emerging disease outbreak such as this, options for validation of assays for clinical testing are limited. 22 discrepancies between assays can be explained by diff erences in detection limits, or by diff erences in specifi city of the primer sets. in our study, n gene rt-pcr yielded four additional weak positive results that could not be confi rmed by any other method, and an individual orf1a rt-pcr identifi cation was made for only one animal. these cases could have resulted from mispriming, or represent presence of levels of virus around the detection limit, as can be expected at the end of the infection cycle. nevertheless, our results suggest a widespread and recent outbreak in this herd, coinciding with infection in two people. our data should not be taken as evidence for infection of people from the camels. comparison of sequence data from the orf1a gene and a concatenated 4·2 kb sequence from the human and camel viruses showed that these viruses are very similar, but distinct. notably, samples from camels and people were analysed in diff erent laboratories in the netherlands and the uk. the data provided show proof that camels can be infected with mers-cov. the sequence diff erence between human and animal viruses from the farm, based on the available overlapping sequence, is so small that we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels were infected from a third as yet unknown source. although additional sequencing might provide improved resolution, it probably will not provide conclusive evidence. the most important unknown is the exact timing of infections, both in the infected people and camels. if the virus entered the farm through either the people or the camels, the infections observed would probably have resulted from a chain of transmissions that introduced mutations. because we did not identify any seronegative animals that were pcr positive, and because all animals had mers-cov neutralising antibodies, our sampling probably took place relatively late in the outbreak on this farm. a more detailed analysis of the outbreak, including testing of additional animals and environmental samples is ongoing, as are attempts to obtain full mers-cov genomes of the human and animal specimens. we cannot exclude the possibility that other common livestock species including cattle, sheep, and goats, or other animals including wild species were involved in the spread of mers-cov. while confi rmation of the source is awaited, we recommend that a detailed case history is taken of any cases of mers-cov, including review of any animal exposures (including animal products), and targeted (prospective) serosurveys to determine what risk factors are associated with human infection. we searched pubmed for articles published in english up to dec 1, 2013, with the search terms "novel coronavirus emc" or "mers-cov". we identifi ed 121 reports linked to the middle east respiratory syndrome coronavirus (mers-cov). one report 21 described the detection in one bat specimen of a single 181 bp fragment in a highly conserved area of the coronavirus genome, identical to the human mers-cov laboratory isolate emc. our report describes the fi rst detection of mers-cov in dromedary camels on a farm in qatar that had been linked to human cases of the disease. our study provides proof that mers-cov has infected dromedary camels in qatar. our results suggest the simultaneous occurrence of a mers-cov outbreak in people and camels. on the basis of the present data, we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels could have been infected from a third as yet unknown source. we recommend that detailed case histories be taken, including review of any animal exposures including animal products, and targeted (prospective) serosurveys to determine what risk factors-other than contact with an individual with the infection-are associated with human infection. blh wrote the report, drew the fi gures, and generated, analysed, and interpreted data. shsad, mma, and mpgk coordinated the study, wrote the report, and interpreted data. cbemr wrote the report, drew the fi gures, did the literature search, and analysed and interpreted data. vsr drew the fi gures, and generated, analysed, and interpreted data. mg, rm, and ab generated and interpreted data. gjg generated, analysed, and interpreted data. mj generated and analysed data. ef led the fi eld investigation, obtained samples, and collected and interpreted data. ad supervised fi eld outbreak management and collected and interpreted data. hg and fa supervised animal health fi eld investigations. ma-t coordinated the study and interpreted data. saa-m coordinated the study. hear led outbreak management and collected and interpreted data. aak coordinated the study and interpreted data. admeo wrote the report and interpreted data. blh, admeo, and vsr hold a pending patent for mers-cov. admeo is scientifi c adviser of viroclinics biosciences. all other authors declare that they have no confl icts of interest. doi:10.1371/ currents.outbreaks.0bf719e352e7478f8ad85fa30127ddb8. 2 who. global alert and response (gar): novel coronavirus infection-update ecology, evolution and classifi cation of bat coronaviruses in the aftermath of sars close relative of human middle east respiratory syndrome coronavirus in bat human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt alert and response (gar): novel coronavirus infection-update alert and response (gar): novel coronavirus infection-update detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building specifi c serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus: data gaps for laboratory preparedness we thank who and the food and agriculture organization of the united nations for their generous help in organisation of the study, theo bestebroer for providing middle east respiratory syndrome coronavirus specifi c primer sets, berend jan bosch for providing antigens for the microarray, and jeroen cremer for technical support. contributions to the study were funded through the european union fp7 projects emperie (contract number 223498; to blh and admeo) and antigone (contract number 278976; to mpgk and admeo). key: cord-268074-9mact9br authors: bi, qifang; wu, yongsheng; mei, shujiang; ye, chenfei; zou, xuan; zhang, zhen; liu, xiaojian; wei, lan; truelove, shaun a; zhang, tong; gao, wei; cheng, cong; tang, xiujuan; wu, xiaoliang; wu, yu; sun, binbin; huang, suli; sun, yu; zhang, juncen; ma, ting; lessler, justin; feng, tiejian title: epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study date: 2020-04-27 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30287-5 sha: doc_id: 268074 cord_uid: 9mact9br background: rapid spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in wuhan, china, prompted heightened surveillance in shenzhen, china. the resulting data provide a rare opportunity to measure key metrics of disease course, transmission, and the impact of control measures. methods: from jan 14 to feb 12, 2020, the shenzhen center for disease control and prevention identified 391 sars-cov-2 cases and 1286 close contacts. we compared cases identified through symptomatic surveillance and contact tracing, and estimated the time from symptom onset to confirmation, isolation, and admission to hospital. we estimated metrics of disease transmission and analysed factors influencing transmission risk. findings: cases were older than the general population (mean age 45 years) and balanced between males (n=187) and females (n=204). 356 (91%) of 391 cases had mild or moderate clinical severity at initial assessment. as of feb 22, 2020, three cases had died and 225 had recovered (median time to recovery 21 days; 95% ci 20–22). cases were isolated on average 4·6 days (95% ci 4·1–5·0) after developing symptoms; contact tracing reduced this by 1·9 days (95% ci 1·1–2·7). household contacts and those travelling with a case were at higher risk of infection (odds ratio 6·27 [95% ci 1·49–26·33] for household contacts and 7·06 [1·43–34·91] for those travelling with a case) than other close contacts. the household secondary attack rate was 11·2% (95% ci 9·1–13·8), and children were as likely to be infected as adults (infection rate 7·4% in children <10 years vs population average of 6·6%). the observed reproductive number (r) was 0·4 (95% ci 0·3–0·5), with a mean serial interval of 6·3 days (95% ci 5·2–7·6). interpretation: our data on cases as well as their infected and uninfected close contacts provide key insights into the epidemiology of sars-cov-2. this analysis shows that isolation and contact tracing reduce the time during which cases are infectious in the community, thereby reducing the r. the overall impact of isolation and contact tracing, however, is uncertain and highly dependent on the number of asymptomatic cases. moreover, children are at a similar risk of infection to the general population, although less likely to have severe symptoms; hence they should be considered in analyses of transmission and control. funding: emergency response program of harbin institute of technology, emergency response program of peng cheng laboratory, us centers for disease control and prevention. since emerging in wuhan, china, in december, 2019, 1 the coronavirus disease 2019 (covid-19) epidemic caused by severe acute respiratory syndrome corona virus 2 (sars-cov-2) has progressed rapidly into a pandemic. covid-19 is characterised by fever, cough, fatigue, shortness of breath, pneumonia, and other respiratory tract symptoms, [2] [3] [4] and in many cases progresses to death. as of april 15, 2020, there have been 1 914 916 confirmed cases and 123 010 deaths reported worldwide. 5 most cases were initially confined to hubei province in china, but there has since been substantial spread not only elsewhere in china but worldwide. a rapid and robust response by the global scientific community has described many important aspects of sars-cov-2 transmission and natural history, 1,2,6-8 but key questions remain. if well tracked, early introductions of an emerging pathogen provide a unique opportunity to characterise its transmission, natural history, and the effectiveness of screening. careful monitoring of cases and low probability of infection from the general community enables inferences, important to modelling the course of the outbreak, that are difficult to make during a widely disseminated epidemic. in particular, we can make assumptions about when and where cases were likely to have been infected that are impossible when the pathogen is widespread. furthermore, during these early phases, uninfected and asymptomatic contacts are often closely tracked, providing important information about transmission and natural history. combined, these data on early introductions can be used to give insights into the natural history of the disease, 9 transmission characteristics, 10 and the unseen burden of infection. 11 here, we use data collected by the shenzhen center for disease control and prevention (shenzhen cdc) on 391 cases of covid-19 and 1286 of their close contacts to characterise key aspects of its epidemiology outside of hubei province. we characterise differences in demographics and severity between cases identified through symptom-based surveillance and monitoring of close case contacts, and estimate the time to key events, such as confirmation, isolation, and recovery. using data from contact tracing, we characterise sars-cov-2 transmission by estimating key values, such as the household secondary attack rate, serial interval, and observed reproductive number (r). on jan 8, 2020, shenzhen cdc identified the first case of pneumonia with unknown cause and began monitoring travellers from hubei province for symptoms of covid-19. over the next 2 weeks this surveillance programme expanded to include travellers from hubei regardless of symptoms, patients at local hospitals, and individuals detected by fever screening in neighbourhoods and at local clinics. suspected cases and close contacts were tested for sars-cov-2 by rt-pcr of nasal swabs at 28 qualified local hospitals, ten district-level cdcs, and two third-party testing organisations, with final confirmation done at the guangdong provincial center for disease control and prevention (guangdong cdc) or shenzhen cdc (appendix 2 p 11). close contacts were identified through contact tracing of a confirmed case and were defined as those who lived in the same apartment, shared a meal, travelled, or socially interacted with an index case 2 days before symptom onset. casual evidence before this study the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been accompanied by a substantial increase in research and publications ranging from transmissibility of sars-cov-2 to clinical characteristics of the infection. as of march 23, 2020, our search of pubmed using keywords ("covid-19" or "sars-ncov-2" or "novel coronavirus") and ("serial interval" or "incubation period" or "attack rate") yielded 14 articles that have estimated either the serial interval or incubation period of coronavirus disease 2019 (covid19) and one that investigated the secondary attack rate. however, most of these estimates have come from either wuhan or publicly available case data; in both instances, incomplete capture of infections and cases might have produced biased estimates. this study is, to our knowledge, the first analysis of sars-cov-2 transmission and covid-19 natural history based on a large primary dataset of cases and close contacts, for which the mode of surveillance (ie, symptom-based versus contact-based) was sufficiently documented and rt-pcr testing was nearly universal. we present one of the first estimates of the serial interval, secondary household attack rate, and dispersion (ie, tendency towards super spreading) for sars-cov-2 based on active surveillance data. we found that the attack rate does not differ significantly by age, with on average 7% of close contacts becoming infected, around 80% of these contacts showing any symptoms, and 3% of infections manifesting severe disease at initial assessment. we also found that contactbased surveillance in shenzhen reduced the duration an infected individual transmits in the community by 2 days. these findings are important for understanding the burden of covid-19 and for strategic planning across the world. these results shed further light on how sars-cov-2 is transmitting, how severe it is, and how effective control measures can be in specific contexts. we provide a key piece of evidence supporting intensive contact tracing and highlighting that children might be an important target for interventions aimed at reducing transmission, even if they do not get sick. contacts (eg, other clinic patients) and some close contacts (eg, nurses) who wore a mask during exposure were not included in this group. symptomatic cases were isolated and treated at designated hospitals regardless of rt-pcr test results. asymptomatic individuals who tested positive were quarantined at centralised facilities. close contacts and travellers from hubei who tested negative were quarantined at home or a central facility, and monitored for 14 days. rt-pcr testing was required for all close contacts at the beginning of isolation, and release was conditional on a negative rt-pcr result. basic demographics, signs and symptoms, clinical severity, and exposure history were recorded for all confirmed cases. here, we analyse confirmed cases identified by the shenzhen cdc between jan 14 and feb 12, 2020, and close contacts of cases confirmed before feb 9, 2020. this work was done in support of an ongoing public health response, and hence was determined not to be human subjects research after consultation with the johns hopkins bloomberg school of public health institutional review board. data collection is part of the continuing public health investigation of an emerging outbreak and therefore the individual informed consent was waived. the study was approved by the ethics committees of shenzhen cdc. analytical datasets were constructed in an anonymised manner, and all analysis of personally identifiable data took place onsite at the shenzhen cdc. we defined symptom-based surveillance to include symptomatic screening at airport and train stations, community fever monitoring, home observation of recent travellers to hubei, and testing of patients admitted to hospital. contact-based surveillance is the identification of cases through monitoring and testing of close contacts of confirmed cases, independently of their symptom presentation. by protocol, those in the contact-based group were tested for sars-cov-2 infection regardless of symptoms, whereas those in the other categories were tested only if they showed signs or symptoms of disease. at the first clinical assessment, data were recorded on 21 signs and symptoms (appendix 2 p 1), and disease severity was assessed. cases with fever, respiratory symp toms, and radiographic evidence of pneumonia were classified as having moderate symptoms. cases were classified as having severe symptoms if they had any of the following: breathing rate 30 breaths per min or higher; oxygen saturation level 93% or lower at rest; oxygen concentration level pao 2 /fio 2 (ratio of arterial oxygen partial pressure to fractional inspired oxygen) 300 mm hg (1 mm hg=0·133 kpa) or lower; lung infiltrates higher than 50% within 24-48 h; respiratory failure requiring mechanical ventilation; septic shock; or multiple organ dysfunction or failure. all other symptomatic cases were classified as mild. relationships between demographics, mode of detection, and symptom severity were assessed and characterised with χ² tests, and simple and multiple logistic regression. distributions were fit to the timing of key events in each confirmed case's course of infection and treatment. the time from infection to symptom onset (incubation period) was assumed to be log-normally distributed and estimated as previously described. [12] [13] [14] we determined the left and right boundaries on the possible exposure and symptom onset times. cases who recently travelled to hubei were assumed to have been exposed while there. cases without a recent travel history but with exposure to a confirmed case were assumed to be exposed from the time of earliest to latest possible contact with that case. only cases for whom we could identify the earliest and latest possible time of exposure and who had a date of symptom onset were included in the analysis. time between symptom onset and recovery was estimated by use of parametric survival methods. patients who had not recovered were considered to be censored on feb 22, 2020, or at the time of death. all other delay distributions were estimated by directly fitting parametric distributions to time between symptom onset or arrival in shenzhen, and confirmation, isolation, or admission to hospital. confidence intervals were calculated with bootstrapping or standard parametric estimators. 15 transmission was characterised by examining the relation ship between confirmed cases and their infected and uninfected close contacts. the household secondary attack rate was calculated as the percentage of household contacts (those sharing a room, apartment, or other sleeping arrangement) who were later confirmed to have sars-cov-2 infection. the distribution of serial intervals (the time between symptom onset in the confirmed case and their infected contacts) was calculated by fitting parametric distributions to the time of symptom onset in clear case-contact pairs. the mean r and distribution of individual reproductive numbers (ie, the number of secondary infections caused by each case) were calculated from the number of secondary infections observed among close contacts of each index case, with ambiguities resolved through multiple imputation (appendix 2 p 11). the relative odds of transmission among contacts of various types were estimated by use of conditional logistic regression and random-effects models, to account for differing numbers of possible infected individuals in each risk group. when assessing the impact of characteristics of infected individuals, we only included risk sets where a single potential infected individual was clearly identi fiable. confidence intervals were estimated by use of bootstrapping or standard parametric approaches. the median incubation period of covid-19 is estimated to be 4·8 days (95% ci 4·2-5·4). 5% of cases who develop symptoms will do so by 1·6 days (95% ci 1·3-2·0) after infection, and 95% by 14·0 days (12·2-15·9). we estimated that the median serial interval of covid-19 is 5·4 days (95% ci 4·4-6·5). 5% of infected cases who develop symptoms will do so by 1·3 days (95% ci 0·9-1·9) after symptom onset of the index case, and 95% by 14·3 days (11·1-17·6) . the sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. shenzhen cdc had full access to all the data in the study, and all corresponding authors share the final responsibility for the decision to submit for publication. between jan 14 and feb 12, 2020, the shenzhen cdc confirmed 391 cases of sars-cov-2 infection ( of 87) than were those detected through contact-based surveillance (tables 1, 2). at the time of the first clinical assessment, 25 (29%) of 87 cases in the contact-based surveillance group did not have fever, and 17 (20%) of 87 had no symptoms. by contrast, 258 (88%) of 292 in the symptom-based surveillance group had fever, and only eight reported no symptoms. in multiple logistic regression, male sex was associated with severe symptoms (odds ratio [or] 2·5 [95% ci 1·1-6·1]). the probability of severe symptoms increased slightly with age, although only individuals aged 60-69 years had a significantly increased risk compared with the reference category, individuals aged 50-59 years (or 3·4 [95% 1·4-9·5]). based on 183 cases with a well defined period of exposure and symptom onset (appendix 2 p 8), we estimated the median incubation period for covid-19 to be 4·8 days (95% ci 4·2-5·4; figure 1, appendix 2 p 2), the maximum-likelihood estimates for the parametric distribution of the cumulative distribution function are shown, along with 1000 parametric bootstrap estimates of the cumulative distribution function. panel a shows estimates of the proportion of cases who are confirmed by rt-pcr, according to the number of days after symptom onset. we estimated that 50% of cases detected through symptom-based surveillance were confirmed by rt-pcr within 4·6 days (95% ci 4·2-5·0) after symptom onset, and 95% were confirmed by rt-pcr within 12·7 days (11·5-13·8) after symptom onset. contact-based surveillance reduced the days from symptom onset to rt-pcr confirmation to 2·9 days (95% ci 2·4-3·4) in 50% of cases and to 6·6 days (5·3-8·0) in 95% of cases. panel b shows estimates of the proportion of cases who were admitted to hospital, according to the number of days after symptom onset. we estimated that 50% of the cases detected through symptom-based surveillance were admitted to hospital by 3·4 days (95% ci 3·1-3·8) after symptom onset, and 95% by 12·4 days (10·9-13·8). contact-based surveillance reduced the days from symptom onset to hospital admission to 2·1 days (95% ci 1·7-2·6) in 50% of cases, and 6·0 days (95% ci 4·5-7·5) in 95% of cases. panel c shows estimates of the proportion of cases isolated, according to number of days after symptom onset. we estimated that 50% of cases detected through symptom-based surveillance were isolated by 3·4 days (95% ci 3·1-3·7) after symptom onset, and 95% by 12·2 days (95% ci 10·8-13·6). contact-based surveillance reduced the days from symptom onset to isolation to 2·2 days (95% ci 1·7-2·6) in 50% of cases, and to 6·5 days (4·7-8·2) in 95% of cases. and estimated that 95% of those who develop symptoms will do so within 14·0 days (95% ci 12·2-15·9) of infection. we estimated that about 5·0% of cases who develop symptoms would not show symptoms until 14 days after infection. based on 228 cases with known outcomes, we estimated that median time to recovery was 20·8 days (95% ci 20·1-21·5). we estimated that the median time to recovery was 22·4 days (95% ci 20·8-24·1) in individuals aged 50-59 years, and was estimated to be significantly shorter in younger adults (eg, 19·2 days in individuals aged 20-29 years; appendix 2 pp 3, 10). in multiple regression models including sex, age, baseline severity, and method of detection, in addition to age, baseline severity was associated with time to recovery (appendix 2 p 3). compared to cases with mild symptoms, those with severe symptoms had a 41% (95% ci 24-60) longer time to recovery (appendix 2 p 3). as of feb 22, 2020, three cases had died. these deaths occurred 35-44 days from symptom onset and 27-33 days from confirmation. cases detected through symptom-based surveillance were confirmed on average 5·5 days (95% ci 5·0-5·9) after symptom onset (figure 2, appendix 2 p 2), whereas those detected by contact-based surveillance were confirmed on average 3·2 days (95% ci 2·7-3·8) after symptom onset. 17 (5%) of 342 cases with a known onset date and start date of quarantine were isolated before developing symptoms. among those isolated after developing symptoms, the symptom-based surveillance group was, on average, isolated 4·6 days (95% ci 4·1-5·0) after symptom onset, whereas the contact-based sur veillance group was isolated 2·7 days (2·1-3·3) after symptom onset. hence, contact-based surveillance was associated with a 2·3-day (95% ci 1·5-3·0) decrease in time to confirmation and a 1·9-day (1·1-2·7) decrease in time to isolation. the mean time between symptom onset and admission to hospital was similar to time between symptom onset and isolation in both the symptom-based and contact-based surveillance groups (figure 2, appendix 2 p 2). 191 (64%) of 298 travellers developed symptoms after arriving in shenzhen, with a mean time from arrival to symptom onset of 4·9 days (95% ci 4·2-5·5; appendix 2 p 2). those developing symptoms before arrival or on the day of arrival were confirmed as cases on average 4·5 days (95% ci 3·8-5·1) after arrival, and isolated on average 3·1 days (2·5-3·7) after arrival. sars-cov-2=severe acute respiratory syndrome coronavirus 2. inf=infinity. *15 confirmed close contacts were excluded from this analysis because contact tracing reports for the negative close contacts in the same clusters were missing. close contacts with missing data on sex, age, contact types, or contact frequency not shown. overall, 1286 close contacts were identified for index cases testing positive for sars-cov-2 between jan 14 and feb 9, 2020, with 244 (84%) of 292 cases having at least one close contact. 622 (95%) of 653 close contacts with known dates for the period when they were under quarantine were followed up for 12 days or longer. 98 of the close contacts tested positive for sars-cov-2 infection by rt-pcr, and one had presumptive infection. assuming those with a missing test result were uninfected, we found that the secondary attack rate was 11·2% (95% ci 9·1-13·8) among household contacts and 6·6% (5·4-8·1) overall (the secondary attack rate increased to 14·9% [12·1-18·2] among household contacts and 9·7% [7·9-11·8] overall if those with missing results were removed from the denominator). in multiple conditional logistic regression analysis of contact types, household contact (or 6·3; 95% ci 1·5-26·3) and travelling together (or 7·1; 1·4-34·9) were significantly associated with infection (table 3) . reporting contact that occurred often was also associated with increased risk of infection compared with moderate-frequency contact (or 8·8; 95% ci 2·6-30·1; table 3) . attack rates were similar across all age categories of infected contacts (table 3) , although we observed some indication of elevated attack rates in older age groups ( figure 3) . notably, the rate of infection in children younger than 10 years (7·4%) was similar to the population average (6·6%). there was no significant association between probability of infection and age of the index case. surprisingly, in univariate analysis a longer time in the community before isolation was associated with a reduced risk of causing infections (data not shown). however, this association was no longer significant after adjusting for contact frequency and type. based on 48 pairs of cases with a clear relationship between the index case and secondary case and time of symptom onset, we estimated that the serial interval is gamma distributed with a mean of 6·3 days (95% ci 5·2-7·6) and an sd of 4·2 days (95% ci 3·1-5·3; figure 1b, appendix 2 p 2). hence, 95% of secondary cases were expected to develop symptoms within 14·3 days (95% ci 11·1-17·6) of their infector. this estimate includes the effect of isolation on truncating the serial interval. stratified results show that if the infected individual was isolated less than 3 days after infection the average serial interval was 3·6 days, increasing to 8·1 days if the infected individual was isolated on the third day after symptom onset or later (appendix 2 p 3). the mean number of secondary cases caused by each index case (ie, the observed reproductive number, r), was 0·4 (95% ci 0·3-0·5). the distribution of individual r values was highly over-dispersed, with 80% of infections being caused by 8·9% (95% ci 3·5-10·8) of cases (negative binomial dispersion parameter 0·58; 95% ci 0·35-1·18). we examined the potential impact of surveillance and isolation through truncating the infectious period. because of the scarce understanding of infectious periods following sars-cov-2 infection, we considered a range of possible infectious periods where infectiousness varies over time and follows a gamma distribution (appendix 2 p 11). we defined the mean infectious day (ie, the average number of days after symptom onset that an infected individual is expected to infect a secondary case) as the weighted mean of the infectious period, where each day is weighted by relative infectiousness. we considered periods where the mean infectious day is less than 15 days after symptom onset (roughly the period of sars and early sars-cov-2 reports 16, 17 ) , and assumed that r=2·6 and that isolation effectively ends the infectious period. under these assumptions we found that if the mean infectious day is greater than 5 days, then it might be possible to bring r below one in cases detected by symptom-based surveillance, and the same can be accomplished by contact-based surveillance if the mean infectious day is greater than 3 days (appendix 2 p 9). for the impact of passive surveillance alone to achieve our observed r of 0·4, we projected that the mean infectious day must be at least 5·5 days (and likely greater) after symptom onset. even if transmission is completely eliminated in the group captured by surveillance (eg, if we could get perfect surveillance on the day of symptom onset), assuming r=2·6, the cases captured by surveillance must, if not isolated, be expected to cause 61% of onward transmission to achieve local elimination by surveillance and isolation alone (appendix 2 p 9). this analysis of early sars-cov-2 cases and their close contacts in shenzhen, china, provides insight into the natural history, transmission, and control of this disease. attack rate proportion with severe symptoms proportion without fever figure 3 : attack rate among close contacts, baseline severity, and proportion of cases without fever at initial assessment by age group *proportion of close contacts for attack rate; proportion of all cases for those with severe symptoms or no fever at initial assessment. the values estimated provide the evidentiary foundation for predicting the impact of this virus, evaluating control measures, and guiding the global response. analyses of how cases are detected, and use of data on individuals exposed but not infected, indicate that infection rates in young children are not lower than the population average (even if rates of clinical disease are). we were able to directly estimate important transmission parameters, and show that, at least among observed contacts, transmission rates are low. estimates of the distribution of time between symptom onset and case isolation by surveillance type reveal that heightened surveillance combined with case isolation could plausibly account for these low rates of transmission. these results paint a positive picture of the impact of heightened surveillance and isolation in shenzhen. however, uncertainty in the number of asymptomatic cases missed by surveillance and their ability to transmit sars-cov-2 must temper any hopes of stopping the covid-19 pandemic by these measures. this work further supports the understanding of covid-19 as a disease with a fairly short incubation period (mean 4-6 days) but a long clinical course, 2,7,18 with patients taking many weeks to die or recover. notably, however, we estimate a higher proportion of cases taking 14 days or more to develop symptoms (5%) than estimated in the study by lauer and colleagues (1%). 6 focusing on cases detected through contact-based surveillance adds nuance to previous characterisations of covid-19. since rt-pcr testing of contacts is near universal, we can assume these cases are more reflective of the average sars-cov-2 infection than cases detected through symptomatic surveillance. in the contact-based surveillance group, any tendency for cases to be male or older (beyond the underlying population distribution) disappears. furthermore, in this group, 20% of cases were asymptomatic at the time of first clinical assessment and nearly 30% did not have a fever. this observation is consistent with a reasonably high rate of asymptomatic carriage, but lower than that suggested by some modelling studies, 19 although rt-pcr has imperfect sensitivity. 20 in shenzhen, sars-cov-2 transmission most probably occurred between very close contacts, such as individuals sharing a household. however, even in this group fewer than one in six contacts (ie, secondary attack rate 11-15%) were infected, and overall we observed far fewer than one (0·4) onward transmission per primary case. as noted above, low transmission levels might in part be due to the impact of isolation and surveillance, but it is equally likely that unobserved trans mission has some role. we also estimated reasonably high rates of overdispersion in the number of cases caused by each infected individual, leaving open the possibility that large covid-19 clusters can occur even if surveillance and isolation are forcing r below one-events that could potentially overwhelm the surveillance system. this work has numerous limitations. as in any active outbreak response, the data were collected by multiple teams under protocols that, by necessity, changed as the situation developed. hence, there might be noise and inconsistency in definitions. notably, the definition of a confirmed case changed to require symptoms near the end of our analysis period (feb 7), but sensitivity analyses show that truncating the data at this point does not qualitatively influence results (appendix 2 pp 5-6). it is, likewise, impossible to identify every potential contact an individual has, so contact tracing focuses on those close contacts who are most likely to be infected; hence our r is assuredly lower than the true reproductive number in the population. asymptomatic travellers will be missed by symptom-based surveillance and, even if they are tested, some asymptomatic contacts might be missed because of the imperfect sensitivity of the rt-pcr test. 20 recovery time in shenzhen is likely to be inflated because cases are required to be isolated for 2 weeks and release is conditional on a negative rt-pcr test. although guidelines for contact tracing and case detection are supposed to be implemented across the country, whether the impact of contact tracing can be generalised to other parts of china depends on a range of factors, including local testing capacity and surveillance resources. as sars-cov-2 continues to spread, it is important that we continue to expand our knowledge about its transmission and natural history. data from the early phase of local outbreaks, when detailed contact tracing is possible and sources of infection can still be reliably inferred, are particularly powerful for estimating critical values pertinent to describing transmission and the natural history of a disease. this is especially true when information about uninfected contacts and mode of detection is used, as we have done here. the resulting estimates provide important inputs for interpreting surveillance data, evaluating interventions, and setting public health policy. yow, sm, xz, zz, xl, lw, wg, cc, xt, xw, yuw, sh, and tf collected the data. jl, qb, sat, and cy did statistical analyses, and drafted the manuscript and figures. tz, bs, ys, jz, tm, and cy collected and cleaned data. qb, tm, jl, and tf conceived the study and supervised data collection. we declare no competing interests. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia 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 2019 novel coronavirus-infected pneumonia in wuhan, china epidemiological and clinical features of the 2019 novel coronavirus outbreak in china covid-19) situation reports the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster outbreak of 2009 pandemic influenza a (h1n1) at a new york city school transmission dynamics and control of severe acute respiratory syndrome estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia analysis of coarsely observed data estimating incubation period distributions with coarse data estimating absolute and relative case fatality ratios from infectious disease surveillance data a leisurely look at the bootstrap, the jackknife, and cross-validation clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study sars-cov-2 viral load in upper respiratory specimens of infected patients time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia report 3: transmissibility of 2019-ncov sensitivity of chest ct for covid-19: comparison to rt-pcr tm, cy, tz, bs, ys, and jz were funded by the emergency response program of harbin institute of technology (hiterp010) and emergency response program of peng cheng laboratory (pclerp001). jl, sat, and qb were funded by a grant from the us centers for disease control and prevention (nu2ggh002000). we thank a azman, d cummings, s lauer, j wallinga, and m mina for advice and input on the manuscript and analyses. we thank all patients, close contacts, and their families involved in the study, as well as the front-line medical staff and public health workers who collected these important data. key: cord-318683-1yxurnev authors: green, manfred s; leduc, james; cohen, daniel; franz, david r title: confronting the threat of bioterrorism: realities, challenges, and defensive strategies date: 2018-10-16 journal: lancet infect dis doi: 10.1016/s1473-3099(18)30298-6 sha: doc_id: 318683 cord_uid: 1yxurnev global terrorism is a rapidly growing threat to world security, and increases the risk of bioterrorism. in this review, we discuss the potential threat of bioterrorism, agents that could be exploited, and recent developments in technologies and policy for detecting and controlling epidemics that have been initiated intentionally. the local and international response to infectious disease epidemics, such as the severe acute respiratory syndrome and west african ebola virus epidemic, revealed serious shortcomings which bioterrorists might exploit when intentionally initiating an epidemic. development of new vaccines and antimicrobial therapies remains a priority, including the need to expedite clinical trials using new methodologies. better means to protect health-care workers operating in dangerous environments are also needed, particularly in areas with poor infrastructure. new and improved approaches should be developed for surveillance, early detection, response, effective isolation of patients, control of the movement of potentially infected people, and risk communication. access to dangerous pathogens should be appropriately regulated, without reducing progress in the development of countermeasures. we conclude that preparedness for intentional outbreaks has the important added value of strengthening preparedness for natural epidemics, and vice versa. the biological weapons convention prohibits the manufacture and use of biological weapons. it came into force in 1975, and has undergone periodic reviews, the last being in 2016. to date, 180 countries are signatories to the convention. unfortunately, terrorist groups or rogue governments are unlikely to feel bound by international agreements. the potential for bioterrorism is of particular concern, since it can cause disease, death, and panic-in great disproportion to the resources expended. 1 there have been a few well documented cases of bioterrorism. in 1984, a religious sect in the usa deliberately contaminated restaurant salad bars with salmonella typhimurium, intending to disrupt local elections. 2 the attack resulted in several hundred cases of salmonellosis and no deaths. the anthrax letters incident in 2001 in the usa resulted in 11 cases of inhalation anthrax, with five deaths, and another 11 cases of cutaneous disease. 3 extensive circumstantial evidence strongly suggests that the perpetrator was a civilian employee of the us military. however, no evidence of a clear motive was found. thousands of workers received prophylactic or post-exposure therapy, and affected buildings were decontaminated at huge expense. 4, 5 in 1993, a cult in japan carried out an attack using anthrax spores with no physical casualties, 6 but later, evidence of post-traumatic stress syndromes was found in victims of the attack. 7 the perpetrators were apparently planning to use other agents such as q fever bacteria, botulinum toxin, and ebola viruses, 8 but they were detained before they could implement further attacks. in this review, we discuss the threat of bioterrorism, potential perpetrators, and general preparedness principles. we examine the special characteristics of biological agents that could potentially be used for bioterrorism, advances in prevention and treatment of diseases caused by these agents, and the remaining deficiencies in the management and control of possible bioterrorist outbreaks. in all respects, the ways in which the resources developed for bioterrorism preparedness could be used for controlling naturally occurring epidemics remain a guiding principle. • preparedness for intentional outbreaks will strengthen the response to naturally occurring epidemics • high level leadership should be maintained with responsibility and authority • health-care providers should maintain awareness of biological agents with bioterrorism potential and consider the presence of unknown pathogens • emergency room and community physicians should be updated regularly about the clinical manifestations of diseases caused by potential bioterrorist agents and emerging infectious diseases. • personal protective equipment should be improved to become more user friendly • improved surge capacity (the ability to rapidly gear up the health system to cope with a sudden, large increase in patients with a serious, contagious disease) is required, particularly in peripheral areas • the capacity of general and reference laboratories should be increased, to keep developing faster, more reliable diagnostic tests • new and improved vaccines (pre-exposure and post-exposure) and treatment regimens should be developed • clinical and environmental surveillance needs to increase • syndromic surveillance systems can be maintained to register suspicious or confirmed cases reported by physicians, and the data can be used to improve risk communication programmes and to monitor the progress of an outbreak • an adequate stockpile of vaccines and medications should be maintained, both nationally and internationally • to improve preparedness for natural and bioterrorist outbreaks, international cooperation should include joint exercises involving multiple countries and constant improvement in the exchange of information on potential bioterrorism threats and management following the breakup of the former soviet union, there was concern that loss of control of their biological weapons programme could allow terrorist groups to gain access to both the weapons and scientific expertise. additionally, in the past few years, developments in the field of microbial genetics have heightened concern about the possible abuse of new technologies. since there are so many unknowns, it is extremely difficult to assess the risks and threats of bioterrorism. 9, 10 the most likely perpetrators could be disgruntled individuals, terrorist organisations, or rogue countries that are believed to support international terrorism. whereas individual attackers are unlikely to cause mass casualties, terrorist organisations could pose a substantial threat if they gain access to sophisticated biological weapons, materials, or scientific expertise. although regulations and safeguards for securing dangerous pathogens in research laboratories now exist in most countries, the scope of these regulations and the extent of the safeguards vary. 11 rogue countries have the necessary capabilities for a bioterrorist attack but might be restrained by the threat of the response of a unified global community. knowledge gained from legitimate research that could also be applied to bioterrorism 12 is considered dual-use. as a result, the regulation of legitimate research on infectious diseases has increased. there will always be a risk of the "insider threat", 12,13 which typically involves a single individual, so it is important to assure that new regulations truly increase security and have minimal negative effect on legitimate research. the cost of regulations applied to research on infectious diseases, in terms of missed opportunities for international collaboration, exchange of pathogens, and sharing of novel agents, is often intangible and overlooked. it is essential to promote healthy organisational cultures to enhance both safety and security in laboratories. 14 since a bioterrorist attack is a low-risk, high-impact event, effective and sustained preparedness is an essential component in both the deterrence and management of an attack. a bioterrorist attack has a lot in common with naturally occurring public health emergencies resulting from infectious diseases. however, there are some important differences. since it is a deliberate act to cause harm, there are the obvious security considerations. the resulting outbreak differs in some important ways from naturally occurring epidemics-for instance, it is more likely to be a point source outbreak initiated by simultaneous exposure to many people. the infectious agent used is likely to be uncommon and possibly not endemic to the region, might have been modified genetically to make it resistant to current medications and vaccines, and produced in a way that enhances its transmission or virulence. therefore, early clinical symptoms and signs after infection with a bioterrorist agent might be unusual, complicating both recognition and management of the disease. these factors could create greater public panic. despite the many similarities with naturally occurring outbreaks of infectious disease, preparedness for bioterrorist attacks is more complex. in many aspects, a bioterrorist attack has the characteristics of a mass casualty event, and thus preparedness involves strengthening of the specialised infrastructure that is required for treatment of seriously ill patients over a very short period of time. new prophylactic and treatment regimens for unusual diseases are required, to ensure their accessibility when needed, along with clear standards for the handling and study of dangerous pathogens. when the proportion of available resources poor international preparedness • delay before the who declared a public health emergency of international concern • delay in implementing coordinated international assistance • logistic challenges in delivering support to assist epidemic response • shortcomings in who's regional and country-level capacity exposed • lack of global plans to address an epidemic of a high-risk pathogen in the least developed urban centres • evaluation of promising vaccine and therapeutic interventions came too late 16 and ebola virus. 17 the local and international responses to the 2014 west african ebola virus epidemic revealed shortcomings that could allow highly contagious epidemics of infectious disease to spread widely before they are terminated (panel 1). 17 during the cold war, agents that could potentially be used as biological weapons were identified on the basis of the following characteristics: pathogenicity for humans, animals, or plants; ability to cause disability or death; stability and infectivity as small particle aerosols; and capability of being readily and rapidly produced and weaponised in munitions or delivery systems. more characteristics have been added, to include other features of biological agents such as the relative ease of medical prevention or treatment and the likelihood of harm to the perpetrator. the us centers for disease control and prevention (cdc) identified bacteria, viruses, and toxins that could potentially be weaponised (panel 2). in 2002, they categorised them into three groups-a, b, and cdepending on ease of dissemination, severity of illness caused, and ability to cause death. 18 biological agents can be infectious and contagious, infectious but not usually contagious, or toxins if they are neither. category a agents were considered the greatest risk to public and national security. the more recent classification of tier 1 select agents and toxins is similar to the category a classification (table 1) . other agents, such as naturally occurring pathogens, produce diseases that are considered of intermediate risk to the public (eg, brucellosis, glanders, q fever). they are moderately easy to disseminate, and include emerging and re-emerging infectious diseases. however, genetic modifications could make them more virulent, produce uncharacteristic clinical signs, increase their resistance to treatment and vaccines, and even change their transmissibility or host range. genetic modifications could be made using the tools of synthetic biology; such activities might be an example of dual-use research. 19, 20 for instance, in 2005, the 1918 spanish influenza pandemic virus was reconstructed, 21 and the poliovirus was synthesised nearly 20 years ago. 22 the addition of an immuno-modulatory gene to the mousepox virus genome in 2001, 23 rendered a mousepox vaccine ineffective, and this technology could potentially be applied to the smallpox virus. 24 the recent synthesis of the extinct horsepox virus 24 has been a reminder that the smallpox virus could be reconstructed, and that the regulations that have been put in place to prevent the misuse of powerful, cheap, and globally available tools must be reconsidered. 25 this possibility has also raised the issue of whether research results should sometimes be censored, or even refused publication, if the potential to cause harm is too high. 19 although bioterrorist agents could be disseminated through multiple routes, the aerosol route would likely maximise exposure. contagious agents could produce a large number of second and later generation cases, depending on the number of people initially exposed, the series average number of people who acquire the disease from one infected individual (r 0 ), and the disease generation time in humans. for instance, the r 0 of pneumonic plague has been estimated to be around 1·3, 26 whereas the r 0 for smallpox is likely to be around 5. 27 for diseases that are not contagious, such as inhalational anthrax, the number of cases of disease will depend almost entirely on the size of the population exposed and the timing of post-exposure antibiotic prophylaxis. 28 aerosolised agents remain the threat of most concern, but safety and security of food [29] [30] [31] and water supplies 32 are also important components of primary prevention (panel 3). new methods to detect toxins in food, such as antibody based assays, are being developed. 33 rapid diagnostics take on additional urgency in a bioterrorist event, because of both health and security concerns. 34 since the 2001 anthrax letters, there have been major advances in diagnostic capabilities. some of the greatest advances in the past decade have been in the speed and reduced cost of sequencing capabilities. 35, 36 highly sensitive and specific pcr-based systems, coupled with modern sample preparation technologies, have enabled sequencing technologies to become less costly, more portable, and multiplexed. with fieldable patient-side diagnostics and sequencing outputs directly connected via cloud-based networks, 37 health-care providers globally can make decisions more rapidly and respond more quickly for individual care or outbreak detection. a rapid, cartridge-based assay for francisella tularensis has been developed for use at point of care. 38 a system that uses a sensitive microsphere technology to detect both antibodies and antigens is now available to diagnose infections with ebola virus and lassa virus. 39 although diagnostic elisa tests are available for anthrax antibodies, 40,41 a compact system (genexpert) that includes both sample processing and pcr amplification can produce a result in about 90 minutes. 42 a rapid and sensitive method to detect smallpox virus has been developed for use at point of care, based on antibody immuno column for analytical processes (abicap) immunofiltration, that produces results in about 45 minutes. 43 however, diagnostic electron microscopy is also still considered a fast and efficient method 44 to identify smallpox and other viral agents. ebola virus was rapidly sequenced during the outbreak in sierra leone to link sporadic cases with the transmission chains. 35 advanced proteomics are also being developed as reference assays 45 and a new method for simultaneous immunodetection of anthrax, plague, and tularaemia from blood cultures has recently been reported, 46 using multiplexed suspension arrays. next generation safety and security of food [29] [30] [31] and water supplies 32 are important components of primary prevention: • intentional contamination of food should be considered, particularly during a large foodborne epidemic with a common source • salmonella and shigella species, enterohaemorrhagic escherichia coli (all serotypes), vibrio cholerae, cryptosporidium parvum, and noroviruses are all potential candidates for intentional contamination of food • contamination of water with biological agents should still be considered, even though it is unlikely to be the major target of bioterrorism, due to chlorination, dilution, and the need for large quantities of the agent to cause a substantial outbreak • c parvum and noroviruses are more resistant to chlorination than other agents, so can be a threat to the water supply • food-borne or water-borne dissemination of these biological agents might lead to higher rates of morbidity and case fatality than previously observed, if the population has been exposed to substantially higher infectious doses • algorithms could be developed to measure the likelihood that outbreaks of disease were a consequence of intentional contamination of food or water, using descriptive, analytical, and molecular epidemiologic tools (none are known to be available so far) 47 increased networking and collaboration of laboratories will also improve the response to intentional outbreaks. effective global surveillance of infectious diseases is essential to control both intentional and naturally occurring epidemics. 48 surveillance data can be used to monitor the progress of an outbreak, and for risk communication. to obtain information rapidly, the ongoing collection of health-related data (termed syndromic surveillance) has been introduced, to monitor patterns of symptoms and signs that are suggestive of an outbreak. [49] [50] [51] [52] although it was hoped that syndromic surveillance would be a more sensitive method for early detection of an epidemic, frequent reports of unusual increases in incidence of non-specific illnesses can desensitise and paralyse the system. 53 in fact, early detection will depend largely on alert, prepared clinicians. for example, when the 2001 anthrax attacks occurred, an astute clinician identified the index case. 34 emergency room and community physicians should be updated regularly on the clinical signs and symptoms associated with the most common bioterrorist agents. 54 the syndromic surveillance system would be more useful after suspicious or confirmed cases have been reported by physicians. a focused analysis of the surveillance data against non-specific, background disease rates could detect changes and provide information about the dynamics of the disease. special legislation might then be necessary, to gain access to medical records. 55 the internet facilitates other potential forms of surveillance and communication about infectious diseases. 56, 57 one such system is promed, which was established by the user community and has proven effective in connecting clinicians and scientists around the world; it has already served as an early warning system 58 standard contact and airborne precautions should be taken. 65 supportive therapy and antibiotics can be provided for secondary infections. there is some evidence of the potential efficacy of thiosemicarbazones. cidofovir has shown in vitro efficacy against variola, and has shown efficacy against other diseases caused by human orthopoxviruses, notably diseases caused by vaccinia viruses. it has also shown efficacy in animal models of orthopoxvirus infections. since 2012, the us food and drug administration (fda) has approved drugs and biologic agents developed under the animal rule. this rule allows for approval of a drug that cannot be tested for efficacy in humans, but is effective in animals and safe in humans. the first drug approved under this rule was the monoclonal antibody raxibacumab for treatment of inhalation anthrax. tecovirimat is a drug that inhibits all orthopoxviruses tested in vitro. it was found to be highly effective in treating monkeypox and rabbitpox in animals and is considered safe in humans. 66 tecovirimat is being considered by the fda for approval for use in humans to treat smallpox under the animal rule. standard and droplet precautions should be taken. 65 ciprofloxacin, levofloxacin, and doxycycline have been approved for the treatment of pneumonic plague. streptomycin and gentamicin have been found to be effective in treatment, although there is some evidence of the development of multiple resistance. 67, 68 tularaemia isolation of the patients is not necessary and standard precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline are all approved for the treatment of tularaemia. streptomycin and gentamicin have been found to be effective. 68, 69 haemorrhagic fevers standard contact and airborne precautions should be taken until diagnosis is confirmed. subsequently, droplet precautions can be considered. supportive care and treatment of secondary infections can be provided. ribavirin is now approved for treatment of lassa fever and it also appears to be effective against new world arenaviruses and crimean-congo haemorrhagic fever. protective n95 respirators and clothing should be provided to health-care personnel. clothing of patients should undergo decontamination and thorough handwashing. supportive therapy is available, with antibiotics such as ciprofloxacin, doxycycline and ampicillin. if the bacteria are resistant to some of the antibiotics, the treatment regimen will depend on sensitivity testing. 60 the regulations require immediate reporting of serious health risks by all member countries. additionally, who has established the global outbreak alert and response network, and the european union has a programme called bichat to improve cooperation between member states in preparedness and response to biological and chemical attacks. they operate the early warning and response system for outbreaks of communicable diseases. the world organisation for animal health has developed plans to identify and deal with a bioterrorism attack on populations of food-producing animals. canada has established the global public health intelligence network for worldwide monitoring of threats to public health. 61 a major benefit of a less formal global collaboration is the development of networks of trust among knowledgeable scientists and clinicians, who are considered early warning posts for both natural and intentional outbreaks. 62 the one health initiative encourages collaboration between health professionals and is as important for bioterrorism preparedness as it is for management of emerging infectious disease and the global spread of antimicrobial resistance. 63 the management of patients that have been infected during incidents of bioterrorism can be challenging. 64 precautions and treatment regimens for several bioterrorist agents are summarised in panel 4. although supportive care serves as the basis of management for all agents, treatments for some of the relevant diseases have substantially progressed. the management of inhalation anthrax has advanced since the 2001 attack, 64, 70, 71 with improvements in critical care, and in treatment of acute lung injury and acute respiratory distress syndrome, severe sepsis, and septic shock. pleural effusions are routinely drained and there are more options for antimicrobial therapy. antibiotics are still recommended for 60 days after exposure or diagnosis, together with the anthrax vaccine. if the vaccine is given concurrently with antibiotic treatment, the period of treatment could be shortened. antibiotics for treatment of other bacterial infection are usually given for shorter periods, since the causative agents do not sequester spores. tularaemia is treated with ciprofloxacin or doxycycline. 72 for smallpox, antivirals such as cidofovir or a related acyclic nucleoside phosphonate analogue appear to be more effective than post-exposure vaccines in preventing mortality, according to experiments in non-human primates infected with monkeypox virus. 73 this suggests that antivirals might play an important role when preparing for a smallpox outbreak. for viral haemorrhagic fevers, ribavirin might have some efficacy in post-exposure prophylaxis. a small-molecule antiviral drug, gs-5734, has been developed that appears to be effective in treating ebola virus infection. 74 for intentional and other sudden outbreaks of contagious disease, isolation of patients and controlling risks to health-care workers remains extremely challenging, as was noted in the mers coronavirus, sars, ebola virus disease, and avian influenza epidemics. 75 hospital units adequately equipped for isolation are needed, similar to those equipped to care for filovirus-infected patients, with negative pressure air filtration. 76 if facilities are too small for the number of patients, a lower level of isolation with strict barrier nursing should be implemented, and in the event of a very large outbreak, there might be a need to set up isolation facilities in public buildings. in regions with poor infrastructure, it might be necessary to consider treating patients in their homes. people who have died should be regarded as infectious and handled with the same precautions used for patients. burial procedures might have to be modified, but every effort should be made to respect religious practices and traditions of the local culture. quarantining of people who might have been exposed to the infectious agent can be problematic, 77 as was the case in the sars and west african ebola virus epidemics. 78 since the quarantined population includes both people who were exposed and people who were not, the risk of disease transmission is higher. during the ebola virus outbreak in west africa, suspect cases were held until they could be cleared as negative, which took about 3 days pending the pcr results. on a national level, reducing the movement of populations is a sensitive issue, potentially interfering with commerce. 79 closure of schools is an important means of achieving social distancing to reduce spread. whether the public should use masks during an outbreak of a contagious disease is less clear, 80 and the efficacy of the masks is extremely variable. the most important reasons for variable efficacy are differences in facial shape, incorrect application, and duration of use. a substantial proportion of the cases and fatalities in the sars and ebola virus epidemics were among healthcare workers. clear guidelines specific to each agent are available to health-care personnel, public health workers, and emergency workers for the use of masks and personal protective equipment. the national ebola virus training and education center has been established in the usa to train health-care workers and assist hospitals in preparing for patients infected with high hazard virus in the usa and other countries. laboratory workers must be trained to work with dangerous pathogens and wear protective gear. designated threat pathogens must be stored, handled, and transported under a different set the role of vaccines in pre-exposure and post-exposure prophylaxis measures should be in place to protect the population from biological agents likely to be used in an attack before an incident occurs. however, since a bioterrorist incident is likely to be caused by biological agents not covered by routine immunisation, pre-exposure prophylaxis is generally confined to vaccines for military forces, health-care workers, and emergency response personnel. to the majority of the population, only postexposure prophylaxis is relevant. post-exposure prophylaxis for an intentional outbreak would include both those people known to be exposed during the incident and those people who were infected by others. the prophylaxis itself might consist of both vaccines and antimicrobials. when using live, attenuated vaccines, the relatively large proportion of the population who has some form of immunodeficiency has to be taken into account. 81, 82 monoclonal antibody preparations are now being considered for prophylaxis in select, high risk groups. 83 table 2 summarises pharmacological prophylaxis for tier 1 pathogens. currently, the vaccines that would most likely be used for pre-exposure or post-exposure prophylaxis are the smallpox and anthrax vaccines. since routine vaccination against smallpox was stopped in the 1980s, less than 50% of the world's population has been vaccinated, and antibody titres usually decline markedly after 5 to 10 years. 84 residual cellbased immunity can persist for many years. 85, 86 postexposure prophylaxis involves ring vaccination, which requires intensive tracing and vaccination of primary contacts, followed by vaccination of secondary contacts, 86 and finally, vaccination of all people in a defined affected region. for post-exposure prophylaxis of those directly exposed during the incident, vaccination can be effective if given within 3 to 4 days of exposure. 87 since it might take that amount of time to detect the first cases after exposure, the vaccine will generally only be effective for secondary and subsequent contacts. immune-boosting adjuvants 88 and toll-like receptor agonists have the potential to improve the immune response to post-exposure vaccination. 89 serious side-effects are relatively rare 90,91 but can affect com pliance. 92 in those cases, lower doses of vaccine might be administered, to provide adequate protection with fewer side-effects. 93 newer smallpox vaccines are in development, 94 including those that could immunise people who have atopic dermatitis. 95 because it is produced in small quantities by collecting antiserum from immunised humans, there might be a shortage of vaccinia immune globulin, used to treat people who would have serious side-effects with the vaccine. one possible solution to this shortage would be to use antibodies against other poxviruses, such as cowpox and monkeypox, 96 because of their cross-protective properties. since naturally occurring inhalation anthrax is extremely rare, there have been safety and immunogenicity profiles of the anthrax vaccine in humans, 97 but the efficacy has only been tested in animal models, and not in clinical trials. [98] [99] [100] the current anthrax vaccine is made from culture filtrates of a toxigenic, avirulent, nonencapsulated mutant of the bacillus anthracis vollum strain, and is administered in five intramuscular doses, followed by annual boosters. the protective, antigen specific memory b cells persist for many years after vaccination and are associated with humoral immunity. 99 serum igg response to the vaccine has been 100% after the fourth dose. 101 for post-exposure prophylaxis in unvaccinated people, the vaccine should be administered as a three-dose subcutaneous series (at 0, 2, and 4 weeks), in conjunction with a 60-day course of appropriate antimicrobial drugs. it has been given to thousands of us military personnel, and notable adverse events have been rare. 102 the anthrax vaccine is not recommended for pregnant women, although one study 103 with women in the us military, inadvertently vaccinated during pregnancy, did not show evidence of an increase in birth defects. in this study, 4418 women received the vaccine in the first trimester and 423 in the second and third trimeseter. more effective anthrax vaccines that require fewer doses are constantly being tested, 104, 105 such as the neat protein anthrax vaccine, 106 a dual purpose influenza vaccine that protects against anthrax, 107 and a combined anthrax-plague vaccine. 108 apart from the 17d yellow fever live attenuated vaccine and the junin virus vaccine, no vaccines for haemorrhagic fevers have been licensed. since the west african ebola virus epidemic, new ebola virus vaccines that have been long under development are being used successfully in the 2018 epidemic in the democratic republic of the congo. 109 essentially no other licensed vaccines are available for other tier 1 select agents. the previously licensed, formalin-inactivated, whole-bacilli plague vaccine has not proven effective against primary pneumonic plague in non-human primate models, 110 series us military. the vaccine has not been used widely for preexposure prophylaxis and has no place in post-exposure prophylaxis. new vaccines against tularaemia are under development 113, 114 including one that might provide crossprotection between plague and tularaemia. 115 the investigational pentavalent (abcde) botulinum toxoid vaccine was provided by the cdc for laboratory workers at high risk of exposure to botulinum toxin and it has also been given to military members that are at risk. the botulinum toxoid vaccine produces effective immunity after several months and has no value for postexposure prophylaxis because of the short latent period of the toxins. this vaccine was discontinued in 2012, 116 because of declines in immunogenicity and adverse events. new recombinant botulism vaccines are being developed, in addition to vaccines against glanders and rift valley fever. [117] [118] [119] efforts are ongoing to greatly shorten the time required to develop and produce new vaccines and other immune approaches. improved technologies are important for rapid scale-up and production of new treatment regimens, particularly following an attack with a contagious agent. the largely unpredictable nature of an epidemic initiated intentionally is likely to increase uncertainty and reduce public trust in the authorities. public education and effective risk communication are essential to increase public confidence and improve cooperation and compliance with recommended medical counter-measures. 120 the anthrax vaccine in military populations has caused considerable scepticism regarding the need for, safety, and efficacy of the vaccine. [121] [122] [123] clinicians and public health personnel should have access to up-to-date information, and the general public should be provided with nontechnical information and simple instructions on how to act during an emergency. sandman 120 has proposed that "one should not over-reassure, ack nowledge uncertainty, and share dilemmas". this behaviour would only cause overreaction or panic when new information about the risk is made public. risk communication will be necessary at all stages: before a bioterrorist incident occurs, when an incident is suspected, when it is confirmed, while it is taking place, and in the aftermath. credible and trusted spokespersons, including respected clinicians, scientists, and public servants for a country, should be adequately informed before an incident. during an outbreak, there could be unexpected events, such as atypical presentation of cases, varying responses to treatment (including unusual side-effects), and false positive and false negative diagnoses. the public might lose trust in the authorities if apparently unexposed people become ill. the advent and global distribution of social networking increases the risk of the dissemination of false or misleading information. lastly, a major infectious disease incident will also require flexibility and possible changes of established government policy. environmental detection of biological agents is another area of research that should be developed. to date, most systems of environmental detection have focused on anthrax, as a result of the anthrax attacks. 124, 125 however, a sensitive and specific set of recombinase polymerase amplification assays for fast screening, detection, and identification of b anthracis in a field setting has recently been developed. 126 the rare occurrence and likely small effect of an aerosol bioterrorist attack limits the practical use of environmental detection to special event venues, public transportation systems, and possibly some government buildings thought to be likely targets. many countries have national stockpiles of drugs and vaccines, for use in the event of a biological or chemical attack, or for serious outbreaks that might achieve epidemic proportions. the usa, for instance, maintains a strategic national stockpile of vaccines and other medical countermeasures. global stores of smallpox vaccines are held by who, in addition to stores held by individual countries. some countries have undertaken active vaccination programmes against smallpox and anthrax in the military and first responder populations. preparedness for bioterrorist incidents requires constant re-evaluation of policies. 127 although there is no evidence that the 2009 h1n1 influenza pandemic or the 2014-16 ebola virus epidemic in west africa were initiated intentionally, the local and international responses revealed strengths and weaknesses in the current state of preparedness for bioterrorist incidents. 128 the ebola virus epidemic spread to a number of countries, with more than 20 000 cases reported worldwide and a case fatality rate of more than 60%. imported cases of ebola virus disease were identified in the usa and spain. locally, in the affected countries in west africa, 10% of the people who died because of ebola virus disease were health-care workers. various shortcomings in the response to the epidemic have been identified since (panel 1). accurately predicting the intentional misuse of a biological agent to cause harm is difficult without intelligence data, but several attempts have been made to rationally predict the categories of risk: man-made, natural, accidental, contagious, and non-contagious. 129 series risks. 132 the risk of bioterrorism has called into question some of the dogmas related to eradication of diseases such as poliomyelitis and measles. 133 for example, if polio is successfully eradicated, universal vaccination might have to continue because of the risk of poliovirus being used as a bioterrorist agent. [134] [135] [136] emerging and reemerging infectious diseases will continue to be a threat, 137 but preparedness for bioterrorism is, in many ways, similar to preparedness for naturally emerging disease. all countries should collaborate to address the root causes of terrorism, and develop appropriate preventive strategies. effective preparedness is, in itself, a deterrent to bioterrorism, since it reduces the incentive to use biological weapons by making a country or region a hard target. it is also the cornerstone of consistent and effective responses to naturally occurring epidemics. the abuse of biological agents can be further reduced or discouraged with reliable intelligence and an effective response if it does occur. national and regional resources and capabilities will vary, but all will require infrastructures that are capable of recognising and dealing with a variety of biological agents. 138 the needs of specific populations, such as the paediatric population, pregnant women, 139 elderly people, and people with immunological disorders, must also be addressed. 140 funding for biodefence is crucial to adequate preparation and response to bioterrorist threats. 141 international preparedness for bioterrorism has the dual benefit of strengthening the infrastructure for responding to naturally occurring epidemics of highly pathogenic organisms. lessons from the 2014 west african ebola virus epidemic show that health-care providers must always be watchful for unusual presentations of disease, and new and improved approaches must be developed for early detection and response. health-care providers need more effective means of isolating infected patients, and better methods to control the movement of potentially infected people outside of the affected areas. personal protective equipment should be inexpensive and effective, and available to use with minimal training and under harsh environments. protection of health-care workers against infection remains particularly prob lematic, and should be a focus of research and development. the ebola virus epidemic has highlighted the importance of improving the logistics of moving human and material resources in areas with relatively poor infrastructure. risk communication and public education before and during an outbreak need to be improved. more clinical trials should be fast-tracked during development of new vaccines and antiviral drugs. preparedness for a low-risk, high-impact event that is bioterrorism should be monitored constantly, tested in tabletop exercises, 142, 143 and integrated into the routine functioning of the health system. here it would serve the dual purpose of ensuring that countries are prepared to meet the challenges of controlling epidemics of emerging and re-emerging infectious diseases. msg designed the review, did the literature search and was responsible for writing the manuscript. jld assisted in the literature search, revised the manuscript, and supplied technical expertise. dc assisted in the literature search and revised the manuscript. drf helped design the review, contributed source material, did the literature search, and helped write and revise the manuscript. we declare no competing interests. we searched pubmed and google scholar using the terms "bioterrorism", "sustainable bioterrorism preparedness", "all-hazards infectious disease preparedness", "biological threat agents", "bioterrorism preparedness", "emerging infectious diseases", "smallpox", "anthrax", "plague", "tularemia", "botulism", "hemorrhagic fevers", and "risk communication". we searched national and international reports from the who and us centers for disease control using the terms "bioterrorism", "bioterrorism preparedness", and "emerging infectious diseases". we also completed web searches for "disease surveillance", "infectious disease diagnostics", "medical countermeasures", "emergency healthcare delivery", and "risk management". we focused on academic literature in english and restricted most of our searches to documents published since 1990, with an emphasis on those published after 2001, and included mainly those published since 2008. federal funding for health security in fy2016 a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, 2001: epidemiologic findings remediation of bacillus anthracis contamination in the u.s. department of justice mail facility total decontamination cost of the anthrax letter attacks bacillus anthracis incident post-traumatic stress disorder symptoms in victims of tokyo subway attack: a 5-year follow-up study biological warfare and bioterrorism: a historical review the threat of bioterrorism: identifying the unknown. ffi focus biodefense in the 21st century department of health and human services (hhs). possession, use, and transfer of select agents and toxins-addition of bacillus cereus biovar anthracis to the hhs list of select agents and toxins. interim final rule and request for comments destruction of microbial collections in response to select agent and toxin list regulations implementing the select agent legislation: perfect record or wrong metric? gaps remain in china's ability to detect emerging infectious diseases despite advances since the onset of sars and avian flu health system resource gaps and associated mortality from pandemic influenza across six asian territories ebola: lessons learned and future challenges for europe public health assessment of potential biological terrorism agents responsible conduct by life scientists in an age of terrorism mitigating the risks of synthetic biology. council on foreign relations characterization of the reconstructed 1918 spanish influenza pandemic virus chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox labmade smallpox is possible, study shows the de novo synthesis of horsepox virus: implications for biosecurity and recommendations for preventing the reemergence of smallpox epidemiologic determinants for modeling pneumonic plague outbreaks risk assessment and risk communication strategies in bioterrorism preparedness. nato security through science series a: chemistry and biology estimating time and size of bioterror attack threat of a biological terrorist attack on the us food supply: the cdc perspective a large food-borne outbreak of group a streptoccocal pharyngitis in an industrial plant: potential for deliberate contamination german outbreak of escherichia coli o104:h4 associated with sprouts water and bioterrorism: preparing for the potential threat to u.s. water supplies and public health a proteomics assay to detect eight cbrn-relevant toxins in food index case of fatal inhalational anthrax due to bioterrorism in the united states rapid outbreak sequencing of ebola virus in sierra leone identifies transmission chains linked to sporadic cases real-time pcr to identify variola virus or other human pathogenic orthopox viruses pocket dna sequencers make real-time diagnostics a reality sensitive detection of francisella tularensis directly from whole blood by use of the genexpert system comparison of magpix assays and enzyme-linked immunosorbent assay for detection of hemorrhagic fever viruses the early humoral immune response to bacillus anthracis toxins in patients infected with cutaneous anthrax specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin g antibodies to anthrax toxin protective antigen rapid detection of bacillus anthracis bloodstream infections by use of a novel assay in the genexpert system rapid and sensitive point-of-care detection of orthopoxviruses by abicap immunofiltration rapid viral diagnosis of orthopoxviruses by electron microscopy: optional or a must? striking against bioterrorism with advanced proteomics and reference methods simultaneous immunodetection of anthrax, plague, and tularemia from blood cultures by use of multiplexed suspension arrays the changing face of pathogen discovery and surveillance public health surveillance and infectious disease detection evaluation of a syndromic surveillance system using the wsare algorithm for early detection of an unusual, localized summer outbreak of influenza b: implications for bioterrorism surveillance syndromic surveillance during pandemic (h1n1) 2009 outbreak internet-based surveillance systems for monitoring emerging infectious diseases digital surveillance for enhanced detection and response to outbreaks if syndromic surveillance is the answer, what is the question? clinical recognition and management of patients exposed to biological warfare agents public health. ethics and the conduct of public health surveillance digital disease detectionharnessing the web for public health surveillance factors influencing performance of internet-based biosurveillance systems used in epidemic intelligence for early detection of infectious diseases outbreaks the internet and the global monitoring of emerging diseases: lessons from the first 10 years of promed-mail social and news media enable estimation of epidemiological patterns early in the 2010 haitian cholera outbreak geneva: world health organization the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health with the changing biological threat…smart international engagement policy would lower cost and increase national security animals as sentinels of bioterrorism agents clinical management of potential bioterrorism-related conditions wisconsin department of health services. infection control and prevention-standard precautions oral tecovirimat for the treatment of smallpox plague: recognition, treatment, and prevention the sanford guide to antimicrobial therapy new therapeutic approaches for treatment of tularaemia: a review centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults antitoxin treatment of inhalation anthrax: a systematic review consequences of delayed ciprofloxacin and doxycycline treatment regimens against francisella tularensis airway infection a single cidofovir treatment rescues animals at progressive stages of lethal orthopoxvirus disease therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu implementing a negative-pressure isolation ward for a surge in airborne infectious patients large-scale quarantine following biological terrorism in the united states: scientific examination, logistic and legal limits, and possible consequences ebola and fda: reviewing the response to the 2014 outbreak, to find lessons for the future assessment of the potential for international dissemination of ebola virus via commercial air travel during the 2014 west african outbreak transmission of ebola viruses: what we know and what we do not know estimating the size of the u.s. population at risk of severe adverse events from replicating smallpox vaccine influence of population immunosuppression and past vaccination on smallpox reemergence next-generation monoclonal antibodies: challenges and opportunities analysis of historical data suggests long-lasting protective effects of smallpox vaccination duration of neutralizing antibody persisting in thai individuals after childhood vaccination against smallpox a model for a smallpox-vaccination policy can postexposure vaccination against smallpox succeed? immune-boosting adjuvants tlr3 and tlr9 agonists improve postexposure vaccination efficacy of live smallpox vaccines risks of serious complications and death from smallpox vaccination: a systematic review of the united states experience adverse events following smallpox vaccination with acam2000 in a military population factors associated with healthcare worker acceptance of vaccination: a systematic review and meta-analysis reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses comparing new-generation candidate vaccines against human orthopoxvirus infections long-term safety of replication-defective smallpox vaccine (mva-bn) in atopic eczema and allergic rhinitis cross-neutralizing and protective human antibody specificities to poxvirus infections randomized, double-blind, placebo-controlled, safety and immunogenicity study of 4 formulations of anthrax vaccine adsorbed plus cpg 7909 (av7909) in healthy adult volunteers select human anthrax protective antigen epitope-specific antibodies provide protection from lethal toxin challenge protective antigen-specific memory b cells persist years after anthrax vaccination and correlate with humoral immunity lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated serum igg antibody response to the protective antigen (pa) of bacillus anthracis induced by anthrax vaccine adsorbed (ava) among u.s. military personnel an overview of adverse events reported by participants in cdc's anthrax vaccine and antimicrobial availability program safety of inadvertent anthrax vaccination during pregnancy: an analysis of birth defects in the u.s. military population progress and novel strategies in vaccine development and treatment of anthrax host immunity to bacillus anthracis lethal factor and other immunogens: implications for vaccine design progress toward the development of a neat protein vaccine for anthrax disease a dual purpose universal influenza vaccine candidate confers protective immunity against anthrax a bivalent anthrax-plague vaccine that can protect against two tier-1 bioterror pathogens, bacillus anthracis and yersinia pestis experimental treatment of ebola virus disease with tkm-130803: a single-arm phase 2 clinical trial plague vaccines: current developments and future perspectives plague vaccines: status and future intranasal delivery of a protein subunit vaccine using a tobacco mosaic virus platform protects against pneumonic plague protective immunity against lethal f. tularensis holarctica lvs provided by vaccination with selected novel cd8+ t cell epitopes francisella tularensis live vaccine strain deficient in capb and overexpressing the fusion protein of igla, iglb, and iglc from the bfr promoter induces improved protection against f. tularensis respiratory challenge yopp-expressing variant of y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins recombinant botulinum neurotoxin hc subunit (bont hc) and catalytically inactive clostridium botulinum holoproteins (cibont hps) as vaccine candidates for the prevention of botulism burkholderia pseudomallei and burkholderia mallei vaccines: are we close to clinical trials? safety and immunogenicity of a mutagenized, live attenuated rift valley fever vaccine, mp-12, in a phase 1 dose escalation and route comparison study in humans bioterrorism risk communication policy why do uk military personnel refuse the anthrax vaccination? a longitudinal study of uk military personnel offered anthrax vaccination: informed choice, symptom reporting, uptake and pre-vaccination health notes from the field: compliance with postexposure prophylaxis for exposure to bacillus anthracis among u.s. military personnel-south korea rapid detection of bacillus anthracis spores using immunomagnetic separation and amperometry rapid detection of viable bacillus anthracis spores in environmental samples by using engineered reporter phages sensitive and specific recombinase polymerase amplification set of assays for fast screening, detection and identification of bacillus anthracis in a field setting the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences pandemic preparedness and response-lessons from the h1n1 influenza of 2009 global catastrophic biological risks: toward a working definition expert views on biological threat characterization for the u.s. government: a delphi study the risk of bioterrorism re-analysed risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication eradicating polio: a balancing act vaccination against polio should not be stopped vaccines should be kept even if polio is wiped out ebola and zika: cautionary tales medical countermeasure development since 2001: a long way yet to go preparing for biological threats: addressing the needs of pregnant women safety of live vaccinations on immunosuppressive therapy in patients with immune-mediated inflammatory diseases, solid organ transplantation or after bone-marrow transplantation-a systematic review of randomized trials, observational studies and case reports biosecurity: assessing the bioweapons threat a plague on your city: observations from topoff shining light on "dark winter key: cord-261533-73721b24 authors: mok, chris ka pun; zhu, airu; zhao, jingxian; lau, eric h y; wang, junxiang; chen, zhao; zhuang, zhen; wang, yanqun; alshukairi, abeer n; baharoon, salim a; wang, wenling; tan, wenjie; liang, weiwen; oladipo, jamiu o; perera, ranawaka a p m; kuranga, sulyman a; peiris, malik; zhao, jincun title: t-cell responses to mers coronavirus infection in people with occupational exposure to dromedary camels in nigeria: an observational cohort study date: 2020-10-06 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30599-5 sha: doc_id: 261533 cord_uid: 73721b24 background: middle east respiratory syndrome (mers) remains of global public health concern. dromedary camels are the source of zoonotic infection. over 70% of mers coronavirus (mers-cov)-infected dromedaries are found in africa but no zoonotic disease has been reported in africa. we aimed to understand whether individuals with exposure to dromedaries in africa had been infected by mers-cov. methods: workers slaughtering dromedaries in an abattoir in kano, nigeria, were compared with abattoir workers without direct dromedary contact, non-abattoir workers from kano, and controls from guangzhou, china. exposure to dromedaries was ascertained using a questionnaire. serum and peripheral blood mononuclear cells (pbmcs) were tested for mers-cov specific neutralising antibody and t-cell responses. findings: none of the participants from nigeria or guangdong were mers-cov seropositive. 18 (30%) of 61 abattoir workers with exposure to dromedaries, but none of 20 abattoir workers without exposure (p=0·0042), ten non-abattoir workers or 24 controls from guangzhou (p=0·0002) had evidence of mers-cov-specific cd4(+) or cd8(+) t cells in pbmc. t-cell responses to other endemic human coronaviruses (229e, oc43, hku-1, and nl-63) were observed in all groups with no association with dromedary exposure. drinking both unpasteurised camel milk and camel urine was significantly and negatively associated with t-cell positivity (odds ratio 0·07, 95% ci 0·01–0·54). interpretation: zoonotic infection of dromedary-exposed individuals is taking place in nigeria and suggests that the extent of mers-cov infections in africa is underestimated. mers-cov could therefore adapt to human transmission in africa rather than the arabian peninsula, where attention is currently focused. funding: the national science and technology major project, national institutes of health. middle east respiratory syndrome coronavirus (mers-cov) is one of eight emerging pathogens identified in the who research and development blueprint requiring urgent action for development of effective vaccines and antiviral drugs. 1 the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as a pandemic virus emphasises the threat posed by zoonotic coronaviruses. mers-cov causes a zoonotic disease, middle east respiratory syndrome (mers), with out breaks in health-care facilities associated with trans mission between humans. as of november, 2019, 2494 laboratoryconfirmed cases of mers, including 858 associated deaths (case-fatality ratio of 34·4%), were reported globally; the majority of these (2102 cases, including 780 deaths) occurred in saudi arabia. 2 travel-associated outbreaks led to 186 cases and 39 deaths in south korea. 2 dromedary camels are the source of zoonotic mers-cov disease. 3 the majority (>70%) of dromedaries are found in africa. they have comparable seroprevalence and virus shedding to those in the arabian peninsula, 4 but no zoonotic disease has been reported in africa. humans with prolonged close exposure to dromedaries in the arabian peninsula have serological evidence of mers-cov infection, sometimes having seroprevalence as high as 50%, 5, 6 but serological evidence is rare in africa, even in dromedary-exposed individuals. 7, 8 however, virologically confirmed infection, especially if it is asymptomatic or mild, might not lead to a serological response. 9 thus, alternative and more sensitive methods for detection of past human mers-cov infection are needed. specific t-cell responses have been shown to be longlasting in sars-cov and mers-cov infected humans, 10, 11 and persist longer than antibodies in sars. we therefore aimed to test peripheral blood mononuclear cells (pbmc) in workers from an abattoir in kano, nigeria, for mers-cov-specific t-cell responses to understand if the dromedary-exposed individuals in africa have been infected by mers-cov. in this observational cohort study, workers at an abattoir in kano, nigeria, consenting to participate in the cohort study in march 13-26, 2018 , were recruited. nonabattoir workers were also recruited randomly from the city of kano during the same period, and blood donors aged 18-65 years sampled in may 10-aug 31, 2018, at guangzhou blood center, guangzhou, china, were randomly included as healthy controls from a region with no dromedary camel exposure. convalescent blood samples collected from 14 people with symptomatic or asymptomatic virologically confirmed mers-cov infections detected at the king abdulaziz medical city, riyadh, and king faisal specialist hospital, jeddah, saudi arabia, collected as part of a previously reported study 11 were included as positive controls. the clinical, serological and t-cell responses (using only interferon [ifn]-γ as a readout of positive cells) of this patient cohort have been previously reported. 11 pbmcs were collected at 6 months (patients 1-6, 8-9, 11-14 as reported in the previous publication) or 24 months (patients [18] [19] after infection. written informed consent was obtained from all study participants in nigeria and the study was approved by the health research ethics committee of the ministry of health, nigeria (moh/off/797/t.i/630). we obtained institutional review board approval from the health commission of guangdong province to use the anonymised blood donor samples for this study. written informed consent was obtained from all recovered patients with mers to participate in this study and approval obtained from the institutional review boards of the national guard hospital, riyadh, and king faisal specialist hospital, jeddah. 12 procedures 8 ml of blood were collected from each study participant from the abattoir and from donors from guangzhou. pbmcs were isolated from blood using leucosep tubes (greiner, kremsmünster, austria) and ficoll-paque plus (ge healthcare, chicago, il) according to the manu facturer's instructions. pbmcs were stored in liquid nitrogen and plasma at -80°c or lower before and during shipping before analysis. plasma was heat inactivated for 30 min at 56°c before the serology testing. anti-mers-cov antibody titres were determined using plaque reduction neutralisation tests. 9, 13 a set of 20-mer peptides overlapping by ten amino acids en comp assing the four mers-cov (hcov-emc/2012) structural proteins (peptides s1, s2, n, and me encompassing the n-terminal and c-terminal portions of the spike [ evidence before this study middle east respiratory syndrome coronavirus (mers-cov) is recognised as one of eight emerging pathogens of greatest threat to global public health, and dromedary camels are the source of human zoonotic infection. the emergence of sars-cov-2 highlights the pandemic potential of zoonotic coronaviruses. although zoonotic disease has been restricted to the arabian peninsula, the largest number (>70%) of mers-cov infected camels are found in africa. we searched pubmed for articles published between nov 8, 2012, and dec 15, 2019, in english with the search terms "mers" and "coronavirus" and "human" and "africa" and manually screened all retrieved articles. there was one mers outbreak reported in tunisia initiated by a traveller returning from the arabian peninsula but no reports of zoonotic disease in africa. there were six sero-epidemiological studies of camel-exposed or other humans in kenya, egypt, nigeria, and morocco and only two (two of 1122 in kenya and three of 476 tested in morocco) found any evidence of mers-cov infection. because there was evidence that serological assays for mers-cov had suboptimal sensitivity for past infection and because we had previous data showing that t-cell assays for mers-cov are specific and potentially more sensitive than antibody detection, we investigated t-cell responses in dromedary-exposed abattoir workers and controls in nigeria. we found that 18 (30%) of 61 abattoir workers with exposure to dromedaries had mers-cov specific t-cell responses, but of 20 abattoir workers without exposure to dromedaries and ten non-abattoir workers from kano, none had such t-cell responses. no individuals with mers-cov t-cell responses had detectable antibody. by contrast, t-cell responses to endemic human coronaviruses were detected comparably in abattoir workers with and without exposure to dromedaries and control groups. we document that dromedary-exposed individuals in africa are frequently infected with mers-cov without evidence of severe disease. our findings indicate that there is substantial zoonotic transmission of mers-cov to people with dromedary exposure in parts of africa. the contribution of mers-cov to zoonotic respiratory disease remains to be established. our findings have implications for global mers-cov control policy. there is a need to confirm our findings elsewhere in africa and to include molecular testing for mers-cov in the investigation of patients with severe acute respiratory infections in dromedary-exposed populations in africa. orf4b, orf5 and orf8b) were synthesised by sino biological (shanghai, china), and used for stimulation of pbmcs. t-cell responses were measured using intracellular cytokine staining assays for interferon-γ (ifn-γ) and tumour necrosis factor (tnf). structural proteins peptide libraries of hku1-cov, oc43-cov, nl63-cov, and 229e-cov were also synthesised by sino biological to detect viral-specific t-cell responses. to enhance specificity, only cells with dual expression of both ifn-γ and tnf after peptide stimulation were considered as positive. flow cytometry was used to determine the phenotype and function of t cells. the following anti-human monoclonal antibodies were used: bv510-cd3 (hit3a; bd, san jose, ca), percp-cy5. 5 in a previous study of dromedary abattoir workers in saudi arabia, ten of 30 workers sampled had detectable t-cell responses to mers-cov. 14 on the basis of this finding, and the assumption that abattoir workers without dromedary exposure and the other control groups would have no detectable t-cell responses, eight abattoir workers would be the minimal sample size required to detect a positive result with 95% probability, where the detection probability is given by: 1 -(1 -p)n with p equivalent to 10/30 and n being the sample size. we aimed at sampling all abattoir workers who consented to participate, as long as we successfully sampled at least eight dromedary-exposed abattoir workers. association of t-cell responses with different exposure to dromedaries was done using fisher's exact test. in univariate analysis, we estimated the crude odds ratio (or) for each potential epidemiological exposure factor in relation to mers-cov t-cell positivity using a logistic regression model. independent risk factors for t-cell positivity were identified using multivariable logistic regression. we included a-priori variables, such as years of work in abattoir and whether other household members frequently visited camel farms, and other variables with a crude or of more than 2 or less than 0·5 in the univariate analysis. due to small sample size and cross-related practices of drinking camel milk and camel urine, we first fitted a logistic regression model which considered all four combinations of the two practices (eg, drinking camel milk only, drinking camel urine only, drinking both camel milk and urine or not drinking either), adjusted for potential confounding factors (model 1). then we further assessed the effect of drinking camel milk and camel urine separately in two models (models 2 and 3) . missing data were handled using multiple imputation with 50 imputations by predictive mean matching using the aregimpute function in r. all statistical analyses were done using r version 3.5.1. the funder of the study 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. we recruited 81 volunteers working in an abattoir in kano, nigeria. dromedaries, sheep, goats, and cattle were slaughtered in different areas of this abattoir (appendix p 4), and workers usually restrict themselves to work with one animal type. 61 (75%) workers had occupational exposure to dromedaries, whereas 20 (25%) were only involved in the slaughtering of sheep, goats, or cattle. ten people residing in kano not involved in abattoir-work and 24 volunteers from guangzhou, china, with no exposure to dromedaries, were also recruited as additional controls. 14 patients with confirmed mers from saudi arabia were included in this study as positive controls. 11 all participants were adults (aged ≥18 years). boots were the main protective equipment used by abattoir workers with (48 [79%] of 61) and without (14 [70%] of 20) exposure to dromedaries, whereas other protection, such as gloves, coveralls, masks, or goggles, were rarely used. there was no significant difference in the demographic characteristics between the three groups recruited in kano (table 1) . none of the sera collected neutralised the nig1657 virus (previously isolated at the same abattoir) at the dilution of 1:10 to levels of greater than 50% of control, the lowest threshold for a positive result (data not shown). pbmcs possessed good viabilities in all groups from which they were collected (appendix p 5) and responded to anti-human cd3 stimulation (appendix p 6). 18 (30%) of 61 samples from workers with exposure to dromedaries contained cd4+ or cd8+ t cells that responded to at least one peptide pool, particularly s1 and s2 pools ( figure 1a, b; appendix p 7) . no mers-cov specific cd4+ or cd8+ t-cell responses were detected in the three groups without exposure to dromedaries ( figure 1c, d) . the proportion of individuals with both cd4+ and cd8+ t-cell responses was significantly larger among dro medaryexposed abattoir workers than in workers without exposure (cd4+ p=0·0014; cd8+ p=0·0009), non-abattoir workers (cd4+ p=0·0038; cd8+ p=0·0018), or the ghuangzhou control group (cd4+ p=0·0005; cd8+ p=0·0003). the magnitude of the cd4+ t-cell responses in abattoir workers with exposure to dromedaries was similar to individuals in the saudi arabian positive control group with a subclinical condition (p=0·094), whereas the cd8+ t-cell responses were comparable to the symptomatic group (p=0·49). for stimulation with peptide pools derived from mers-cov accessory proteins (orf3, orf4a, orf4b, orf5 and orf8b), pbmcs were available from 11 workers with exposure to dromedaries who had t-cell responses to mers-cov structural proteins, from 11 who had negative t-cell responses to mers-cov structural proteins, and from four each from abattoir workers without exposure to dromedaries and non-abattoir workers. eight of the 11 dromedary-exposed workers who had t-cell responses to structural proteins also had t-cell responses to accessory proteins ( figure 1e ). none of the abattoir workers with dromedary exposure who did not have t-cell responses previously, nor those without dromedary exposure and non-abattoir workers had t-cell responses to accessory proteins ( figure 1e ). all the t-cell responses detected to accessory proteins were cd4+ t-cell responses and no cd8+ t-cell responses were detected (data not shown). taken together, of 61 workers with exposure to dromedaries in our cohort, six had both cd4+ and cd8+ t-cell responses against mers-cov structural proteins, four had only cd4+, and eight had only cd8+ t-cell responses. the mers-cov-specific cd4+ and cd8+ t cells were multifunctional with dual expression of two cytokines (ifn-γ and tnf). the majority of mers-cov-specific cd4+ t cells from dromedary-exposed workers were phenotypically effector memory (cd45ra-ccr7-) cells (figure 1f), whereas cd8+ t cells consisted of effector memory (cd45ra-ccr7-) and effector (cd45ra+ ccr7-) cells ( figure 1g, h) , comparable to the temra subset (effector memory t cells expressing cd45ra) described in mers survivors. 11 thus, these multifunctional cells are expected to rapidly and efficiently respond to subsequent mers-cov reinfection. 61 (53%) of the 115 participants had pbmcs available for additional testing for four endemic human coronaviruses (229e, hku1, nl63, and oc43), including 18 dromedary-exposed workers positive and ten negative for a mers-cov t-cell response and 33 from the negative control groups who were all mers-cov t-cell negative. 47 (77%) of 61 were t-cell positive to one or more of the human coronaviruses, with cd4+ t-cell responses being detected in all four groups (figure 2a), whereas cd8+ t-cell responses were found less often (figure 2b). in this group of 61 people, mers-cov t-cell responsiveness was not significantly associated with t-cell responses to any of the other coronaviruses (fisher's exact test; 229e p=0·57, hku1 p=0·58, nl63 p=0·37, and oc43 p=0·40). of the 47 with t-cell response to any of the other coronaviruses, ten (21%) had t-cell responses to mers-cov. by contrast, seven (50%) of 14 with no detectable t-cell response to any other coronavirus had t-cell responses to mers-cov, the negative association being statistically significant (fisher's exact test p=0·047) . human coronaviruses did not differ between the exposure groups and this was in marked contrast with the observations with mers-cov, which was observed exclusively in the dromedary-exposed group. drinking unpasteurised camel milk (or 0·24, 95% ci 0·07-0·83) and drinking camel urine (0·30, 0·09-0·94) were significantly and negatively associated with t-cell positivity (table 2). in the multivariate analysis, drinking both camel milk and urine was significantly negatively associated with t-cell responses (0·07, 95% ci 0·01-0·54; model 1; table 3). similar findings were obtained from a model without adjustment for potential confounders (data not shown). we further assessed the effect of each practice separately (models 2 and 3; table 3) and found that drinking unpasteurised camel milk (0·14, 0·02-0·81) and camel urine (0·19, 0·04-0·84) remained a significant factor for t-cell negativity. the two practices of drinking camel milk and camel urine were closely cross-related; 48 (79%) of 61 dromedary-exposed workers drank camel milk or urine, 15 drank milk without drinking urine, and two drank urine without drinking milk. our results indicated that drinking camel milk or camel urine was associated with a protective effect against mers-cov infection, but we could not separate their independent effects in the analysis. dromedaries in africa have comparable seroprevalence of mers-cov and virus shedding to those in the arabian peninsula, but zoonotic disease has not been reported. 4, 12, 15 even serological evidence of mers-cov infection in dromedary-exposed populations is uncommon. we previously found no serological evidence of mers-cov infection in 261 dromedary-exposed abattoir workers in an abattoir in kano, nigeria, although virus rna was repeatedly detected in the camels slaughtered during the winter months, with a peak of 11% of animals shedding virus in some weeks. 16 the negative serological results in workers from the same abattoir in this study were thus consistent with those of other studies of dromedary-exposed populations in kenya and egypt, which also did not find mers-cov-specific antibodies. 7, 16, 17 one study in kenya 18 found two seropositive individuals among 1010 people tested, and our study 8 in morocco detected three seropositive individuals among 476 people living in dromedary herding areas. because some patients with confirmed mers disease might not manifest neutra lising antibody responses and because such antibody responses can wane over time, serological studies could underestimate the extent of mers-cov infections in africa. 9 furthermore, antibody responses might not be positive in those with mild or asymptomatic infection, 9, [19] [20] [21] and t-cell responses are known to be more sensitive and long-lasting following sars-cov infections. 22 we have therefore previously analysed t-cell responses to mers-cov. 11 in these studies, both mers survivors (symptomatic and asymptomatic) and camel workers one abattoir worker with exposure to dromedaries had missing data for years working in abattoir, one for other household members frequently visited camel farms, two for travel outside kano in the past 6 months, and one for participated in mass gathering. *mean for age was 27·7 years (sd 8·9). †mean for years working in abattoir was 11·4 years (sd 9·8). (asymptomatic) identified in saudi arabia were shown to have mers-cov specific t cells in their blood, and some of those with t-cell responses did not have neutralising antibodies. comparable findings were observed in the korean outbreak; some patients with mild mers did not produce neutralising antibodies but had mers-cov-specific t cells in their peripheral blood. 21 we have shown that mers-cov-specific t cells were present in 18 (30%) of 61 dromedary-exposed workers but not in controls without exposure to dromedaries, and we conclude that mers-cov infections in people with occupational contact with dromedaries is underestimated in nigeria, and probably elsewhere in africa. t-cell responses in these workers recognised the highly variable s1 region and unique accessory proteins found in mers-cov, arguing for the mers-cov specificity of the t-cell responses. by contrast, t-cell responses to human coronaviruses nl63, hku1, 229e, and oc43 were found equally distributed in the dromedary-exposed worker group and the control groups (abattoir workers without dromedary exposure, non-abattoir workers, and ghuangzhou negative control). cross-reactive t-cell responses to other human endemic coronaviruses were not likely to be an explanation for the mers-cov t-cell responses in the dromedary-exposed workers, the association being a negative one. the observation that dromedary-exposed individuals with mers-cov t-cell responses did not have antibody responses is consistent with previous studies on mers and the underlying mechanisms needs further investigation. a question of relevance to public health is why no human zoonotic mers has been documented in africa even though zoonotic infection seems to be taking place as assessed by specific t-cell responses. the perception that mers does not occur in africa might reduce the use of mers-cov diagnostics in patients who have travelled to the arabian peninsula, precluding detection of zoonotic mers in africa. our finding that zoonotic mers-cov infection is occurring in dromedaryexposed populations in africa highlights that mers-cov needs to be considered in the differential diagnosis of patients with severe acute respiratory infections in these regions. an alternative hypothesis is that mers-cov strains in africa differ in pathogenic potential to those circulating in the arabian peninsula-ie, causing infection but less likely to cause severe disease. we have shown that mers-covs identified from africa (clade c), including those isolated in nigeria (clade c1), are phylogenetically distinct from contemporary viruses causing disease in the arabian peninsula (clade b). 12, 23, 24 viruses from the african clade c1-lineage were found to replicate less efficiently in human respiratory epithelial cell lines, in ex-vivo cultures of the human lung and in experimentally infected human dpp4 transgenic mice, possibly suggesting impaired pathogenic potential. 12 the absence of antibodies in individuals with t-cell responses might also be indicative of less severe infections, because patients with mild or asymptomatic mers-cov infections often do not have detectable antibody in both the acute and convalescent stages of infection. 9, 21 irrespective of whether mers-cov in africa is less pathogenic than the virus strains in the arabian peninsula, our findings argue for more intensive investigation of mers-cov in both humans and camels in africa. if repeated unsuspected zoonotic transmission of mers-cov continues to take place in africa as our findings indicate, given the much larger number of mers-cov-infected dromedaries in africa, the possibility of the virus adapting and efficiently transmitting between humans is probably more likely here than in the arabian peninsula where mers control efforts have been focused. the phylogenetic diversity of clade c viruses in africa suggests that these are the precursors that gave rise to the potentially more pathogenic clade b viruses currently enzootic in the arabian peninsula. 12, 25 if so, similar pathogenic mers-cov might independently emerge in africa. overall, our findings suggest that the mers control in the arabian peninsula needs to be extended to africa. occupational contact with camels was found to be a key risk factor for mers-cov infection, as defined by the positive t-cell responses against mers-cov. a univariate analysis of exposure factors associated with mers-cov infection (ie, mers-cov t cell reactivity) in the dromedary-exposed worker group revealed that drinking unpasteurised camel milk and drinking camel urine for medicinal purposes were significantly and negatively associated with infection risk. because the practices of drinking raw camel milk and urine were often associated and because of the small sample size, it was not possible to estimate their independent effects in a multivariate analysis in which both factors were concurrent variables. the finding that drinking unpasteurised camel milk was negatively correlated with infection risk is surprising and requires independent confirmation. camel milk has been previously thought of as a potential risk factor for mers-cov infection because mers-cov virus has sometimes been detected in camel milk. however, camel milk contains high titre antibodies to mers-cov, which is likely to neutralise any infectious virus particles, and viable mers-cov was not isolated from milk samples in which mers-cov rna was detected. 26 thus, mers-cov antibody present in camel milk could provide protection against mers-cov infection. our study had some limitations. exposure and risk factors associated with t-cell positivity were self-reported and the details on frequency or intensity for different modes of contacts with dromedaries were not collected. a small sample size reduced the power of the multivariable logistic regression analysis, although we were still able to identify a large protective effect of drinking unpasteurised camel milk or urine on t-cell positivity. in conclusion, we have shown that detection of virusspecific t-cell responses was a more sensitive method for detecting past infection compared with the serological tests being used hitherto, findings that may be also relevant to assessment of population-based infection attack rates of sars-cov-2 using seroprevalence that are currently under way. our findings suggest that the incidence of mers infections taking place in africa is underestimated. these findings have implications for policies on global mers prevention and control and highlight the need for attention towards camel-herding regions in africa as well as the arabian peninsula. ckpm, jincz, and mp designed the study. ckpm, joo, and sak coordinated and carried out the field work. az, jingz, and jincz designed and performed the experiments. jw, zc, zz, and rapmp participated in the experiments. ckpm, az, jingz, and mp analysed the data. ehyl and wl did the statistical analysis. yw collected pbmc from guangzhong blood donors. ana and sab provided mers patients samples from saudi arabia. ww and wt contributed new reagents. ckpm, az, jincz, and mp drafted the manuscript. all authors critically reviewed and commented on the manuscript. we declare no competing interests. review of emerging infectious diseases requiring urgent research and development efforts middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation risk factors for mers coronavirus infection in dromedary camels in burkina faso presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study occupational exposure to dromedaries and risk for mers-cov infection no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study middle east respiratory syndrome coronavirus (mers-cov) neutralising antibodies in a high-risk human population mers-cov antibody responses 1 year after symptom onset, south korea lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses mers coronaviruses from camels in africa exhibit region-dependent genetic diversity seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt high prevalence of mers-cov infection in camel workers in saudi arabia who mers global summary and assessment of risk. world-health-organization lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria mers coronaviruses in dromedary camels mers-cov antibodies in humans antibody response and disease severity in healthcare worker mers survivors transmission of mers-coronavirus in household contacts immune responses to middle east respiratory syndrome coronavirus during the acute and convalescent phases of human infection memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in africa and middle east middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria enzootic patterns of middle east respiratory syndrome coronavirus in imported african and local arabian dromedary camels: a prospective genomic study middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels key: cord-320548-oigyut2k authors: zumla, alimuddin; memish, ziad a; maeurer, markus; bates, matthew; mwaba, peter; al-tawfiq, jaffar a; denning, david w; hayden, frederick g; hui, david s title: emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options date: 2014-09-01 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70828-x sha: doc_id: 320548 cord_uid: oigyut2k the emergence and spread of antimicrobial-resistant bacterial, viral, and fungal pathogens for which diminishing treatment options are available is of major global concern. new viral respiratory tract infections with epidemic potential, such as severe acute respiratory syndrome, swine-origin influenza a h1n1, and middle east respiratory syndrome coronavirus infection, require development of new antiviral agents. the substantial rise in the global numbers of patients with respiratory tract infections caused by pan-antibiotic-resistant gram-positive and gram-negative bacteria, multidrug-resistant mycobacterium tuberculosis, and multiazole-resistant fungi has focused attention on investments into development of new drugs and treatment regimens. successful treatment outcomes for patients with respiratory tract infections across all health-care settings will necessitate rapid, precise diagnosis and more effective and pathogen-specific therapies. this series paper describes the development and use of new antimicrobial agents and immune-based and host-directed therapies for a range of conventional and emerging viral, bacterial, and fungal causes of respiratory tract infections. the emergence of diffi cult-to-treat known and novel bacterial, viral, and fungal respiratory tract pathogens with epidemic potential is of major global concern. treatment options are limited by increasing antimicrobialdrug resistance. however, new viral infections causing severe respiratory tract disease with pandemic potential have focused global attention. 1 a substantial rise in the number of patients with multidrug-resistant pulmonary tuberculosis 2 and pan-drug-resistant bacteria 3 has been noted. increasing use of immunosuppressive agents, broad-spectrum antibiotics, and anticancer agents, coupled with resistance to azoles, has led to an increase in the number of invasive pulmonary fungal infections 4 with resultant high morbidity and mortality. successful treatment outcomes for patients with respiratory tract infections across all health-care settings require appropriate, eff ective, and pathogen-specifi c drug or alternative treatments. we describe a range of conventional and emerging viral, bacterial, and fungal causes of respiratory tract infections for which new antimicrobial drugs and immune-based and host-directed therapies are being developed and studied. the outbreak of severe acute respiratory syndrome coronavirus (sars-cov), 5 re-emergence of avian infl uenza a h5n1, 6 global circulation of oseltamivirresistant seasonal infl uenza a h1n1, 7 and subsequent emergence of the pandemic infl uenza a h1n1 strain pdm09 virus (which continues to circulate), 8 have shown the potential limitations of current antiviral treatments for severe respiratory viral infections. epidemic waves of avian infl uenza a h7n9, 9 sporadic cases of avian infl uenza a h10n8, 10 the ongoing outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection, and the burden of common respiratory viruses 11 -such as seasonal infl uenza, respiratory syncytial virus, rhinoviruses, and adenoviruses-show that the development of more eff ective therapies to reduce morbidity and mortality is urgently needed. research is focused on the repurposing of available antiviral drugs for generic or specifi c use and for two classes of antiviral drugs are approved for the prevention and treatment of infl uenza in most countries: m2 inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir; table 1). in general, antiviral treatment is indicated as early as possible for any patient with confi rmed or suspected infl uenza who has severe, complicated, or progressive illness or is admitted to hospital, and in outpatients at higher risk of infl uenza complications. 12, 13 time to treatment after onset of symptoms, illness severity, and extent of viral replication are key variables with respect to response. starting of treatment should not be delayed for diagnostic testing. m2 inhibitors-also known as adamantanes-are ineff ective against infl uenza b viruses and recently circulating infl uenza a h3n2 and 2009 pandemic infl uenza a h1n1 viruses, which are resistant because of an s31n mutation in the m2 ion channel. 12 however, a proportion of avian infl uenza a h5n1 strains will be susceptible, 14 and the combined use of an adamantane and a neuraminidase inhibitor improves antiviral activity for susceptible isolates. 15 two neuraminidase inhibitors are approved for use in most countries: oseltamivir and zanamivir. laninamivir is approved for use in japan only, and peramivir in china, japan, and south korea. several observational studies have shown that when adults admitted to hospital with severe infl uenza are given oseltamivir, mortality falls and clinical outcomes improve, especially when treatment is initiated within 2 days of the onset of symptoms (but positive eff ects are noted when it is begun as late as 4-5 days after onset). 12, 13, 16, 17 oseltamivir reduces mortality in infl uenza a h5n1 infection when given before the onset of respiratory failure, 18 and might be benefi cial when started as late as 6-8 days after symptom onset. 19 in patients admitted to hospital with severe infl uenza a h7n9 infection, reduction of viral load after treatment with oseltamivir correlated with improved outcome, whereas the emergence of virus resistant to neuraminidase inhibitors that harbours an arg292lys substitution is associated with poor outcomes and poor response to oseltamivir and peramivir. 20 the standard duration of oseltamivir treatment is 5 days; longer treatment is recommended for critically ill patients with respiratory failure, who often have prolonged viral replication in the lower respiratory tract despite treatment. 13 whether increased doses provide greater antiviral eff ects in such patients is under investigation. a randomised controlled trial 21 of patients in hospital (76% of whom were children) showed no virological or clinical advantages when a double dose of oseltamivir was given rather than a standard dose. no additional benefi t was noted with high-dose oseltamivir in adults admitted with infl uenza a, although a faster virological response was noted in those with infl uenza b. 22 however, in a randomised controlled trial 23 of 18 critically ill patients with 2009 pandemic infl uenza a h1n1, a triple-dose oseltamivir regimen was associated with signifi cantly higher proportions of viral clearance at 5 days than was standard therapy (78% vs 11%; p=0·015). 23 studies of intravenous neuraminidase inhibitors that are underway should provide further data on the value of high-dose therapy. zanamivir and laninamivir have generally similar profi les of susceptibility. for example, the his275tyr mutation confers high-level resistance to oseltamivir carboxylate and reduced susceptibility to peramivir in n1containing viruses but does not substantially diminish susceptibility to zanamivir and laninamivir. 30 inhaled zanamivir has not been studied in detail in severely ill patients or those admitted to hospital, in whom eff ective delivery to sites of viral replication and tolerability could be an issue. by contrast, intravenous zanamivir has been used widely on a compassionate basis since the 2009 h1n1 pandemic, particularly for late treatment of critically ill adults with 2009 pandemic infl uenza a h1n1 virus infection and those with suspected or proven oseltamivir resistance. 31 one trial 32 has shown no drug-related trends in safety measures, and a subset of 93 patients positive at baseline for infl uenza showed a median decrease in nasopharyngeal viral rna load of 1·42 log 10 copies per ml after 2 days of treatment. a phase 3 trial in patients who have been admitted to hospital is underway (nct01014988). a phase 2 randomised controlled trial of inhaled laninamivir in uncomplicated infl uenza failed to show superiority in illness alleviation (primary endpoint) compared with placebo. the trial, involving 639 patients, tested 40 mg and 80 mg doses of the inhaled drug. the median time to alleviate fl u symptoms was 102·3 h for the 40 mg dose and 103·2 h for the 80 mg dose, compared with 104·1 h for the placebo (nct01793883). das181 has host-directed receptor-destroying action, which is inhibitory for parainfl uenza and infl uenza viruses, including those resistant to amino adamantanes and neuraminidase inhibitors. 15 when delivered topically, it is eff ective in animal models of lethal infl uenza caused by the h5n1 and h7n9 viruses, including the neuraminidase-inhibitor-resistant arg292lys -containing variant. 35 in a phase 2 randomised controlled trial, 36 inhaled das181 reduced pharyngeal viral replication in uncomplicated infl uenza but did not reduce nasal viral loads or improve clinical outcomes. case reports 37 suggest that inhaled or nebulised das181 might be eff ective in immunocompromised hosts with severe parainfl uenza lung disease. favipiravir (t-705; 6-fl uoro-3-hydroxy-2-pyrazinecarboxamide) is active against infl uenza a, b, and c viruses, including strains resistant to approved antivirals, and a broad range of other rna viruses when given at series somewhat higher concentrations. 38 combinations of favipiravir and neuraminidase inhibitors have additive and synergistic eff ects in preclinical models, 39 but clinical trials have been restricted to uncomplicated infl uenza so far. these clinical trials (combination amantadine, ribavirin, and oseltamivir vs oseltamivir mono therapy [nct01227969], nitazoxanide vs oseltamivir vs combination vs placebo [nct01610245], favipiravir vs placebo randomised controlled trial in outpatients [nct02008344, nct2026349]), which have not been published, suggest that favipiravir has antiviral eff ects similar to those of oseltamivir. 40 a randomised controlled trial 41 showed that favipiravir shortened the time to alleviation of infl uenza symptoms by about 15 hours compared with placebo, and further studies are underway. nitazoxanide is an oral antiparasitic drug with immunomodulatory eff ects, including upregulation of interferon and various interferon-inducible genes and a specifi c infl uenza-inhibitory eff ect related to blockade of haemagglutinin maturation. 42 nitazoxanide inhibits infl uenza replication in vitro 43 and in a phase 2 randomised controlled trial 44 had signifi cant antiviral eff ects (1·0 log 10 reduction in nasal viral loads) and resulted in a signifi cantly faster time to alleviation of illness (roughly 20 h diff erence in medians from placebo) in uncomplicated infl uenza. 44 a placebocontrolled randomised trial of nitazoxanide versus oseltamivir-and the com bination thereof-in uncomplicated infl uenza and a hospital-based study of its use in severe respiratory illness are in progress (nct01610245). non-randomly assigned studies and case reports suggest that convalescent plasma with neutralising antibodies is a useful add-on therapy for patients with sars and severe infl uenza pneumonia, including that caused by infl uenza a h5n1. 45 a recently published systematic review of available sars and infl uenza treatment studies employing convalescent plasma or serum found a signifi cant overall mortality benefi t. 46 a prospective observational study 47 showed lower crude mortality and faster nasopharyngeal viral clearance in plasma-treated patients who were admitted with severe 2009 pandemic infl uenza a h1n1 infection, whereas in a randomised controlled trial 48 a reduction in mortality was reported in severe illness when hyperimmune globulin was given within 5 days of the onset of symptoms (table 2) . heterosubtypic haemagglutinin stem-neutralising antibodies, which are highly eff ective in animals, 49 are entering clinical evaluation in human beings. the combination of antivirals with diff erent mechanisms of actions (eg, a neuraminidase inhibitor with a polymerase inhibitor such as favipiravir, 38 a broad-spectrum antihaemagglutinin-neutralising antibody, 49 (20) oral rimantadine and nebulised saline (21) post-hoc analysis showed faster cough resolution but no signifi cant diff erences in the proportion of patients shedding virus by treatment day 3 (57% zanamivir plus rimantadine, 67% placebo plus rimantadine), or in the durations of hospitalisation and supplemental oxygen use underpowered because of low enrolment oseltamivir and corticosteroids (19) more rapid improvement in partial pressure of oxygen, fraction of inspired oxygen, and sequential organ failure assessment scores; shorter ventilator use (median 7 days vs 15 days, p=0·03); and faster viral clearance in the sirolimus than in the control group rct=randomised controlled trial. 51 in a retrospective study 53 of critically ill adults, mortality rates did not diff er between those who received a triple combination of antiviral drugs and those receiving oseltamivir only, and a randomised controlled trial sponsored by the national institute of allergy and infectious diseases in higher-risk outpatients is underway (nct01227969). host-directed therapies aim to reduce the damaging consequences of the host immune response to the pathogen. combinations of antivirals with host-directed therapies such as the immunomodulator sirolimus, an mtor inhibitor that blocks host pathways needed for viral replication (table 2) , 54 might also enhance antiviral activity. other host-directed therapies inhibiting cellular targets needed for effi cient viral replication (eg, the raf-mek-erk mitogenic kinase cascade and the ikk-nf-κb module) might provide future options for clinical testing. 15 the role of adjunctive immunomodulatory therapies in severe infl uenza and other respiratory viral infections remains uncertain. several observational studies show that systemic corticosteroids given for 2009 pandemic infl uenza a h1n1-associated viral pneumonia increased the risk of mortality and morbidity (eg, secondary infections), especially when there was a delay in initiation, or absence of, eff ective antiviral therapy. 45 their use might delay viral clearance and increase the risk of the emergence of resistance 20 and fungal infections. 45 other potential adjunctive therapies for infl uenza include intravenous immunoglobulin, n-acetylcysteine, statins, macrolides, peroxisome proliferator-activated receptor agonists, celecoxib, mesalazine, plasmapheresis, and haemo perfusion. 45 chloroquine was eff ective against infl uenza a h5n1 infection in one animal model 55 but was ineff ective in other animal models and one human randomised controlled trial. 56, 57 interferons mers-cov infection can cause severe respiratory disease, and has higher mortality in those with medical comorbidities. although empirical treatment with a range of antivirals has been tried for severe respiratory tract infections caused by mers-cov and sars-cov, no regimens have been rigorously assessed in clinical trials (panel). 58,59 mers-cov elicits attenuated innate immune responses with delayed proinfl ammatory cytokine induction in cell culture and in vivo. 60, 61 it is also readily inhibited by type 1 interferons (interferon alfa and especially interferon beta), suggesting a potential therapeutic use for interferons. early pegylated interferon alfa therapy was eff ective in a sars primate model, and treatment with interferon-alfa-consensus-1 plus systemic corticosteroids was associated with improved oxygen saturation and more rapid resolution of radiographic lung opacities than were systemic corticosteroids alone in an uncontrolled study of patients with sars patients. 62 further studies of interferons in mers-cov seem warranted. ribavirin was used extensively in patients with sars without any benefi cial eff ects and was complicated by haemolytic anaemia and metabolic disturbances in many cases. 58, 59 a combination of interferon alfa 2b and ribavirin reduced lung injury and moderately decreased viral replication (<1·0 log 10 reduction in lung titres) when given to rhesus macaques within 8 h of inoculation with mers-cov. 63 the treatment combination was given to several severely ill patients with mers, but the infections proved fatal, probably because of late administration in the advanced stage of the disease. 64, 65 ribavirin has in-vitro inhibitory eff ects against mers-cov. 66 the use of protease inhibitors with lopinavir and ritonavir as initial therapy in sars was associated with signifi cantly less death (2·3% vs 15·6%, p<0·05) and intubation (0% vs 11·0%, p<0·05) than was use of ribavirin alone in a matched historical cohort (n=44 for lopinavir and ritonavir as intial treatment vs n=634 for the matched historical cohort). 68 however, one study reported that nelfi navir and lopinavir have high 50% eff ective inhibitory concentrations (ec 50 ) against mers-cov in vitro, 66 whereas another found inhibition with lopinavir at clinically achievable concentrations. 69 several drugs have shown inhibitory eff ects against mers-cov in cell cultures, including interferons, ciclosporin, and mycophenolic acid. 66, 67, 69 mycophenolic acid was inhibitory at clinically achievable concentrations, and the combination of mycophenolic acid and interferon β1b lowered the ec 50 of each drug by one-to-three times. 66 dipeptidyl peptidase 4 (dpp4), also known as cd26, is a functional receptor for mers-cov, and an anti-cd26 polyclonal antibody showed in-vitro inhibitory eff ects on mers-cov. 70 by contrast, inhibitors of the enzymatic action of dpp4 (eg, gliptins) did not inhibit viral replication. timely administration of neutralising antibodies could have a high likelihood of therapeutic success. 46 treatment with convalescent plasma (from patients who have recovered from sars-cov infection) containing high levels of neutralising antibody within 2 weeks of illness onset resulted in a higher proportion of discharges at day 22 than did treatment more than 14 days after onset (58% vs 16%, p<0.001). 71 75 showed worse outcomes when systemic corticosteroids were given in sars. consequently, their use should be avoided unless a carefully controlled prospective study is done to test their eff ectiveness when combined with an antiviral. several observational studies have shown that systemic corticosteroids given for 2009 pandemic infl uenza a h1n1-asssociated viral pneumonia or acute respiratory distress syndrome increased the risk of mortality and morbidity (eg, secondary bacterial or fungal infections), especially if there is delay or lack of eff ective antiviral therapy. 45 use of systemic corticosteroids has probably contributed to delayed viral clearance and emergence of antiviral resistance in patients with severe infl uenza a h7n9 infection requiring extracorporeal membrane oxy genation. 20 infl uenza increases the risk of invasive aspergillosis, especially among immunocompromised patients, and this is often a silent infection in the early stages, 76 so direct surveillance with aspergillus antigen and pcr testing on respiratory secretions is advisable. patients treated for fungal infections will have to undergo antifungal therapeutic drug monitoring. 77 data are insuffi cient to support routine use of any of the immune therapies. better animal data and careful systematic clinical studies, including serial virological measurements of priority treatments such as convalescent plasma and interferons (and randomised controlled trials if case numbers are suffi cient), are needed. currently, clinical management of patients with severe respiratory tract infections due to mers-cov largely relies on meticulous intensive care supportive treatment and prevention of complications. research done in patients with haemopoietic stem-cell transplants shows that adoptive transfer of antigenspecifi c t cells can restore protective immunity and prevent or reverse disease due to opportunist viral infections such as cytomegalovirus. 78 in transplant recipients, transfer of donor-derived t cells can result in resolution of infection through expansion of virusspecifi c t cells, with associated clinical improvement. 79 transfer of donor t cells is associated with the risk of severe acute graft-versus-host disease, and thus most t-cell therapies have been done in patients who have low lymphocyte counts. lymphopenia enables only a very low number of t cells to be transferred, which then proliferate in lymphopenic hosts, most likely as a result of the interleukins 7 and 15 if the patient does not receive immunosuppressive treatment during t-cell therapy. 80 t-cell therapy targeting cytomegalovirus strains resistant to drug treatment is clinically relevant in lung transplant recipients. 81 t-cell expansion requires time to induce clinical regression of viral infection. several other approaches might be applicable in situations that necessitate fast clinical action-eg, use of synthetic mhc antigens loaded with the relevant peptide from the pathogen of interest (so-called tetramer or multimer mhc-peptide complexes), which engage pathogenspecifi c lymphocytes expressing the pathogen-specifi c t-cell receptors. pathogen-specifi c t cells can be isolated through use of soluble mhc-peptide complexes, and can immediately be transferred into patients for salvage treatments for viral infections. 82 t-cell expansion can also be achieved with several stimuli targeting several infectious pathogens. 83 expansion of t cells targeting several antigens of cytomegalovirus, epstein-barr virus, and adenovirus provides broad antiviral specifi city after stem-cell transplantation. 84 an alternative approach to series become independent of ex-vivo expansion of t cells is the identifi cation of t-cell receptors that would recognise viral infected cells that could be transferred into recipient eff ector cells. 85 t cells can also be engineered to produce an antiviral rna that would block viral infection. 86 synthetic antisense molecules, such as phosphorodiamidate morpholino oligomers, are structurally similar to rna but the phosphorodiester linkage is replaced with a neutral phosphorodiamidate linkage and the ribose ring with a six-membered morpho lino ring. 87 they change gene expression by inhibiting translation, disrupting rna secondary structure, and interfering with pre-mrna splicing. 88 the usefulness of phosphorodiamidate morpho lino oligomers coupled to argininerich cell-penetrating peptides has been repeatedly demonstrated against bacterial pathogens 89 and could be a viable option for any microbial gene of interest. specifi c biological therapy for infectious pathogens targets not only drug-resistant pathogens but also their immune evasion mechanisms. 90 an antibody directed against cd19 (a b-cell marker) fused to a t-cell signalling molecule can be expressed in t cells and could kill target cells once they encounter their nominal target antigen. such cd19 chimeric-antigen-receptor cells are used to remove epstein-barr-virus-positive lymphoma cells in the case of post-transplantation proliferative diseases. 91 similar approaches can be used for the eff ective removal of pathogen-infected cells when very specifi c antibodies exist and if target molecules are expressed on infected cells only. 92 the frequency and spectrum of resistance to antibiotics in specifi c bacterial pathogens that cause respiratory tract infections continues to increase worryingly. multidrugresistant streptococcus pneumoniae-with resistance to three or more antibiotics-was initially noted in 1977 in south africa 93 and subsequently in many other countries, with alarming rates of 30-50% of s pneumoniae that are multidrug resistant in the usa and spain. [94] [95] [96] the european antimicrobial resistance surveillance system showed that 22·2% of s pneumoniae were intermediate penicillin susceptible, 10·9% were penicillin resistant, and 21·1% were resistant to erythromycin. 97 concerns about multidrug-resistant and pan-antibioticresistant gram-negative bacteria 98, 99 are focused on klebsiella pneumoniae, enterobacter spp (production of extended spectrum β lactamase, klebsiella pneumoniae carba penemase, ndm1, and ampc), acinetobacter baumannii, and pseudomonas aeruginosa. in one survey of us health centres, 78% of gram-negative bacteria were resistant to all antibiotics except colistin (to which 62% of acinetobacter spp, 59% of pseudomonas spp, and 52% of enterobacter spp were resistant). 98 therapeutic options to treat these infections are limited. 100, 101 carbapenems are recommended for organisms that produce extended-spectrum β lactamases. 101 in a metaanalysis, [102] [103] [104] [105] [106] doripenem was more eff ective for p aeruginosa infections than were comparators in a modifi ed intention-to-treat analyses. polymyxin b and colistin are concentration-dependent bactericidal agents that bind to bacterial cell membranes and have reliable activity against acinetobacter spp. novel β-lactamase inhibitors 107 and antibiotic com bination therapies 108 might provide stopgap measures for fulfi lling clinical need. antibiotic development pipelines remain thin, 109, 110 and global attention is focused on increasing awareness for investments into the development of new antibacterial agents 111 and other antibacterial innovations, coupled to raising global awareness for more prudent use of available drugs. 112 in 2012, an estimated 1·3 million people died worldwide from tuberculosis, 170 000 of whom had multidrugresistant disease. 113 multidrug-resistant tuberculosis, which is caused by mycobacterium tuberculosis bacilli resistant to at least isoniazid and rifampicin, is now widespread globally, with an estimated half a million cases in 2012. 2 extensively drug-resistant tuberculosisresistance to rifampicin, isoniazid, any fl uoroquinolone, and at least one of the three injectable second-line drugs, amikacin, kanamycin, and capreomycin-has been reported in 92 countries. 113 who recommends use of second-line drugs for 18-24 months or longer for extensively drug-resistant or multidrug-resistant disease. 114, 115 treatment success rates are low in both individualised and standard regimens and new drugs and regimens are needed. in the past 5 years, a promising pipeline of new drugs for the treatment of multidrug-resistant and extensively drug-resistant tuberculosis has emerged. 115 progress has been made by repurposing drugs that are already available, including re-engineering existing antibacterial compounds and redesigning scaff olds, leading to discovery of new compounds. 116, 117 two new drugs, delamanid (opc-67683) and bedaquiline (tmc207 or r207910), have been approved by regulatory authorities. these new drugs are combined with older drugs to treat multidrug-resistant disease. 118, 119 host-directed adjunct therapies several approaches to rational development of adjunct immune-based therapies for multidrug-resistant tuberculosis have been developed. 120, 121 non-steroidal antiinfl ammatory drugs can reduce m tuberculosis load and series alleviate lung disease 122 in mice. 123 effl ux pump inhibitors such as verapamil and reserpine reduce macrophageinduced drug tolerance, and thus could be used as adjunct host-directed therapies. 124, 125 phosphodiesterase inhibitors such as cilostazol and sildenafi l improve mycobacterial clearance and decrease time to sterilisation by reducing tissue infl ammation. 126 a range of adjunct immunotherapy approaches implicating cytokines or their inhibitors and other biological immunomodulatory compounds are being assessed as means to limit damage from infl ammatory responses against m tuberculosis. various cytokine regimens, including interferon c or interleukin 2, have been assessed, with variable eff ect. 127, 128 the antiinfl ammatory eff ects of macrolide antibiotics need to be further studied. 129 whole genome sequencing might allow for rapid determination of resistance patterns of m tuberculosis strains, enabling tailored treatment regimens. other immunomodulatory strategies include restoration of eff ective antipathogen-directed immunoresponses-and consequent decreasing of damaging host responses in lung tissues-in multidrug-resistant tuberculosis with infusions of the patient's own bonemarrow-derived stromal cells. a phase 1 trial showed that the procedure is safe, 130 and phase 2 trials are planned to assess the eff ects of mesenchymal stromal cell adjunct therapy on clinical and microbiological outcomes. invasive fungal respiratory tract infections are increasingly reported worldwide (table 3) . 131, 132 the two most common pulmonary fungal pathogens are aspergillus fumigatus and pneumocystis jirovecii. they increasingly represent primary causes of morbidity and mortality in critically ill patients across europe, africa, and asia as a result of more people living with hiv, increased use of immunomodulatory drugs in patients with cancer, transplantations, and use of broad-spectrum antibiotics. some patients with relapsed or micro biologically unconfi rmed multidrug-resistant tuber culosis have alternative diagnoses, including chronic pulmonary aspergillosis, and more com prehensive searches for alternative fungal diagnoses in smear and culture negative cases should be done in patients with multidrug-resistant disease. 133 aspergillus is the most important fungal cause of invasive pulmonary disease, and a fumigatus is the cause in more than 75% of cases. voriconazole is the most eff ective treatment for invasive aspergillosis but resistance has been noted on all continents except south america. 134, 135 widespread use of the azoles as fungicides in agriculture has led to the environmental development of pan-azole resistance. 136 resistance can also emerge during treatment, typically to itraconazole, and is possibly linked to a combination of low blood concentrations of the drug and high fungal loads. [137] [138] [139] modelling suggests that more than 6·5 million people have severe asthma with fungal sensitisations, as much as 50% of adults with asthma who attend secondary care have fungal sensitisation, and an estimated 4·8 million adults have allergic bronchopulmonary aspergillosis. 140, 141 people with asthma who are sensitised to a fumigatus have a much higher rate of bronchiectasis than do those who are unsensitised. reclassifi cation of aspergillosis in adults with cystic fi brosis by aspergillus serology (ige and igg) and both pcr and antigen on sputum showed three distinct classes of aspergillosis. 18% had allergic bronchopulmonary disease, 15% had aspergillus sensitisation, and 30% had aspergillus bronchitis; the remaining patients had no disease. long-term oral antifungal therapy is benefi cial for 60-80% of patients with asthma, but is of unproven benefi t in cystic fi brosis. 142 resistance in a fumigatus has been reported throughout europe in roughly 4% of samples from patients with cystic fi brosis. 143, 144 a new fungus causing disseminated infections in patients with aids was identifi ed in 2009. 145 molecular identifi cation on the basis of its1 and its2 sequencing showed that all isolates of this new species were tightly clustered and were most similar to emmonsia pasteuriana and emmonsia parva, and slightly more distantly related to histoplasma capsulatum. clinical features of infection included fever, loss of weight, anaemia, skin lesions akin to those in disseminated histoplasmosis, and a chest radiograph similar to that noted in pulmonary tuberculosis. the fungus was cultured from skin and blood, but not sputum or csf. signifi cant clinical responses were noted when patients were given intravenous amphotericin b followed by itraconazole. 145 a large combination study 146 series did not reach its primary endpoint of reduced mortality, although patients with positive galactomannan seemed to benefi t most. guidelines for management of invasive aspergillosis still favour voriconazole over all other treatments and combination therapy is not usually recommended. a tablet formulation of posaconazole, which is more bioavailable than the oral suspension, is available and can be given once a day, 147 and the us food and drug administration has approved an intravenous suspension of the drug. the only new drug to be approved is isavuconazole, a broad-spectrum azole, which will be available in intravenous and oral forms (application for approval was submitted in july, 2014). itraconazole seems safe in the fi rst trimester of pregnancy, whereas fl uconazole increases the risk of fallot's tetralogy by a factor of three to one in 1000. 148 drivers for the development of new antifungal drugs include inadequate response rates, the absence of oral preparations of echinocandins, drug interactions, important drug toxic eff ects (especially amphotericin b and voriconazole), and triazole and echinocandin resistance. several drugs are being repurposed for use as antifungals, and new drugs are under development (table 4) . [149] [150] [151] [152] [153] [154] [155] sertraline, which is used for depression, has synergistic activity with fl uconazole in a murine model of cryptococcal infection. 156 calcineurin and targets of rapamycin inhibitors have antifungal activity, which is synergsitic with that of azoles. 157 hsp90 inhibitors initially developed for cancer treatment can improve fl uconazole activity in vitro and in animals. 158 enoxacin, a fl uoroquinolone antibiotic, shows activity in a murine candidiasis model. 159 although azoles are important for the treatment of invasive pulmonary aspergillosis, the degree of immunosuppression and other immunological factors have a role in treatment outcomes. antifungal immune responses could be improved by adaptive transfer of pathogen-specifi c t cells directed against invasive and pulmonary fungal infections, particularly infections with candida, aspergillus, and mucormycetes, especially after allogeneic stem-cell transplantation. t-cell responses are mhc class i restricted (for cd8positive t cells) or mhc class ii restricted (for cd4positive t cells), and thus an eff ective t-cell response needs to match the genetic background of the patient. t-cell transfer was developed on the basis of the promising fi nding that transfer of pathogen-specifi c t-cell clones induces clinically signifi cant responses. 160, 161 several approaches have been used to obtain these pathogen-specifi c t cells. anti-pathogenspecifi c t cells can be expanded ex vivo under appropriate conditions (usually with the help of recombinant cytokines, synthetic peptides, or cellular components representing the pathogen). responder t cells are identifi ed by interferon-γ production, removed via an interferon-capture assay, and transferred into the patient. this approach requires time for expansion of t cells (either the patient's own or those of an mhc-matched donor). this protocol enabled the expansion of aspergillus spp, candida spp, with the terms "respiratory tract", "pneumonia", "infections", "bacteria", "virus", "fungus", and "mycobacteria". we also combined these terms with the words "antibiotic", "antibiotic resistance", "treatment", "drugs", "drug development", "drug pipeline", "antibiotic development", "host-directed", "therapy", "adjunct therapy", "steroids", and "immunotherapy". we complemented the search with publications from who, the us centers for disease control and prevention, http://clinicaltrials. gov, and google scholar. we also reviewed studies cited by articles identifi ed by this search. and mucor spp-reactive t cells defi ned by interferon-γ production. upon re-encounter with the nominal target antigen, the t cells proliferated and increased the antifungal reactivity of phagocytes. 162 new and antimicrobial-resistant species of bacteria, viruses, and fungi continue to emerge because of the remarkable genetic and adaptable plasticity of the microbiota. 163 respiratory tract infections are among the top two causes of death globally. 164, 165 microorganisms do not respect international boundaries, and ease of travel and airborne spread make them a threat to global health security. the increasing frequency of antibiotic resistance and limited therapeutic options emphasise the urgent need for more international cooperation to tackle new emerging microbial threats and multidrug-resistant microbes. development of new therapeutic options needs to be coupled to international regulations on the use and prescription of antimicrobial drugs. dsh and az coordinated the writing of this series paper and wrote the draft outline, and subsequent and fi nal drafts. all authors contributed relevant text and tables on their expert sections or sections and contributed to fi nalising the paper. fgh has served as non-paid consultant for multiple companies engaged in marketing and/or clinical development of antivirals for respiratory viral infections including several whose therapeutics are discussed in this review (adamas, biocryst, gsk, genentech, janssen, roche, romark, toyama/medivector, visterra). dwd holds founder shares in f2g, a university of manchester spin-out company. he acts as a consultant to trinity group, t2 biosystems, glaxosmithkline, sigma tau, oxon epidemiology, and has consulted for merck and astellas and he has been paid to give talks on behalf of astellas, gilead, and pfi zer. all other authors declare no confl icts of interest. interspecies transmission and emergence of novel viruses: lessons from bats and birds clinical features and rapid viral diagnosis of human disease associated with avian infl uenza a h5n1 virus history of swine infl uenza viruses in asia writing committee of the world health organization consultation on northern hemisphere infl uenza vaccine composition for 2013-2014. who recommendations for the viruses used in the 2013-2014 northern hemisphere infl uenza vaccine: epidemiology, antigenic and genetic characteristics of infl uenza a(h1n1)pdm09, a(h3n2) and b infl uenza viruses collected from who. avian infl uenza a(h7n9) virus clinical and epidemiological characteristics of a fatal case of avian infl uenza a h10n8 virus infection: a descriptive study addressing the public health burden of respiratory viruses: the battle against respiratory viruses (brave) initiative for the niaid collaborative antiviral study group. safety and effi cacy of nebulized zanamivir in hospitalized patients with serious infl uenza antiviral agents for the treatment and chemoprophylaxis of infl uenza. recommendations of the advisory committee on immunization practices (acip) antiviral resistance among highly pathogenic infl uenza a (h5n1) viruses isolated worldwide in 2002-2012 shows need for continued monitoring newer infl uenza antivirals, biotherapeutics and combinations complications and outcomes of pandemic 2009 infl uenza a (h1n1) virus infection in hospitalized adults: how do they diff er from those in seasonal infl uenza? eff ectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with infl uenza a h1n1pdm09 virus infection: a metaanalysis of individual participant data determinants of antiviral eff ectiveness in infl uenza virus a subtype h5n1 eff ectiveness of antiviral treatment in human infl uenza a(h5n1) infections: analysis of a global patient registry association between adverse clinical outcome in human disease caused by novel infl uenza a h7n9 virus and sustained viral shedding and emergence of antiviral resistance eff ect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe infl uenza: double blind randomised controlled trial a prospective intervention study on higher-dose oseltamivir treatment in adults hospitalized with infl uenza a and b infections viral clearance with standard or triple dose oseltamivir therapy in critically ill patients with pandemic (h1n1) 2009 infl uenza intravenous peramivir for treatment of infl uenza a and b virus infection in high-risk patients a clinical trial of intravenous peramivir compared with oral oseltamivir for the treatment of seasonal infl uenza in hospitalized adults intravenous peramivir for treatment of infl uenza in hospitalized patients business wire. biocryst announces outcome from the peramivir phase 3 interim analysis early emergence of an h275y mutation in a hematopoietic cell transplant recipient treated with intravenous peramivir rapid selection of oseltamivir-and peramivirresistant pandemic h1n1 virus during therapy in 2 immunocompromised hosts cs-8958, a prodrug of the new neuraminidase inhibitor r-125489, shows long-acting anti-infl uenza virus activity clinical experience with intravenous zanamivir under an emergency investigational new drug program in the united states safety and pharmacokinetics of intravenous zanamivir treatment in hospitalized adults with infl uenza: an open-label, multicenter, single-arm, phase ii study long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of infl uenza: a doubleblind, randomized, noninferiority clinical trial a randomized double-blind controlled study of laninamivir compared with oseltamivir for the treatment of infl uenza in patients with chronic respiratory diseases an investigational antiviral drug, das181, eff ectively inhibits replication of zoonotic infl uenza a virus subtype h7n9 and protects mice from lethality a phase ii study of das181, a novel host directed antiviral for the treatment of infl uenza infection das181 treatment of hematopoietic stem cell transplant patients with parainfl uenza virus lung disease requiring mechanical ventilation favipiravir (t-705), a novel viral rna polymerase inhibitor combinations of favipiravir and peramivir for the treatment of pandemic infl uenza a/california/04/2009 (h1n1) virus infections in mice clinical eff ectiveness and safety of favipiravir, a novel anti-infl uenza drug with a selective inhibition activity against viral rna polymerase. 51st interscience conference on a phase 2, randomized, double blind, placebocontrolled, multicenter study evaluating the safety and pharmacokinetics of diff erent dosing regimens of favipiravir (t-705) in adult subjects with uncomplicated infl uenza, viii options for control of infl uenza a new class of anti-infl uenza molecules targeting viral hemagglutinin at the post-translational level romark announces clinical trial results for new infl uenza drug presented at idsa meeting eff ect of nitazoxanide in adults and adolescents with acute uncomplicated infl uenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial adjunctive therapies and immunomodulatory agents in the management of severe infl uenza the eff ectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral aetiology: a systematic review and exploratory meta-analysis convalescent plasma treatment reduced mortality in patients with severe pandemic infl uenza a (h1n1) 2009 virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 infl uenza a(h1n1) infection an in vivo human-plasmablast enrichment technique allows rapid identifi cation of therapeutic infl uenza a antibodies antiviral combinations for severe infl uenza infections: a review triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant infl uenza virus strains in vitro effi cacy of oseltamivirzanamivir combination compared to each monotherapy for seasonal infl uenza: a randomized placebo-controlled trial triple-combination antiviral drug for pandemic h1n1 infl uenza virus infection in critically ill patients on mechanical ventilation adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h1n1 pneumonia and acute respiratory failure anti-malaria drug chloroquine is highly eff ective in treating avian infl uenza a h5n1 virus infection in an animal model eff ectiveness of chloroquine against infl uenza chloroquine for infl uenza prevention: a randomised, double-blind, placebo controlled trial international severe acute respiratory and emerging infections consortium treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients severe acute respiratory syndrome and coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicenter retrospective matched cohort study mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc use of convalescent plasma therapy in sars patients in hong kong contact investigation of a case of human novel coronavirus infection treated in a german hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection eff ects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study invasive aspergillosis after pandemic (h1n1) pharmacokinetics and pharmacodynamics of a novel triazole, isavuconazole: mathematical modeling, importance of tissue concentrations, and impact of immune status on antifungal eff ect immune reconstitution after allogeneic transplantation and expanding options for immunomodulation: an update monoculture-derived t lymphocytes specifi c for multiple viruses expand and produce clinically relevant eff ects in immunocompromised individuals infusion of cytomegalovirus (cmv)-specifi c t cells for the treatment of cmv infection not responding to antiviral chemotherapy transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation clinical grade generation of hexon-specifi c t cells for adoptive t-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation rapid salvage treatment with virus-specifi c t cells for therapy-resistant disease expansion of t cells targeting multiple antigens of cytomegalovirus, epstein-barr virus and adenovirus to provide broad antiviral specifi city after stem cell transplantation morpholino antisense oligomers: design, preparation, and properties antisense-mediated modulation of splicing: therapeutic implications for duchenne muscular dystrophy viral strategies of immune evasion viral interference with antigen presentation to cd8+ t cells: lessons from cytomegalovirus stimulation of human ebv-and cmv-specifi c cytolytic eff ector function using allogeneic hla molecules t cell allorecognition via molecular mimicry treating b-cell cancer with t cells expressing anti-cd19 chimeric antigen receptors cytotoxic therapy for severe swine fl u a/h1n1 emergence of multiply resistant pneumococci molecular characterization of streptococcus pneumoniae invasive serotype 19a isolates from adults in two spanish regions trends in antibacterial resistance among streptococcus pneumoniae isolated in the usa: update from protekt us years 1-4 streptococcus pneumoniae: does antimicrobial resistance matter? antimicrobial activity of ceftaroline tested against drug-resistant subsets of streptococcus pneumoniae from us medical centers variation in defi nitions and isolation procedures for multidrug-resistant gram-negative bacteria: a survey of the society for healthcare epidemiology of america research network who. antimicrobial resistance-global report and surveillance variation in potency and spectrum of tigecycline activity against bacterial strains from u.s. medical centers since its approval for clinical use new developments in carbapenems meta-analysis of doripenem vs comparators in patients with pseudomonas infections enrolled in four phase iii effi cacy and safety clinical trials current control and treatment of multidrug-resistant acinetobacter baumannii infections polymyxins revisited global assessment of the antimicrobial activity of polymyxin b against 54 731 clinical isolates of gram-negative bacilli: report from the sentry antimicrobial surveillance programme in vitro activity of colistin (polymyxin e) against 3,480 isolates of gram-negative bacilli obtained from patients in canadian hospitals in the canward study determination of in vitro activities of polymyxin b and rifampin in combination with ampicillin/sulbactam or cefoperazone/ sulbactam against multidrug-resistant acinetobacter baumannii by the e-test and checkerboard methods antibiotics in phase ii and iii clinical trials antibiotics currently in clinical development who. who global strategy for containment of antimicrobial resistance discovery and preclinical development of new antibiotics who guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update new antituberculosis drugs, regimens, and adjunct therapies: needs, advances, and future prospects advances in the development of new tuberculosis drugs and treatment regimens tuberculosis drug discovery in the post-post-genomic era provisional cdc guidelines for the use and safety monitoring of bedaquiline fumarate (sirturo) for the treatment of multidrugresistant tuberculosis rational development of adjunct immunebased therapies for drug-resistant tuberculosis: hypotheses and experimental designs host genotype-specifi c therapies can optimize the infl ammatory response to mycobacterial infections ibuprofen therapy resulted in signifi cantly decreased tissue bacillary loads and increased survival in a new murine experimental model of active tuberculosis antitubercular specifi c activity of ibuprofen and the other 2-arylpropanoic acids using the ht-spoti whole-cell phenotypic assay effl ux inhibition with verapamil potentiates bedaquiline in mycobacterium tuberculosis acceleration of tuberculosis treatment by adjunctive therapy with verapamil as an effl ux inhibitor adjuvant host-directed therapy with types 3 and 5 but not type 4 phosphodiesterase inhibitors shortens the duration of tuberculosis treatment diff erential gene expression in response to adjunctive recombinant human interleukin-2 immunotherapy in multidrug-resistant tuberculosis patients totally drugresistant tuberculosis and adjunct therapies anti-infl ammatory activity of macrolide antibiotics autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an openlabel phase 1 safety trial human fungal infections: the hidden killers the frequency of pneumocystis jirovecii in sputum samples of hiv and tb patients received at the central reference laboratory in windhoek, namibia global burden of chronic pulmonary aspergillosis as a sequel to pulmonary tuberculosis voriconazole resistance in aspergillus fumigatus-should we be concerned? risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in aspergillus species clinical implications of azole resistance in aspergillus fumigatus, the netherlands frequency and evolution of azole resistance in aspergillus fumigatus associated with treatment failure azole antifungal resistance in aspergillus fumigatus high frequency triazole resistance found in non-culturable aspergillus fumigatus from lungs of patients with chronic fungal disease global burden of abpa in adults with asthma and its complication chronic pulmonary aspergillosis allergic bronchopulmonary aspergillosis: review of literature and proposal of new diagnostic and classifi cation criteria allergic bronchopulmonary aspergillosis in cystic fi brosis. a european epidemiological study novel immunologic classifi cation of aspergillosis in adult cystic fi brosis high prevalence of azole-resistant aspergillus fumigatus in adults with cystic fi brosis exposed to itraconazole a dimorphic fungus causing disseminated infection in south africa a randomised, double-blind study of combination antifungal therapy with voriconazole and anidulafungin versus voriconazole monotherapy for primary treatment of invasive aspergillosis. 22nd european congress of clinical microbiology and infectious diseases a new solid oral tablet formulation of posaconazole: a randomized clinical trial to investigate rising single-and multiple-dose pharmacokinetics and safety in healthy volunteers oral fl uconazole during pregnancy and risk of birth defects therapy for fungal diseases: opportunities and priorities a phase 1, randomized, open-label crossover study to evaluate the safety and pharmacokinetics of 400 mg albaconazole administered to healthy participants as a tablet formulation versus a capsule formulation enfumafungin derivative mk-3118 shows increased in vitro potency against clinical echinocandin-resistant candida species and aspergillus species isolates activity of mgcd290, a hos2 histone deacetylase inhibitor, in combination with azole antifungals against opportunistic fungal pathogens modeling nikkomycin z dosing and pharmacology in murine pulmonary coccidioidomycosis preparatory to phase 2 clinical trials in vitro and in vivo antifungal activities of t-2307, a novel arylamidine advancing antifungal r&d. www.f2g.com/f2g-ltd-completes-30-million-fi nancing-round-to-fund-pre-clinical-and-clinical-developmentof-novel-anti-fungal-compounds/ (accessed may the antidepressant sertraline provides a promising therapeutic option for neurotropic cryptococcal infections signaling cascades as drug targets in model and pathogenic fungi progress and prospects for targeting hsp90 to treat fungal infections inhibition of candida albicans virulence factors by novel levofl oxacin derivatives restoration of viral immunity in immunodefi cient humans by the adoptive transfer of t cell clones adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specifi c t-cell lines clinical-scale generation of multi-specifi c anti-fungal t cells targeting candida, aspergillus and mucormycetes towards an ecology of the lung: new conceptual models of pulmonary microbiology and pneumonia pathogenesis deaths due to respiratory tract infections in africa: a review of autopsy studies 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 key: cord-303272-1w8epdht authors: reusken, chantal bem; haagmans, bart l; müller, marcel a; gutierrez, carlos; godeke, gert-jan; meyer, benjamin; muth, doreen; raj, v stalin; vries, laura smits-de; corman, victor m; drexler, jan-felix; smits, saskia l; el tahir, yasmin e; de sousa, rita; van beek, janko; nowotny, norbert; van maanen, kees; hidalgo-hermoso, ezequiel; bosch, berend-jan; rottier, peter; osterhaus, albert; gortázar-schmidt, christian; drosten, christian; koopmans, marion pg title: middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study date: 2013-08-09 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70164-6 sha: doc_id: 303272 cord_uid: 1w8epdht background: a new betacoronavirus—middle east respiratory syndrome coronavirus (mers-cov)—has been identified in patients with severe acute respiratory infection. although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of mers-cov. anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. we investigated possible animal reservoirs of mers-cov by assessing specific serum antibodies in livestock. methods: we took sera from animals in the middle east (oman) and from elsewhere (spain, netherlands, chile). cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum igg by protein microarray using the receptor-binding s1 subunits of spike proteins of mers-cov, severe acute respiratory syndrome coronavirus, and human coronavirus oc43. results were confirmed by virus neutralisation tests for mers-cov and bovine coronavirus. findings: 50 of 50 (100%) sera from omani camels and 15 of 105 (14%) from spanish camels had protein-specific antibodies against mers-cov spike. sera from european sheep, goats, cattle, and other camelids had no such antibodies. mers-cov neutralising antibody titres varied between 1/320 and 1/2560 for the omani camel sera and between 1/20 and 1/320 for the spanish camel sera. there was no evidence for cross-neutralisation by bovine coronavirus antibodies. interpretation: mers-cov or a related virus has infected camel populations. both titres and seroprevalences in sera from different locations in oman suggest widespread infection. funding: european union, european centre for disease prevention and control, deutsche forschungsgemeinschaft. in 2012, a new betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-was identifi ed in patients with severe respiratory disease in the middle east. as of aug 2, 2013, 94 laboratory-confi rmed cases, including 46 deaths, have been reported to who. 1 illness associated with mers-cov infection is characterised primarily by mild-to-severe respiratory complaints, most requiring hospital admission for acute respiratory distress syndrome. comorbidities and immunosuppression seem to predispose for infection and severe disease, [2] [3] [4] [5] [6] and unpublished serological studies suggest that asymptomatic infections occur. 7 all cases reported so far have been linked to jordan, qatar, saudi arabia, and united arab emirates. humanto-human transmission has been reported, particularly in health-care settings, but on the basis of available evidence the basic reproduction number (r 0 ) is thought to be low, suggesting that the virus is not transmitted readily. 6, 8 therefore, the primary reservoir of mers-cov is probably animals. diff erent coronaviruses have various hosts including wildlife, livestock, poultry, pets, and human beings. coronaviruses can adapt to new host species, as shown by the zoonotic origin of several human coronaviruses. 9 human coronavirus oc43 has recent common ancestry with bovine coronaviruses. 10 rhinolophid bats were identifi ed as a likely reservoir for severe acute respiratory syndrome coronavirus (sars-cov), which emerged in people in 2002-03, through intermediate carnivorous hosts. 11 molecular clock analysis 12 showed that bat and civet strains of viruses closely related to sars-cov only diverged a few years before the outbreak. human coronavirus 229e has a common ancestor with coronaviruses found in ghanaian hipposideros spp bats. 13 mers-cov is able to replicate in various bat cell lines 14 and phylogenetic analyses show that it is closely related to betacoronavirus lineage c viruses from pipistrellus spp bats in europe and asia. [15] [16] [17] [18] molecular clock dating of epidemiologically unlinked isolates of human mers-cov estimated their divergence from a common ancestor in mid-2011, 4, 19 with a cluster of isolates from the eastern arabian peninsula diverging in late 2012. 4 this fi nding could suggest that the diversity of mers-cov in people is the result of multiple independent, geographically structured, zoonotic events in the middle east. 4, 19 possible animal reservoirs need to be identifi ed to determine how circulation of mers-cov is maintained and to break the chain of transmission. 20 mers-cov can infect cells of several species, including human beings and bats. 14 the functional receptor is conserved between species, suggesting that receptor use is not an important barrier to cross-species transmission. 21 data for exposure history of patients are scarce, but suggest contact with livestock, including dromedary camels and goats. 2, 4, 5 food and agriculture organization data from 2011 show that cows, goats, sheep, and dromedary camels are the main sources of meat and milk in jordan, saudi arabia, and united arab emirates. 22 serological studies are best suited to screen animal populations, but have not yet been reported for mers-cov in animals, although several methods have been described for testing antibodies of people. 23, 24 for specifi city, who recommends use of a combination of screening assays with recombinant spike protein, and confi rmatory testing by neutralisation assays. here, we describe antibody profi ling of serum samples from major livestock species that might be relevant to the epidemiology of mers-cov in the middle east, using samples collected from herds inside and outside the region. we sampled a cohort of 105 dromedary camels (camelus dromedarius) from two herds on the canary islands. 50 were male, 55 were female, 88 were adults, nine were age 3-4 years, seven were age 2 years, and one was age 3 months. both herds had the same owner, with frequent exchange of animals between the herds. one herd is from a coastal dune habitat with no other livestock, while the other herd is in an inland valley close to a tropical fruit farm, in particular mango and papaya-which could attract fruit bats-and nearby (roughly 500 m) to horse and goat farms with 25 and 300 animals, respectively. the camels were born in the canary islands except for three adults, which were imported from morocco. 25 the camels are used in the tourist industry. 80 sera were taken april-june, 2012, nine in may, 2013, and 16 paired sera were taken in these months in 2012, and 2013, all for routine veterinary purposes. samples were obtained by jugular puncture. 50 female dromedary camels from oman were sampled in march, 2013. the camels were aged 8-12 years and belonged to diff erent owners from separate locations. the camels are retired racing camels now used for breeding, and blood was taken by jugular puncture for routine screening for brucellosis. omani dromedaries sera were collected for veterinary purposes from two llamas (lama glama), six alpacas (vicugna pacos), and two bactrian camels (camelus bactrianus) in the netherlands. sera were collected for veterinary purposes from two bactrian camels, 18 alpacas, fi ve llamas, and two guanaco (lama guanicoe) in buin zoo in chile. sera from cattle (n=40), domestic goats (n=40), and sheep (n=40) were from routine submission to the dutch animal health service. sera from spanish domestic goats (n=40) were provided by the instituto de investigación en recursos cinegéticos (ciudad real, spain) from submissions for tuberculosis control in 2011. all sera were obtained in agreement with local regulations and dutch import regulations with regard to animal disease legislation. positive human control sera for the three antigens used on the microarray were taken as described previously. 24 all samples were stored at -20°c until testing. we tested the sera for the presence of igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc43 s1 antigens in a protein microarray. the receptor-binding domains, which contain the s1 subunit of spike proteins of mers-cov (residues 1-747), sars-cov (residues 1-676), and human coronavirus oc43 (residues 1-760) were expressed, purifi ed, and spotted on glass slides. slides were incubated with serum and species-specifi c conjugates, as previously described. 24 goat sera were incubated with alexa fluor 647-conjugated rabbit anti-goat igg fc fragment (jackson immuno research, west grove, pa, usa); combinations of the mean fl uorescent intensities of reactions of sera with mers-cov and human coronavirus oc43 antigens from 105 spanish dromedary camels (a). plaque reduction neutralisation tests for bovine coronavirus and mers-cov (b): two representative sera are shown (numbers 15 and 5, corresponding to camel id numbers in table 2) in dilutions of 1/40, 1/160, and 1/640 as well as the virus input control. all samples were tested in duplicates (only one well shown) and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90%. igg reactivity of both camel sera to mers-cov antigen and human coronavirus oc43 antigen in a two-step dilution series in the microarray (c). igg reactivity of two two-step serially diluted omani dromedary camel sera with human coronavirus emc antigen and human coronavirus oc43 antigen in the microarray (d). rfu=relative fl uorescence units. mers-cov=middle east respiratory coronavirus. cow sera with alexa fluor 647-conjugated goat antibovine igg (h+l; jackson immuno research); sheep sera with alexa fluor 647-conjugated donkey anti-sheep igg (h+l; millipore, temecula, ca, usa); and camelid sera with dylight 650-conjugated goat anti-llama igg (h+l; agrisera, vännas, sweden). fluorescence signals were quantifi ed as described previously. 24 we tested the sera for igg reactivity at a dilution of 1/20 and set an arbitrary cutoff at an average signal intensity of 20 000 relative fl uorescence units (rfu). this high cutoff was chosen to reduce cross-reactive false positives. 24 we present results as rfu for each set of quadruplicate spots per antigen. negative fl uorescent intensities (caused by background correction) were assigned to 0. analyses were done with graphpad prism (version 6.02). sera were heat-inactivated before virus neutralisation by incubation for 30 min at 56°c. two-fold serial dilutions of sera were prepared using 96-well plates, starting dilution 1/10. mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with clemizole penicillin (penicillin g), streptomycin, and 1% fetal bovine serum, to a dilution of 2000 tissue culture infective dose 50 per ml. 50 μl virus suspension was added to the plates and the plates were incubated at 37°c for 1 h. the mixtures of virus and serum were then incubated on 96-well plates containing vero cells for 1 h followed by washing with phosphate buff ered saline and incubation with imdm and 1% fetal bovine serum for 3-4 days, after which endpoint titres were measured. all tests were repeated twice independently. we tested neutralisation activity of sera against mers-cov (erasmus mc isolate) and bovine coronavirus (nebraska strain) by plaque reduction neutralisation test (90% plaque reduction) with african green monkey kidney cells (cell line vero b4; dsmz acc 33) or bovine kidney cells (cell line pt; cclv-rie11) in a 24-well plate format. virus (30-60 plaqueforming units) and heat-inactivated sera (diluted from 1/40 to 1/640) were pre-incubated in 200 μl of serumfree optipro medium (life technologies, karlsruhe, germany) at 37°c for 1 h. virus adsorption was done at 37°c for 1 h. supernatants were removed and overlaid with avicel resin (fcm biopolymer, brussels, belgium). 5 assays were stopped after 3 days by fi xation with 8% paraformaldehyde for 30 min. all samples were tested in duplicate and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90%. the sponsors 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 fi nal responsibility for the decision to submit for publication. sera were tested for igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc43 s1 antigens in a protein microarray (fi gure 1). human coronavirus oc43 is serologically closely related to bovine coronavirus, 26 diverging at the end of the 19th century. 10 bovine coronavirus circulates in cows, sheep, goats, and old and new world camelids. [27] [28] [29] [30] because bovine coronavirus s1 was not available, human coronavirus oc43 s1 antigen was used as a proxy. sera from three llamas, four alpacas, one guanaco, and two bactrian camels reacted with human coronavirus oc43 antigen. one cow and one goat serum reacted with human coronavirus oc43 antigen as did sera from 16 of 105 (15%) spanish dromedary camels. all sera from cattle, sheep, and goats tested negative for mers-cov antigen, but sera from 15 spanish camels (14%) did react with mers-cov antigen (fi gure 1). the reactivity was highly specifi c-the same sera did not bind to sars-cov antigen but a positive control specimen did. no correlation existed between the reactivity of sera with mers-cov antigen and human coronavirus oc43 antigen (fi gure 2). all but one serum sample that reacted with mers-cov antigen were from adult animals. one reactive serum was from a 2-year-old animal. to confi rm the presence of mers-cov specifi c igg in the spanish camel sera, we used a mers-cov neutralisation assay to test a subset of 49 camel sera with diff erent degrees of reactivity with mers-cov and human coronavirus oc43 antigen according to microarray. nine spanish camels had mers-cov neutralising antibodies with titres varying between 1/20 and 1/320 (table 1). three of the 12 sera reacted with mers-cov spike antigen but did not neutralise mers-cov, most likely because of recognition of nonneutralising epitopes. all mers-cov neutralising sera had (almost) saturating reactivity with mers-cov antigen on the microarray, whereas reactivity with human coronavirus oc43 antigen varied from negative to 50% of saturating reactivity (table 1). the variable human coronavirus oc43 signals suggest that mers-cov did not generally cross-react with human coronavirus oc43 or bovine coronavirus antigens. all nine camels with mers-cov neutralising antibodies were born and raised on the canary islands; seven were female, two were male. eight camels were adults, one was 2 years old. to show that the reactivity of the camel sera with human coronavirus oc43 antigen according to the microarray was caused by the presence of bovine coronavirus igg and to further exclude mers-cov neutralising activity caused by cross-neutralisation by the bovine coronavirus antibodies, we tested camels that had suffi cient serum left (n=15) in a comparative mers-cov and bovine coronavirus plaque reduction neutralisation test (fi gure 2, table 2). all camel sera neutralised bovine coronavirus, but with varying titres, suggesting a lower cutoff than 20 000 rfu for oc43 in the microarray (fi gure 1). five camels had high neutralising antibody titres against bovine coronavirus (and a mean signal intensity of greater than 50 000 rfu for human coronavirus oc43 antigen on microarray) but were negative for mers-cov neutralisation, suggesting that cross-neutralisation in this direction did not occur and that the mers-cov neutralising activity was not caused by the presence of bovine coronavirus neutralising antibodies. a serum sample from a patient who had mers, neutralised mers-cov with a high titre (1/640) but neutralised bovine coronavirus less effi ciently (titre 1/80). the latter fi nding was most probably caused by previous infection with human coronavirus oc43-this patient had a high titre (1/>5120) in a human coronavirus oc43 recombinant spike immuno fl uorescence assay and a saturating signal with human coronavirus oc43 antigen in the microarray. two human serum samples positive for human coronavirus oc43 did not neutralise mers-cov, one of which neutralised bovine coronavirus at a titre of 1/80 (table 2) . we tested 50 sera from dromedary camels in oman at a dilution of 1/20 by microarray and mers-cov neutralisation test. all the sera showed saturating reactivity with mers-cov antigen on the microarray, no sars-cov antigen reactivity, and human coronavirus oc43 antigen reactivity varying between negative (below the cutoff of 20 000 rfu) and saturating signals (fi gure 1, table 1). serial dilution of two sera with saturating reactivity for both antigens at the initial dilution of 1/20 showed that mers-cov antigen reactivity was still above the cutoff at 1/5120, whereas human coronavirus oc43 antigen reactivity fell below the cutoff at dilutions of 1/80 to 1/320 (fi gure 2d). consistent with the microarray data, all sera had high mers-cov neutralising capacity, with titres varying between 1/320 (seven of 50 samples) and 1/2560 or more (16 or 50 samples). in this study we describe the presence of mers-cov neutralising antibodies in dromedary camels both in a mers-cov linked (oman) and unlinked regions (canary islands). all the sera from dromedary camels from oman and some from spain had specifi c igg reactivity with the mers-cov receptor binding domain s1. we confi rmed our expectation that another betacoronavirus-bovine coronavirus-circulated in these camelids. 29 spanish camels (9%) had specifi c neutralising antibodies against mers-cov that were clearly not caused by cross-neutralisation by bovine coronavirus antibodies. our study is the fi rst in which animals have been tested for the presence of antibodies specifi c to mers-cov (panel). animal screening is necessary to understand the epidemiology of mers-cov. at present, bats are thought to be the ultimate reservoirs for several established human coronaviruses as well as sars-cov. accordingly, phylogenetic analysis has shown that mers-cov is related to betacoronavirus lineage c viruses found in pipistrellus spp bats. 15, 16 however, direct transmission of mers-cov to people from bats seems unlikely. 4, 19 the identifi cation of possible intermediate hosts that are probably in closer contact with people (eg, livestock) is urgently needed. common livestock species in the middle east include dromedary camels but also cattle, sheep, and goats. based on the available data, we cannot rule out circulation of a mers-related coronavirus in these species-sera were not available from epi demi ologically linked regions. the high prevalence of mers-cov neutralising antibodies in dromedary camels from oman suggests circulation of mers-cov or a closely related virus in this population. however, attempts to identify viral sequences in spanish camel sera and faecal samples using pancoronavirus and specifi c betacoronavirus 2c pcr methods 15, 31, 32 were unsuccessful (unpublished data), as was untargeted amplifi cation followed by deep sequencing of faecal samples (unpublished data). these results imply that the camels were not actively shedding the virus at the time of sampling. less than 10% of the animals in the canary islands had mers-cov neutralising sera with titres up to 1/320. this low seroprevalence means either that exposure of the animals to other putative reservoirs is rare 33 or that the virus is absent in this closed-off population of roughly 2000 animals. 25 we cannot rule out that the population might have once had an outbreak but that by the time of sampling, antibody titres had waned and no new introductions of the virus had occurred. the camels have contact with wild rodents, rabbits, pigeons, and doves and possibly also with bats. seven insectivorous bat species, including three pipistrellus spp, are native to the canary islands, while egyptian fruit bats (rousettus aegyptiacus) have been introduced. 34 the 100% seroprevalence with high titres in omani camels from diff erent owners and locations suggests a diff erent situation in the middle east, with widespread circulation of mers-cov or a closely related virus. this diff erence of epidemiology might be because the virus circulating in the middle east is diff erent to that circulating in spain, with increased animal transmissibility and human infections. 35 in addition, the omani camels were once racing camels now held for breeding and might be kept in circumstances that favour virus transmission. for cattle, a relation has been established between the incidence and eff ects of respiratory diseases, management practices, and animal transport. 36, 37 to our knowledge, the camel populations in oman and the canary islands are not connected. camels on the canary islands were originally imported in the 15th century from the horn of africa for labour and transport. nowadays, import of animals from africa is banned because of the risk of foot-and-mouth disease. only three camels in our study were originally imported from morocco, more than 18 years ago. because the closest relatives of mers-cov were identifi ed very recently in neoromicia zuluensis bats from africa, 38 the introduction of mers-cov or related viruses into some african camel populations could have occurred decades ago, giving a possible explanation for mers-cov antibodies in camels from the canary islands. in the middle east, huge numbers of camels are imported from africa to meet the demand for meat. the top fi ve camel breeding countries are all african, and saudi arabia and united arab emirates are in the top fi ve camel meat producing countries. 22 this increased turnover of animals in the middle east we searched pubmed for "novel coronavirus emc" or "mers-cov", we identifi ed 43 reports in english linked to the middle east respiratory syndrome coronavirus (mers-cov) published before july 22, 2013. none of these reports described a serological study for mers-cov-specifi c antibodies in animals. our report describes the fi rst mers-cov serological study of major livestock relevant to the middle east. our study shows that mers-cov or a related virus has infected dromedary camel populations. both titre levels and seroprevalences in sera from diff erent locations in oman suggest widespread infection of camelids with mers-cov or a closely related virus. targeted studies are needed to confi rm our fi ndings and their possible relevance to human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels from diff erent regions for which epidemiological background information is available, as well as virological assessment of samples from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime, we recommend a detailed case history of confi rmed mers-cov cases, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. compared with the canary islands could also aff ect the epidemiology of a virus, through more frequent infl ux of immunologically naive animals. targeted studies are needed to confi rm our fi ndings and their possible relevance in relation to the human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels for which epidemiological background information is available, as well as virological assess ment of specimens from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime we recommend a detailed case history of people with mers-cov, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. who. global alert and response (gar): novel coronavirus infection recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov)-update interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk coronavirus diversity, phylogeny and interspecies jumping evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc43 severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats ecoepidemiology and complete genome comparison of diff erent strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus circulation of group 2 coronaviruses in a bat species common to urban areas in western europe comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections specifi c serology for emerging human coronaviruses by protein microarray camel trypanosomosis in the canary islands: assessment of seroprevalence and infection rates using the card agglutination test (catt/t. evansi) and parasite detection tests antigenic and biological relationships between human coronavirus oc43 and neonatal calf diarrhoea coronavirus serum antibodies to bovine coronavirus in swedish sheep serosurveillance of viral diseases in korean native goats (capra hircus) enteric coronavirus infection in a juvenile dromedary analysis of the genome sequence of an alpaca coronavirus the diff erential clinical impact of human coronavirus species in children with cystic fi brosis generic detection of coronaviruses and diff erentiation at the prototype strain level by reverse transcription-pcr and nonfl uorescent low-density microarray forecast and control of epidemics in a globalized world la fauna de quiropteros del archipelgo canario coronavirus genomics and bioinformatics analysis risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds associations between the distance traveled from sale barns to commercial feedlots in the united states and overall performance, risk of respiratory disease, and cumulative mortality in feeder cattle during 1997 to 2009 close relative of human middle east respiratory syndrome coronavirus in bat we thank prof mc horzinek for helpful suggestions. rds was funded by the european public health training program (euphem), ecdc, stockholm, sweden. contributions to the study were funded through the european union fp7 projects emperie (contract number 223498; to blh, sls, ao, cd) and antigone (contract number 278976; to cg, cd, mpgk, ao). work in bonn was also funded by deutsche forschungsgemeinschaft (dfg grant dr772/3-1 to cd). key: cord-317795-689at1qx authors: bielicki, julia a; duval, xavier; gobat, nina; goossens, herman; koopmans, marion; tacconelli, evelina; van der werf, sylvie title: monitoring approaches for health-care workers during the covid-19 pandemic date: 2020-07-23 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30458-8 sha: doc_id: 317795 cord_uid: 689at1qx health-care workers are crucial to any health-care system. during the ongoing covid-19 pandemic, health-care workers are at a substantially increased risk of becoming infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and could come to considerable harm as a result. depending on the phase of the pandemic, patients with covid-19 might not be the main source of sars-cov-2 infection and health-care workers could be exposed to atypical patients, infected family members, contacts, and colleagues, or live in communities of active transmission. clear strategies to support and appropriately manage exposed and infected health-care workers are essential to ensure effective staff management and to engender trust in the workplace. these management strategies should focus on risk stratification, suitable clinical monitoring, low-threshold access to diagnostics, and decision making about removal from and return to work. policy makers need to support health-care facilities in interpreting guidance during a pandemic that will probably be characterised by fluctuating local incidence of sars-cov-2 to mitigate the impact of this pandemic on their workforce. an adequate level of staffing is crucial to maintain patient care during the ongoing covid-19 pandemic. 1 frontline health-care staff assess and manage patients with covid-19, patients presenting with emergencies not related to covid-19, and patients with essential routine care needs. one of the greatest risks to the health-care system is a high rate of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection among health-care workers and the consequent lack of skilled staff to ensure a functioning local or regional response to the pandemic. 2 this risk has been increased by the need for rapid scaling up of intensive care unit (icu) capacity in affected regions, the redeployment of clinical staff to frontline positions (eg, icus or covid-19 wards), and the recruitment of less experienced staff (eg, newly qualified students or health-care staff moving from their specialism) to the workforce in response to the pandemic. 3, 4 health-care workers could acquire sars-cov-2 at work through direct or indirect contact with infected patients or other health-care workers, or as a result of ongoing community transmission. community transmission of sars-cov-2 is targeted by public health measures, whereas infection by patient or health-care worker contact is primarily addressed by facility-based infection prevention and control (ipc) measures. however, sources of infection might not be clear and this uncertainty can have negative effects on the clinical workforce. ipc measures are extensive in hospitals managing patients infected with sars-cov-2 and, broadly speaking, include rigorous cleaning and dis infection to reduce environmental contamination and the use of personal protective equipment (ppe), isolation, and cohorting. 5 national and international recommendations for risk assessment and management of hospital health-care staff working with patients infected with sars-cov-2 are detailed and publicly available. [6] [7] [8] [9] however, recommendations might not be easily transferrable because health-care systems are highly variable in terms of their structure and workforce composition. 10 available guidance can become rapidly unsuitable when the situation at the frontline of health-care delivery is continuously changing. therefore broad recommendations need to be translated into locally applicable and pragmatic solutions. in this personal view, we outline and discuss possible approaches to inform develop ment of local policy related to health-care worker exposure and management during the covid-19 pandemic. several emerging viral diseases are known to have had a major effect on health-care workers, which is currently being observed also with sars-cov-2. 11, 12 in an early case series from wuhan, china, 29% of patients with sars-cov-2 were health-care workers and were assumed to have acquired the infection in hospital. 13 deaths among health-care workers infected with sars-cov-2 are rare and have mostly affected those older than 50 years. 14, 15 tragically, health-care workers rehired from retirement to help at the frontline have commonly experienced the highest mortality when compared with their working-age counterparts. 16, 17 with an increasing understanding of the disease, the proportion of healthcare workers contracting covid-19 in hospital has decreased, but stringent ipc measures and continued vigilance are needed. 18 the risk profile for sars-cov-2 exposure and infection among health-care workers differs substantially from other groups. in designated covid-19 wards or hospitals, health-care workers are at high risk of infection. potential exposure to sars-cov-2 is inherent to their work and is prevented only by excellent adherence to all ipc measures, including the use of appropriate ppe. there is uncertainty about what is optimal ppe, but it is clear that standardised and rigorous application of ppe and other ipc measures can dramatically reduce nosocomial transmissions. 19, 20 personal view health-care workers are likely to be in contact with patients and colleagues who have atypical, few, or no symptoms while still being highly contagious. [21] [22] [23] a high proportion of such individuals will be present in the hospital, including in areas with insufficient awareness or identified need of ipc measures, as the virus spreads (figure). particular attention is needed for health-care workers looking after patients who are highly dependent and live in long-term care facilities, which may be built to resemble home-like environments, compro mising the ability to apply stringent ppe and other ipc measures. 24 similarly, the presence of oligosymptomatic health-care workers infected with sars-cov-2 in situations during which ppe is not usually applied, such as scheduled meetings, grand rounds, educational events, and break times, will become more likely as the pandemic progresses. 25 finally, with increasing community transmission, the highest risk of sars-cov-2 exposure for health-care workers could be outside of the hospital. many health-care workers will contract sars-cov-2 through interactions with infected family members or other close contacts, or from the community in areas with active, unmitigated transmission. 26, 27 improper ppe use, suboptimal adherence to ipc measures, and having a family member with covid-19 can double or triple the risk of subsequent health-care worker sars-cov-2 infection. 28 a detailed study of the prevalence of sars-cov-2 among mildly symptomatic health-care workers in dutch hospitals shows that many infections were most likely acquired in the community. 29, 30 defining the risk of a health-care worker being infected with sars-cov-2 can be the first step towards selecting the most appropriate monitoring and evaluation approach. 6, 8, 9, 31, 32 risk categories for in-hospital exposures are frequently based on the type of contact that has taken place and whether ppe was used consistently and appropriately. additional specifications are sometimes included in risk assessment algorithms-eg, presence during aerosol-generating procedures or exact distancing from patients with covid-19 (usually closer or further than 2 m). 31, 33 focusing on adherence to ppe implies that the optimal ppe for all potential contact situations is known and available. however, the effect of optimal ppe and other ipc measures is being debated because robust evidence to match ppe and ipc interventions to the risk profile of a given exposure is scarce. [34] [35] [36] exposures to sars-cov-2 via community cases and infected colleagues can be frequent depending on the phase of the outbreak. risk assessment of health-care worker exposure, in our opinion, is going to be most useful in epidemic phases with low rates of community transmission. in all other situations, all health-care workers should be considered at moderate to high risk of contracting sars-cov-2, especially when extended ipc measures, including some use of ppe, cannot be implemented for all patient contacts and staff interactions. data showing that viral shedding and potential sars-cov-2 trans mission could occur 2-3 days before symptom onset highlight the importance of wearing adequate ppe in hospitals during phases of high sars-cov-2 incidence. 37 therefore, risk-appropriate ppe and optimal adherence to ipc measures will reduce the risk of health-care worker infection to that encountered in the community. guidance provided by peking union medical college hospital (beijing, china) suggests that all health-care workers in close contact with patients with covid-19, regardless of ppe use, should undergo nasopharyngeal and oropharyngeal pcr testing and a full blood count after an unspecified block period of work in the designated area. 38 further management decisions are determined by the results of these tests but, if negative, health-care workers are monitored for 1 week and could resume work after this time if asymptomatic. calls have been made by health-care workers to improve availability of testing for asymptomatic healthcare staff and allow screening. 39 in our opinion, this approach has the distinct disadvantage of requiring very frequent evaluation, given that intermittent testing might not capture asymptomatic sars-cov-2 positive individuals. for example, in a case series of 13 patients with asymptomatic sars-cov-2 infection, eight were rt-pcr negative up to 14 days after first identification of sars-cov-2 and could well have been missed by fortnightly screening. 40 we therefore do not favour regular general sars-cov-2 testing of health-care workers by pcr as an effective monitoring approach. an alternative to intermittent pcr testing is to adopt a responsive approach to moni toring health-care workers. most national monitoring systems incorporate some form of daily (self)screening for fever and assessment of respiratory symptoms. 6, 9, 33, 41, 42 stringent documentation and reporting requirements are an additional burden on health-care workers who are already stretched by the demands of patient care. active symptom monitoring by public health authorities or their delegates of healthcare workers deemed at risk of sars-cov-2 infection in occupational health is not feasible once an epidemic is in the exponential phase. self-monitoring and reporting are more feasible but must be combined with excellent communication from occupational health officers to ensure that health-care workers feel adequately supported and have a point of contact to discuss any concerns or questions. very low threshold access to occupational health to report any feelings of illness is crucial. health-care workers might be concerned about whether such symptoms could indicate sars-cov-2 infection and might be reluctant to report mild symptoms because they feel that they are burdening the system. furthermore, even mild symptoms can be indicative of sars-cov-2 infection, as shown when enhanced access (whereby all people with any respiratory symptoms or generalised symptoms suggestive of an infection are invited for testing) to testing was made available at a group of hospitals in the netherlands. 43 direct access to occupational health has the additional advantage of enabling some psychosocial screening of the effect of working during the covid-19 pandemic. testing should be made available widely to symptomatic health-care workers and auxiliary acute health-care staff. the importance of supporting health-care worker access to sars-cov-2 testing in the case of symptoms cannot be overemphasised, particularly when the source of infection shifts from individual patients who are clearly identifiable to widespread viral transmission. interactions with colleagues who are also at increased risk of exposure and infection could become classed as high-risk pro cedures. during the period of unmitigated community transmission in the uk, access to testing for health-care workers, including those with symptoms, could not be guaranteed at a time when the medical workforce was under severe pressure from soaring sars-cov-2 cases. after roll-out in a single uk national health service trust, 18% of symptomatic staff tested positive for sars-cov-2 in the first 2 weeks of the test being available, showing that there is potentially a large pool of infected individuals working in hospitals in a setting with sustained commu nity transmission. 44 many countries prioritise health-care workers for sars-cov-2 testing, often on the basis of reported sympt oms and regardless of a confirmed exposure. for example, switzerland and the netherlands recommend rapid access to sars-cov-2 pcr testing and results for health-care workers because this information is used for decision making about deployment of medical staff. 45, 46 personal view decision making on health-care worker removal from and return to work the most suitable approach towards managing removal from and return to work of health-care workers depends on the pursued public health strategy (ie, containment or mitigation) and the current pressures on the health-care system. during containment, standard quarantine and isolation should also be applied to health-care workers given that a special provision for health-care workers is unlikely to be necessary or helpful. premature redeployment of quarantined or isolated health-care workers will probably be needed only in exceptional cases-eg, for highly specialised staff. when testing of all symptomatic individuals cannot be guaranteed, as is often the case in a mitigation phase, pcr testing of symptomatic health-care workers should be prioritised and can be used to reduce workforce depletion caused by quarantine and isolation of sympto matic health-care workers. the pressures on a given health-care system are considerable; however, it is difficult to justify a special status for health-care workers from a public health perspective because of the bidirectional nature of sars-cov-2 infections among this group. although health-care workers can acquire sars-cov-2 at work, introducing transmission into the community, they may also bring sars-cov-2 into the hospital following community exposures. pcr testing of asymptomatic quarantined health-care workers will provide false reassurance for exposed indi viduals with early negative results who go on to develop disease later on in the defined quarantine period. the role of pcr testing is different for symptomatic individuals. home isolation periods range from a minimum of 7 days (under certain conditions) in france and the uk, to 14 days in germany and italy, and isolation is often recommended independently of whether sars-cov-2 has been identified by testing. in most cases, an additional requirement of at least 48 h without symptoms before ending isolation is also specified. in the netherlands, infected health-care workers who are considered crucial for the care of patients with covid-19 can return to work after 24 h without symptoms, so shorter isolation periods are concei vable. 6, 8, 31, 42, 45, 46 pcr testing of health-care workers should be used to ensure that isolation of symptomatic staff is limited to individuals who have been confirmed as sars-cov-2 positive. 39, 45 in some cases, pcr testing is recommended to support rapid return to work of infected health-care workers if they become negative on pcr before the isolation period has elapsed. for example, german guidance recommends that health-care workers who required hospital treatment can return to work immediately if two pcr tests at least 24 h apart are negative. 31, 47 in switzerland, re-testing of health-care workers infected with sars-cov-2 at the end of the isolation period is proposed for those working in high-risk areas (haemato-oncology, icus, transplant units) and those with prolonged disease. 45 there is considerable uncertainty about the relevance of prolonged detection of sars-cov-2 on pcr testing for transmissibility; therefore, the role of repeat testing to determine redeployment of health-care workers after sars-cov-2 infection is unclear. for health-care workers with confirmed sars-cov-2 infection, testing at the end of the isolation period is sometimes used to confirm suitability for return to work, often with two pcr tests at least 24 h apart. 42, 48 however, in practice, these recommendations are problematic. a study comparing rt-pcr testing and virus culture found that patients with mild symptoms were positive by rt-pcr for up to 28 days, whereas no infectious virus could be recovered after day 10 post illness onset. 49 therefore, a symptom-based algorithm that informs when isolated health-care workers should return to work appears to be best when exposed or infected health-care workers are considered crucial to service maintenance and extended periods of quarantine or isolation are not feasible. studies are ongoing to assess the possible role of serology as a marker for viral clearance in people with mild illness. the exact algorithm for managing exposed and infected health-care workers should aim to safeguard staff wellbeing and reduce onwards transmissions to colleagues and patients without undermining the ability to maintain an adequate service, which is often a difficult balance. the exact configuration of the health-care setting and ipc strategies will affect the success of the management strategy and might require different approaches during different phases of the pandemic, especially when the use of ppe and sources of health-care worker infection are shifting (table) . with proper use of ppe and good adherence to ipc measures, the risk of sars-cov-2 infection of health-care workers caring for patients with covid-19 is considered to be very low. physical distancing should be encouraged for contact with colleagues, such as during meetings, joint meals, and in office spaces. monitoring and identifying health-care workers with symptoms compatible with or suggestive of sars-cov-2 infection is essential to ensure appropriate triaging of staff for duty, further evaluation, and follow-up. there are considerable psychological and social strains on health-care workers because they work in a highly stressful and demanding environment and could have negative psychological effects caused by concerns over ppe availability; therefore, monitoring policies should con sider how to incorporate the rapid assessment of psychosocial needs of health-care workers. rapid and low-threshold access to sars-cov-2 testing and results for health-care workers are key to maintaining personal view an adequate workforce. regular screening of health-care workers by pcr is unlikely to be an effective means of workforce management until evidence-based algorithms to define target staff and frequency of testing are developed, and even then negative tests might offer a false sense of reassurance. clear algorithms must exist can identify hcws at considerable risk of acquiring sars-cov-2 in the health-care setting and focus resources on active monitoring or proactive laboratory testing; can support implementation of quarantine measures for a specific group of hcws, minimising the effect on the workforce and maximising containment of sars-cov-2 within the health-care environment can reduce awareness that interactions with any patients with covid-19 (known or unknown) carry some risk of nosocomial transmission to hcws; can be confusing when understanding of the optimal ppe remains unclear; can undermine hcw engagement with key ipc measures other than ppe (eg, hand hygiene and physical distancing) in the erroneous belief that these are ineffective; might not be relevant in settings where some level of ppe is universally recommended (eg, wearing of surgical masks for all patient contacts) and there is high adherence to other ipc measures use of (self)quarantine after contact can maximise containment of sars-cov-2 within the health-care environment, especially in hcws who may have no, few, or atypical symptoms; can reduce hcw anxiety about contracting sars-cov-2 in the workplace from colleagues with known exposure can rapidly deplete the workforce, particularly in cases of hcws infected with sars-cov-2 exposing many colleagues or when there is uncontrolled community transmission, with hcws exposed outside of the hospital; might not be relevant in settings where some level of ppe is universally recommended (eg, wearing surgical mask for all patient contacts) and there is high adherence to other ipc measures active (eg, at the start of shifts or through regular telephone or email reporting) can support the reliable reporting of signs and symptoms compatible with sars-cov-2 infection; can lead to earlier identification of symptomatic hcws, and therefore support targeted timely testing to reduce exposure of colleagues and patients; can be an opportunity to interact with hcws about their general psychological and physical wellbeing to provide wider support can present a considerable administrative and resource challenge, depending on the exact method of active monitoring and selection of hcws who undergo active monitoring; can lead to a rapid depletion of staff if minor symptoms lead to (self)isolation without sars-cov-2 testing; might be a drain on resources, especially in cases of clusters involving multiple hcws and in settings where large sections of a hospital are dedicated to the care of patients with covid-19 self-monitoring can reduce the barrier to hcw sars-cov-2 testing, if a simple algorithm is combined with clear advice on how to access testing; can involve the majority of hcws in one facility, thereby detecting sars-cov-2 cases among personnel resulting from known and unknown exposures within and outside of the health-care setting; can be supported using digital tools, such as symptom monitoring apps can support rapid identification of hcws infected by sars-cov-2 to provide adequate clinical support and inform self-isolation; can provide a sense of security to staff working in close proximity with colleagues (eg, icus, operating theatres, emergency departments); can represent an efficient use of resources, especially if the threshold for accessing testing is low, sampling is carried out rapidly after onset of symptoms, and results are available in a timely fashion can lead to delays in identification of symptomatic sars-cov-2 positive hcws by relying on (self-) identification of symptoms if pathways to accessing testing are unclear or cumbersome, or if hcws feel uncomfortable with accessing testing because of fear or stigma; might not identify asymptomatic or oligosymptomatic sars-cov-2 positive hcws who could theoretically represent a source of infection for other staff or patients can prevent a difficult to justify disconnect between public health measures and special provisions for hcws; could ensure that the risk of introduction of sars-cov-2 from the health-care setting to the community is minimised for handling the possible scenarios from testing of sympto matic health-care workers, in principle those who are sars-cov-2 negative and those who are sars-cov-2 positive with or without clinical illness. these algorithms need to detail the exact pathway to inform return to work and include advice and support for household contacts of health-care workers who are sars-cov-2 positive. one logical consequence of offering testing to symptomatic health-care workers is to support rapid return to work of those who are sars-cov-2 negative and clinically able to work. when adequate staffing cannot be maintained and rapid redeployment of sars-cov-2 infected health-care workers is necessary, re-testing could identify health-care workers no longer shedding the virus, but the relevance of ongoing shedding is unclear. the roles of serological testing and prophylaxis (responsive or long term) for health-care workers remain to be defined. it is hoped that evidence of previous infection will correlate with the presence of neutralising antibodies and could identify health-care workers at a low risk of reinfection for voluntary supported deployment in covid-19 wards. trials of (chemo)prophylaxis for health-care workers have started recruitment in various countries-eg, covidaxis in france (nct04328285) and bcg-corona in the netherlands (nct04328441)but are not expected to report for some time. specific recommendations for monitoring health-care workers for potential sars-cov-2 infection should be available for all staff who are expecting to see or currently managing patients with covid-19. we feel that in a strict containment phase with low levels of community circulation, management strategies should closely align with those defined for exposed and infected members of the general public, meaning that quarantine and isolation will be stringently applied. given that out breaks put excess pressure on the health-care system, special provisions for health-care workers are unlikely to be needed or justifiable. however, beyond this stage, algorithms for accelerated redeployment of mildly symptomatic health-care workers might be necessary to safeguard adequate staffing levels for patient care, and a very low threshold for access to testing should be instituted to support this. clearly, health-care workers returning to work must prioritise their clinical and psychological wellbeing and consequent ability to reenter the workspace. on the whole, health-care staff have been observed to be extremely dedicated to ensure that their patients are adequately cared for under very difficult circumstances. supporting health-care workers in selfmonitoring and self-care, providing them with easy access to diagnostics and medical and psychosocial support, and offering clear guidance for safe and timely transitioning back to work will strengthen patient care as a whole and could ultimately improve outcomes for many patients and health-care workers. all authors contributed to the concept of the manuscript and identified relevant data. jab wrote the first draft of the manuscript, which was critically revised and approved by all authors. we declare no competing interests. covid-19 and italy: what next? the art of war" in the era of coronavirus disease 2019 (covid-19) intensive care during the coronavirus epidemic intensive care management of coronavirus disease 2019 (covid-19): challenges and recommendations escalating infection control response to the rapidly evolving epidemiology of the coronavirus disease 2019 (covid-19) due to sars-cov-2 in hong kong contact tracing: public health management of persons, including healthcare workers, having had contact with covid-19 cases in the european union covid-19) pandemic: increased transmission in the eu/eea and the uk-sixth update health workers exposure risk assessment and management in the context of covid-19 virus interim us guidance for risk assessment and public health management of healthcare personnel with potential exposure in a healthcare setting to patients with coronavirus disease (covid-19) health workforce development in the european union: a matrix for comparing trajectories of change in the professions risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures clinical characteristics of 138 hospitalised patients with 2019 novel coronavirus-infected pneumonia in wuhan, china characteristics of health care personnel with covid-19-united states 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 death from covid-19 of 23 health care workers in china epidemia covid-19-aggiornamento nazionale timely mental health care for the 2019 novel coronavirus outbreak is urgently needed protecting health care workers during the coronavirus disease 2019 (covid-19) outbreak: lessons from taiwan's severe acute respiratory syndrome response personal protective equipment for preventing highly infectious diseases due to exposure to contaminated body fluids in healthcare staff protecting chinese healthcare workers while combating the 2019 novel coronavirus protecting health-care workers from subclinical coronavirus infection temporal dynamics in viral shedding and transmissibility of covid-19 epidemiology of covid-19 in a long-term care facility in king county, washington beyond the assistance: additional exposure situations to covid-19 for healthcare workers covid-19 in australian healthcare workers: early experience of the royal melbourne hospital emphasises the importance of community acquisition epidemiological, clinical characteristics and outcome of medical staff infected with covid-19 in wuhan, china: a retrospective case series analysis epidemiology of and risk factors for coronavirus infection in health care workers: a living rapid review covid-19 in healthcare workers in three hospitals in the south of the netherlands rapid assessment of regional sars-cov-2 community transmission through a convenience sample of healthcare workers, the netherlands kontaktpersonennachverfolgung bei respiratorischen erkrankungen durch das coronavirus sars-cov-2 risk assessment of healthcare workers with potential workplace exposure to covid-19 case (v8.1) public health management of cases and contacts associated with coronavirus disease medical masks vs n95 respirators for preventing covid-19 in health care workers: a systematic review and meta-analysis of randomised trials a systematic risk-based strategy to select personal protective equipment for infectious diseases risk of nosocomial transmission of coronavirus disease 2019: an experience in a general ward setting in hong kong asymptomatic and presymptomatic sars-cov-2 infections in residents of a longterm care skilled nursing facility diagnosis and clinical management of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: an operational recommendation of peking union medical college hospital (v2.0) covid-19: the case for health-care worker screening to prevent hospital transmission follow-up of asymptomatic patients with sars-cov-2 infection covid-19, france et monde interim guidance for coronavirus-healthcare worker management by occupational health (version 16) sars-cov-2 infection in 86 healthcare workers in two dutch hospitals in roll-out of sars-cov-2 testing for healthcare workers at a large nhs foundation trust in the united kingdom public health management of healthcare workers, having had unprotected contact with covid-19 cases in switzerland uitgangspunten inzetten en testen zorgmedewerkers optionen zum management von kontaktpersonen unter medizinischem personal (auch bei personalmangel) in arztpraxen und krankenhã¤usern) covid-19: kriterien zur entlassung aus dem krankenhaus bzw aus der hã¤uslichen isolierung virological assessment of hospitalized patients with covid-2019 this manuscript is part of the output from recover (rapid european covid-19 emergency research response), which has received funding from the eu horizon 2020 research and innovation programme (grant agreement number 101003589). the funder had no role in the writing of the manuscript or the decision to submit for publication. the views and opinions expressed in this personal view are those of the authors. key: cord-313615-cts45n3j authors: tam, john s; barbeschi, maurizio; shapovalova, natasha; briand, sylvie; memish, ziad a; kieny, marie-paule title: research agenda for mass gatherings: a call to action date: 2012-01-15 journal: lancet infect dis doi: 10.1016/s1473-3099(11)70353-x sha: doc_id: 313615 cord_uid: cts45n3j public health research is essential for the development of effective policies and planning to address health security and risks associated with mass gatherings (mgs). crucial research topics related to mgs and their effects on global health security are discussed in this review. the research agenda for mgs consists of a framework of five major public health research directions that address issues related to reducing the risk of public health emergencies during mgs; restricting the occurrence of non-communicable and communicable diseases; minimisation of the effect of public health events associated with mgs; optimisation of the medical services and treatment of diseases during mgs; and development and application of modern public health measures. implementation of the proposed research topics would be expected to provide benefits over the medium to long term in planning for mgs. a mass gathering (mg), as defi ned by who, is "any occasion, either organized or spontaneous, that attracts suffi cient numbers of people to strain the planning and response resources of the community, city or nation hosting the event". 1 mgs can be spontaneous or organised and include sports events, social or cultural functions, gatherings of displaced populations due to natural disasters or war, and political or religious congregations. [2] [3] [4] [5] major mgs are likely to involve communities from diff erent parts of the world. participants and host and home communities face health risks that are of international concern. therefore, the control of infectious diseases and provision of medical services for non-communicable dis eases and other risks to health are increasingly important in the planning for mgs. risks commonly associated with mgs include health systems having to accom modate a surge in capacity; challenges to existing health interventions; introduction and transmission of non-endemic diseases during and after mgs; diffi culties associated with risk communication to participants from diff erent cultures; and those arising because of the high profi le of some events, such as security risks. international mgs can amplify the transmission of infectious diseases, and infections can spread from the home to the host community during travel to and from the event and from the host to the home community on return. 6 such events can pose substantial risks to global health security and present challenges for surveillance of the spread of such diseases to new ecological settings and vulnerable populations. 7 in addition to risks of infectious diseases during mgs, non-communicable risks include cardiovascular diseases, environment-related heat injury, fi re-related injury, illnesses related to use of drugs and alcohol, occupational injuries, trauma or crush injuries associated with stampedes, exacerbation of respiratory diseases, and crowd safety. 8 large mgs can also provide opportunities for terrorist activities. in planning for mgs, international and local health authorities need to ensure that the strengthened public health systems and rapid responses to health risks are integrated with other important components of the overall event management. eff ective public health policy should be based on evidence. the organisation of international mgs generally requires provision of huge amounts of resources by the host country and the dividend of such high visibility is called legacy. this legacy should be measured not just in terms of absolute improvements (eg, new roads and ambulances) but also in terms of improvements to the public health system and society as a whole. for example, the legacies of strengthened integration of clinical or laboratory services, early warning or syndromic surveillance, and fi eld epidemiology or response adopted for the past summer olympics (sydney, australia, athens, greece, and beijing, china) [9] [10] [11] are used routinely in the health systems. the development of eff ective methods for, and improvement of, planning and handling of the health risks associated with mgs will strengthen global health security, prevent excessive emergency health problems and associated economic loss, and mitigate potential societal disruption in host and home communities. such development requires credible evidence to support activities that can reduce the global eff ect of infectious diseases and address local public health issues related to morbidity and mortality resulting from noncommunicable diseases during mgs. however, there are gaps in knowledge about many public health issues that contribute to eff ective planning. 6, 12, 13 therefore, a robust knowledge about illnesses, from basic scientifi c understanding to societal eff ects of infections and noncommunicable diseases, is essential for modern public health practices and policy development related to the planning for mgs. several reports [11] [12] [13] [14] [15] and who planning and guidance documents 1,16-18 have drawn attention to the importance series of research into public health issues associated with mgs and identifi ed those that need immediate attention. despite these eff orts, an overarching research agenda based on public health to address the gaps in knowledge in mg health has not yet been developed. moreover, international coordination to prioritise and enable the funding and implementation of such an agenda has been lacking. the recent recognition and rapid development of mg health can provide focus on such issues. previously identifi ed research priorities for mgs tended to focus on logistical issues relating to site security and emergency management, crowd control, and surge in the need for medical services. 12, 13 other specialties tend to focus on each of the specifi c public health topics that are associated with mgs. much research into existing and emerging infectious diseases is devoted to the development of rapid diagnostic methods, surveillance and response, and treatment and vaccines. although a research agenda based on public health must be underpinned by basic science, applied science and operational research are areas of particular interest to the organisers of mgs and the decision makers for public health, especially those in poorly resourced countries. the modern day idea of mg health has developed from the notion that "mass gatherings medicine is concerned with the provision of emergency medical care at organized events with >1000 people in attendance" 18 to include several specialties (fi gure). the intricate interactions between the diff erent specialties is essential for the planning and success of mgs. new advances or interventions undertaken in other specialties should be integrated with research into mgs. the proposed research agenda is a broad strategy for research into public health, with a focus on issues related to mgs. it is not intended to be an exhaustive compilation of all possible research questions about the strategic planning for and operation of mgs. instead the agenda is an outline of key research into methods to control public health and policy. the results of this research can provide an evidence-based platform for policy decisions and practices to reduce the risks and eff ects of mg-associated health issues and global security risks to public health. the principal objectives of this research agenda are to identify topics for research and underpin and prioritise their importance in achieving interventions for the control of public health; provide a research framework to gather evidence to address health issues associated with mgs and global security risks to public health; ensure focus on less well addressed issues such as operational and implementation research, particularly for under-resourced regions; provide a platform to enable co ordination, discussion, and interaction among organisers of mgs, public health professionals, and researchers; and encourage a multidisciplinary approach to address gaps in knowledge about health risks associated with mgs and their control. the proposed research agenda is organised as a framework of fi ve major public health research directions. although many public health emergencies associated with mgs are not predictable, much can be done to prevent and minimise their eff ects. continuous monitoring of participants' vulnerability to health risks at mgs and understanding trends in risks that are associated with specifi c events (religious, sports, or concerts) can be used to predict what might happen in the future. they are also essential for successful preparedness and management of risk reduction and strengthening the response capacity of host and home communities. recognition and analysis of the changing risks and vulnerabilities during mgs are starting points for raising awareness and communication of pending risks. building global capacity for health intelligence for noncommunicable and infectious diseases is important for the elucidation of the risks associated with mgs. most of this information is available through networks such as the emerging infectious disease networks 19, 20 and the who global non communicable disease network. 21 importantly, the information can be used to implement strategies for risk assessment and mitigation in planning for specifi c mgs (panels 1, 2). morbidity and mortality at mgs can be mitigated through the assessment and management of risks associated with pre-existing non-communicable diseases. mitigation methods such as the provision of essential drugs and information about their availability at the mg can be initiated during pretravel medical care and advice. 5, 26 incidence of trauma and heat-related illness at a site can be reduced with the provision of advice and installations to combat the eff ects of weather, and eff ective crowd control. 5 models for the prediction of the spread of infections and occurrence of other emergency health issues during mgs have yet to be validated. 7 many of the diffi culties in restricting the spread of emerging communicable diseases [6] [7] [8] 23 are not new and have proven diffi cult to resolve. there is also uncertainty global clinical and laboratory surveillance systems for communicable diseases, such as those for seasonal and pandemic infl uenza, 22 are well established. the establishment of country-level surveillance systems for infectious diseases that can be adapted to diff erent epidemiological settings for mass gatherings (mgs) could also provide alerts for the occurrence of non-communicable diseases, such as radiation-related or chemical-related illnesses or those caused by extreme environmental temperatures (eg, program for monitoring emerging diseases). 23 an important component of the alert and response strategy is an integrated event management system that provides a platform for rapid dissemination of devices and procedures required for the management of health risks. research into their development and implementation is needed. syndromic surveillance can potentially provide rapid initial information about the occurrence of both non-communicable and infectious diseases. however, its establishment and assessment of eff ectiveness during mgs might require further assessment. further work is also needed to identify appropriate parameters for assessment of the eff ectiveness of such surveillance systems during mgs. surveillance of non-communicable diseases is a formidable but necessary step for the improvement of the health of the global community. an estimated 63% of global mortality in 2008 was attributable to non-communicable diseases and 80% of such deaths occurred in low-income and middle-income countries. 24 age-specifi c and sex-specifi c profi les of non-communicable diseases by country 25 allow host countries of mgs to estimate possible risks of non-communicable diseases in participants from specifi c countries and plan for mitigation strategies. although the challenges for the organisers of mgs and the eff ects of infectious diseases at such events have been summarised, 6, 14 there are many gaps in our understanding of emerging communicable diseases. integration of information from local and international surveillance of infectious diseases is important for strengthening the intelligence about the global threats before, during, and after mgs. 23 the risks of non-communicable and infectious diseases during mgs are proportional to the probability of occurrence of risk factors during the event. the identifi cation of these risks factors for the diff erent types of mgs will provide a scientifi c basis for planning eff ective prevention. although major risk factors associated with non-communicable and communicable diseases are likely to be similar worldwide, factors specifi c to the type of mg might lead to health problems. systematic risk assessment helps identify potential risks of outbreaks and guides the establishment of eff ective risk management solutions. systematic assessment will also identify potential or deliberate health security risks that require assistance from other authorities and government agencies. the leading causes of morbidity and mortality during the hajj are heat-related illnesses and trauma-related injuries. identifi cation of such risks allowed event planners to instigate preventive measures and rapid response strategies. provision of shaded areas can reduce the incidence of heat-related illnesses and eff ective crowd control reduces the risk of a stampede. drug and alcohol use were identifi ed as health risks for other types of mgs; 26 therefore, restriction of their use can mitigate the associated illnesses. the types and magnitude of health risks associated with spontaneous mgs due to natural disasters and confl icts are diff erent from those of organised mgs. objectives for risk management at such events are focused on facility-based health-care provision in addition to prevention. the potential for importation and subsequent global spread of infectious diseases during mgs are well understood. many emerging human infections are recognised as zoonotic diseases (eg, severe acute respiratory syndrome [sars], infl uenza a h5n1, nipah virus infection). the emergence of novel or rare pathogens in home communities and their subsequent spread to the host community and beyond can be amplifi ed during mgs. planning for the potential risks and hazards that are associated with mgs is essential to ensure success. many reports and manuals are available for planning mgs; 1,27-32 however, their use should be tempered by the results of the risk assessment. importantly, planning should maximise the legacy of the mg. the conceptual model of a lasting public health legacy as a framework for the relation between planning inputs, implementation, and public health outcomes was put forward by who and the international olympic committee to ensure sustainable, positive health eff ects for the host communities after the olympic games. 11 legacy planning should also include passing the knowledge gained to future hosts of similar mgs. series about how observations pertaining to particular pathogens, population groups, or settings can be used to develop public health policies for planning diff erent types of mgs. the development of evidence-based strategies for non-pharmaceutical inter ventions is urgently needed to address infection control and mitigate spread in the absence of available drugs and vaccines. additionally, such strategies are of particular concern for countries that do not have adequate access to pharmaceutical interventions such as vaccines and antimicrobial drugs. in some instances, available data for planning mgs might not have been assimilated in the best way for policy. a balance between basic scientifi c research and operational research is essential to inform the implementation of prevention strategies, best practices, and public health decision making (panel 3). research into how to contain the spread of infectious diseases should have the broadest possible applicability in diff erent settings and at diff erent resource levels. however, some results might not be generalisable to the planning for mgs, such as those from studies of pathogen transmission in health-care settings. eff ective management of health risks for noncommunicable and infectious diseases during mgs requires planning in advance. it is an integral part of planning that consists of risk identifi cation, communication, analysis, assessment, prevention, and monitoring. 23, 28, 29 many of the processes for risk management of non-communicable and infectious diseases during mgs are common. however, each can be specifi c to the type of mg and needs to be addressed accordingly. 8, 26 high visibility of mgs complicates risk management and can lead to political and media pressure and thereby aff ect the decision-making process. prevention of the occurrence of non-communicable and infectious disease at mgs requires coordinated risk assessment and management before, during, and after the event as shown in the planning for the hajj. [2] [3] [4] [5] [6] 26 ideally, the primary prevention of human infections with emerging communicable diseases is the eff ective control of pathogens at their source. since at-source elimination of all emerging pathogens is not possible, secondary interventions (eg, pharmaceutical or nonpharmaceutical) are needed to mitigate the spread of infection during mgs. however, the eff ectiveness of such interventions has not been established. 33, 34 in addition to reduction of the rates of morbidity and mortality associated with human infections during mgs, reduction of both the circulation of pathogens and human exposure might lessen the global health security risks. the eff ect of emergencies and crises on health can be substantially reduced if home and host communities are well prepared and are able to reduce their risks. the main challenge during mgs is the existence of systematic operative capacities such as risk assessment plans, coordinating mechanisms and standard procedures, institutional capacities, legislation and budgets, skilled vaccination is highly eff ective in the prevention of infectious diseases. however, many countries, particularly those with insuffi cient resources, have not developed strategies for vaccinating their populations at risk and people travelling to mgs. the reason is partly related to the lack of information about the transmission of infectious diseases (eg, infl uenza) and the social, economic, and health eff ects to the host and home communities. public health authorities need to decide how to eff ectively prioritise vaccine use on the basis of available information about disease burden and severity, epidemiology, and vaccine eff ectiveness and safety for vaccine-preventable infections associated with mgs. 6 a failure to promote and implement the polio vaccination programme caused the re-emergence of poliovirus in nigeria and subsequent international spread, 35,36 emphasising the risks associated with insuffi cient vaccine coverage for participants at mgs. an outbreak of neisseria meningitides serogroup a (originating from africa) during the hajj in 1987 37 was later successfully controlled with the introduction of mandatory pretravel vaccination and use of fl uoroquinolones among african pilgrims. 38 however, there are infectious diseases, including some of the most important and most dangerous, for which there are no vaccines. risk assessment and management during the planning for mgs can enable the development of eff ective health policies. strategic risk assessments are used to gather, coordinate, and analyse data that are necessary to identify existing risks, anticipate potential diffi culties, establish â�¢ enhance applications of existing vaccines against possible infectious diseases that are associated with mgs â�¢ assess the global vaccine supply and production to improve the processes of rapid response, surge in capacity, and rapid deployment and tracking of vaccine use for planning mgs â�¢ develop innovative clinical trial methods to study the eff ectiveness and safety of novel vaccines before and after licensing â�¢ develop new vaccines, platforms, and formulations that are safe with enhanced immunogenicity, especially in children and elderly people series priorities, and provide the basis for enacting targeted policies and implementation of corrective interventions. a system is needed for the measurement of the eff ect of public health policy and estimation of the probability of success. these interlinking processes are well described for regularly organised events such as the hajj and olympic games. 5, 11, 14 an example of the eff ective development of health policy is the organisation of the hajj-such as a smoke-free environment for the prevention of fi re, structural changes to prevent crowding, and recommendations for vaccination of pilgrims to prevent transmission of infectious diseases (eg, infl uenza, meningitis, poliomyelitis). 5 however, such eff ective policies are not possible for spontaneous mgs such as population displacement as a result of natural disasters or confl icts. development of vaccines for emerging infectious diseases presents substantial challenges and can take many years for diseases that are caused by novel pathogens such as severe acute respiratory syndrome (sars; panel 4). even if a vaccine exists, it might need to be regularly updated, clinically assessed for safety and effi cacy, and promptly produced for immediate use (eg, infl uenza vaccines). the effi cacy and eff ectiveness of a vaccine are dependent on the immune responses that are determined by the age of the recipient and composition of the vaccine (eg, conjugated or adjuvanted). improvements to vaccines and formulations that can provide longer-lasting and broader activity aff ord better protection, increase the applicability of vaccines, and reduce the frequency of vaccination. during an outbreak, the important factors are the rapid production and equitable distribution of vaccines to countries in need. ensuring rapid and eff ective management of patients and prevention of diseases requires robust health services at mgs. providers of emergency services play an important part in ensuring public safety during such events. knowledge and monitoring of medical service provision during mgs has been rapidly increasing in the past decade. 12, 13, 39 however, a lack of consolidated data for diff erent types of mgs means that organisers are not able to plan accordingly for the emergency medical services that might be needed. improved and targeted clinical management and infection control can substantially reduce the incidence and transmission of infectious diseases during mgs. optimum clinical management must be based on an improved under standing of the pathogenesis of these infections, advances in laboratory diagnosis, development and application of eff ective antimicrobial drugs, and other treatment modalities (panel 5). there are many gaps in our basic understanding of how many of the pathogens that are associated with mgs cause disease in people and what factors aff ect severity of illness. host immune responses, underlying comorbidity, age, and the properties of the infecting pathogen can all contribute to severity. the clinical presentation of many infections, such as infl uenza, is not specifi c, which makes diff erential diagnosis and early treatment to reduce further transmission and severe outcomes diffi cult. for example, antibiotics can help control severity and further spread of travellers' diarrhoea caused by bacteria. rapid and reliable diagnostic testing can expedite the initiation of timely and appropriate treatment and infection control. increase in and optimisation of the repertoire of antimicrobial drugs immunomodulator drugs, immunoglobulins, and natural products) that are applicable in low-resource areas and in fi eld conditions (such as availability, whether licensed or not, acceptance, and effi cacy in diff erent ethnic, sex, and age groups) and are easy to administer in paediatric-care and emergency-care settings â�¢ optimise management of people who are at risk of severe disease and complications, including emergency-care practices that are applicable across a range of resource settings health-care capacity and response â�¢ assess the eff ectiveness of global, national, and local responses to outbreaks of communicable diseases and develop new methods for assessment â�¢ undertake operational studies to investigate the surge capacity needs, particularly in host countries for mgs, including development of triage schemes in diff erent health-care and resource settings, and surge planning to maintain adequate resources â�¢ undertake studies to identify evidence-driven clinical-care pathways and principles that optimise health-care delivery in a range of resource settings â�¢ undertake studies to develop principles and practices for rapid assessment and introduction of new interventions during health emergencies, including systems for collation, sharing, and assessment in real time of clinical data series and development of clinical research to assess effi cacy of putative adjuvant treatments such as immunomodulator drugs, passive immuno therapy, and traditional medicine that are suitable for use in under-resourced areas would be most benefi cial in the preparation for mgs (panel 5). the availability and quality of health services contributes to the eff ect of infectious diseases in the source and home countries (panel 5). the same pathogen that might have a small eff ect on the rates of morbidity and mortality in countries with well organised health-care systems can be devastating in countries where health-care systems are suboptimum. new public health methods need to be harnessed to help reduce the eff ect of health problems during mgs. use of innovative communication channels, such as the internet and mobile phone networks, have the potential to aid surveillance, rapid risk assessment, and dissemination of accurate information. 23, 40, 41 mathematical modelling and risk communication have potential applicability in all aspects of research into health risks associated with mgs. some countries and mg organisers use state-of-the-art approaches for early detection and monitoring of diseases such as syndromic surveillance. 3, 29 in some countries computerised health-care and laboratory-based infor mation systems are used for planning mgs and these systems can be adapted for monitoring large-scale outbreaks. other innovative technologies such as mobile phones can be used in remote areas or countries that lack the resources to gather and transmit health-related data in real time, provide rapid feedback, and train health-care workers. 7, 23 applicability and use of these modern methods of monitoring in diff erent settings and contexts require further investigation, with special attention to issues related to integration and interoperability of initiatives for infection control during mgs (panel 6). evidence-based public health decision making in planning and mitigating health risks requires rapid access to information. however, such information is often incomplete, evolving, and derived from an increasingly complex array of sources such as basic science researchers, epidemiologists, social and political scientists, and economists. modelling is useful in that it can incorporate diverse data to inform public health policy and decision making. 42, 43 advances in mathematical modelling for public health are expected to include computational structural biology; integration of epidem iological and geographical data into phylogenetic models; within-host and population-level susceptibility models; behavioural modelling; and assessment of the eff ects of climate change on disease transmission and the use of novel datasets on contact patterns and population mobility. [44] [45] [46] [47] [48] communication is a key strategy in risk management in planning for mgs. the sars outbreak in 2003 reinforced the idea that a timely and transparent public information policy could help reduce excessive and inappropriate public health responses and minimise the social disruption and economic consequences of a fast-moving global epidemic. 49, 50 increased investment in identifying eff ective approaches and developing and assessing new communication methods will benefi t risk prevention and control eff orts. the specifi c challenge is to provide clear, credible, and appropriate communication to meet the needs of diverse communities and retain public trust in a dynamic yet unknown process. 51, 52 some of the main research topics in this specialty include the link between communication and behaviour change models; development and assessment of methods that can be quickly accessed and used in mgs; and assessment of best practices, challenges, and barriers in risk research in early detection and monitoring of disease â�¢ identify, develop, and adapt modern technologies for early detection of outbreaks of communicable diseases and their application in disease surveillance during mgs â�¢ integrate and continuously assess innovative approaches and channels for disease surveillance and monitoring â�¢ develop effi cient mechanisms to address the global challenges to sharing information, data, and details about pathogens identifi ed during outbreaks at mgs in terms of local, ethical, legal, and research perspectives â�¢ defi ne the timeliness and quality of data required for early detection of disease from local to district, regional, national, and global levels â�¢ assess the application of modelling to understand and estimate key parameters for risk management â�¢ investigate the role of modelling to assess eff ect of public health policies for diff erent mgs â�¢ assess modelling in public health policy planning and strategic decision making in mg planning research into health issues related to mgs is at an early stage. research directions outlined here should contribute to the evidence that can be used to formulate risk management guidelines and assist event planning and health-care policy makers. the research agenda presented here is not intended to be restricted to specifi c aspects of health research but rather to encourage a multidisciplinary approach focused on mg health and to help gain more knowledge. in the future, the focus should be on strategies directed towards developing common research frameworks and defi nitions. additionally, the knowledge generated by use of the multidisciplinary approach to research ought to be assessed for direct relevance to mgs in terms of their capacity for integration in legacy building and systemic sharing of information. jst wrote the text. mb planned the outline for the review, and provided and consolidated who policies and guidelines about mgs. zam provided the concept for the review and planned the content with the team. ns provided the outline and wrote the section about risk communication. sb provided information about outbreak control and research agenda for infl uenza. m-pk contributed information about health research directions and vaccine development. all authors reviewed and provided advice on drafting the review. we declare that we have no confl icts of interests. we identifi ed references for this review by searching pubmed, medline, and the internet for articles published in english from january, 1960, to june, 2011, by using the search terms "mass gatherings", "research", "infectious diseases", "communicable diseases", "non-communicable diseases", "public health", "alert and response", "mass gatherings planning", "legacy", "outbreak", "surveillance", "prevention", "treatment", "olympic games", and "hajj". we reviewed the articles and information found during these searches. additional references cited in the articles were also reviewed. communicable disease alert and response for mass gatherings the hajj: communicable and non-communicable health hazards and current guidance for pilgrims public health surveillance for mass gatherings a literature review of the health and safety risks associated with major sporting events: learning lessons for the london 2012 olympic and paralympic games health risks at the hajj global perspectives for prevention of infectious diseases associated with mass gatherings crowd and environmental management during mass gatherings non-communicable health risks during mass gatherings public health legacy: experiences from vancouver 2010 and sydney 2000 olympic and paralympic games mass gatherings and public health: the experience of the athens 2004 olympic games who western pacifi c region. the health legacy of the beijing olympic games: successes and recommendations mass gathering medicine: a review of the evidence and future directions for research the development of conceptual models for mass gathering health preparing for infectious disease threats at mass gatherings: the case of the vancouver 2010 olympic winter games the quest for public health security at hajj: the who guidelines on communicable disease alert and response during mass gatherings asia pacifi c strategy for emerging diseases: technical papers mass-gathering medical care: a review of the literature global capacity for emerging infectious disease detection networks and the epidemiology of infectious disease global noncommunicable disease network (ncdnet): report of the first global forum convened by the world health organization who global technical consultation: global standards and tools for infl uenza surveillance infectious disease surveillance and modelling across geographic frontiers and scientifi c specialties who. global status report on noncommunicable diseases 2010 who. noncommunicable diseases country profi les emergence of medicine for mass gatherings: lessons from the hajj safe and healthy mass gatherings: a health, medical and safety planning manual for public events who. global forum on mass gatherings communicable disease alert and response for mass gatherings: key considerations who. interim planning considerations for mass gatherings in the context of pandemic (h1n1) 2009 infl uenza federal emergency management agency, usa. special events contingency planning manual federal emergency management agency, usa. special events contingency planning: job aids manual world health organization writing group. non-pharmaceutical interventions for pandemic infl uenza, international measures non-pharmaceutical public health interventions for pandemic infl uenza: an evaluation of the evidence base nigerian states again boycott polio-vaccination drive global poliomyelitis eradication: status and implications epidemic group a meningococcal disease in haj pilgrims meningococcal disease and travel measuring emergency services workloads at mass gathering events automated vocabulary discovery for geo-parsing online epidemic intelligence use of unstructured event-based reports for global infectious disease surveillance mass gathering medicine: a predictive model for patient presentation and transport rates the impact of mass gatherings and holiday traveling on the course of an infl uenza pandemic: a computational model modeling the impact of global warming on vector-borne infections computational procedures for optimal experimental design in biological systems deterministic epidemic models on contact networks: correlations and unbiological terms how to make predictions about future infectious disease risks modeling and public health emergency responses: lessons from sars sars revisited: managing "outbreaks" with "communications responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management perceived risk and effi cacy beliefs as motivators of change: use of the risk perception attitude (rpa) framework to understand health behaviors eff ective media communication during public health emergencies. a who handbook communicating uncertainty-how australian television reported h1n1 risk in 2009: a content analysis key: cord-272135-a09bf50o authors: brouqui, philippe; puro, vincenzo; fusco, francesco m; bannister, barbara; schilling, stephan; follin, per; gottschalk, rené; hemmer, robert; maltezou, helena c; ott, kristi; peleman, renaat; perronne, christian; sheehan, gerard; siikamäki, heli; skinhoj, peter; ippolito, giuseppe title: infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease date: 2009-04-22 journal: lancet infect dis doi: 10.1016/s1473-3099(09)70070-2 sha: doc_id: 272135 cord_uid: a09bf50o the european network for infectious diseases (eunid) is a network of clinicians, public health epidemiologists, microbiologists, infection control, and critical-care doctors from the european member states, who are experienced in the management of patients with highly infectious diseases. we aim to develop a consensus recommendation for infection control during clinical management and invasive procedures in such patients. after an extensive literature review, draft recommendations were amended jointly by 27 partners from 15 european countries. recommendations include repetitive training of staff to ascertain infection control, systematic use of cough and respiratory etiquette at admission to the emergency department, fluid sampling in the isolation room, and analyses in biosafety level 3/4 laboratories, and preference for point-of-care bedside laboratory tests. children should be cared for by paediatricians and intensive-care patients should be cared for by critical-care doctors in high-level isolation units (hliu). invasive procedures should be avoided if unnecessary or done in the hliu, as should chest radiography, ultrasonography, and renal dialysis. procedures that require transport of patients out of the hliu should be done during designated sessions or hours in secure transport. picture archiving and communication systems should be used. post-mortem examination should be avoided; biopsy or blood collection is preferred. over the past two decades, many new and re-emerging diseases have posed threats to public health and have provided new challenges for infectious disease researchers worldwide. expansion of human populations has caused both a greater proximity to wildlife habitats, resulting in the emergence of new zoonoses, and a massive urbanisation process, which facilitates the rapid spread of communicable diseases in human beings. travel across the world has become increasingly frequent, resulting in the ever-increasing risk of worldwide contagion spread. these emerging problems were highlighted during the severe acute respiratory syndrome (sars) epidemic. 1 imported highly infectious diseases (hid), such as lassa fever and viral haemorrhagic fever, have been reported many times in the literature but have seldom been at the origin of an outbreak. 2,3 terrorist attacks with biological agents pose a substantial threat to the safety, health, and security of the citizens of every country. as defi ned in the 2007 issue of the biosafety in microbiological and biomedical laboratories manual, 4 "group 3 agents are pathogens that usually cause serious human or animal disease but do not ordinarily spread from one infected individual to another and eff ective treatment and preventive measures are available," whereas "group 4 [agents] are pathogens that usually cause serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. eff ective treatment and preventive measures are not usually available." several laboratoryassociated infections with group 3 and 4 pathogenic agents have already been reported. [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] although laboratories that handle group 3 and 4 agents should comply with biosafety regulations, laboratory leakage can happen at any time, for example, when working with a known agent or when attempting to isolate an unknown infectious agent, such as occurred with mimivirus. 21 experience shows that the recognition and isolation of a new infectious agent is often followed by a reported laboratory-acquired infection caused by the new isolate, as was reported for sars. 22, 23 the infection of a single laboratory worker with a highly infectious agent could be the origin of an outbreak, particularly if the agent has the capability of human-to-human transmission (ie, sars-associated coronavirus). 23 in some situations, such as a cough in so-called "superspreader" patients with extremely drug-resistant tuberculosis, smallpox, or sars-associated coronavirus, or exposure to infected blood of a patient with late-phase haemorrhagic fever, the inoculums to which health-care workers are exposed are likely to be equivalent to those received by a laboratory worker during specimen handling. the care of such patients should consequently be administered in a way to ensure the same level of protection and safety to health-care workers as to laboratory workers exposed to the same agent. the european network for infectious diseases (eunid) comprises 30 national representatives and experts from 16 european member states and is funded by the european commission, within its public health and risk assessment programme. 24 it was created to exchange information, share best practices, develop training, and improve the connection between national and regional review infectious disease experts. this network of clinicians, public health epidemiologists, microbiologists, infectioncontrol and critical-care clinicians, who are experienced in the management of hid, represent national or regional infectious diseases units designated to care for patients with hid. one of eunid's agreed tasks was to develop a consensus statement on the design and operation of high-level isolation units (hliu) in europe. 25 an hid is transmissible from person to person, causes life-threatening illness, presents a serious hazard in health-care settings and the community, and therefore requires specifi c control measures. 25 an hliu is defi ned as a health-care facility that is specifi cally designed to provide safe, secure, high-quality and appropriate care, with optimum infection containment, infection prevention, and control procedures for a single patient or a small number of patients who have, or who may have, an hid. these units have also been described as biocontainment units by us experts. 26 we defi ne an isolation room as a single-bed room with negative pressure. our task was to develop a consensus statement for infection control during clinical management and invasive procedures in hid patients admitted to an hliu. to assess this task, an expert (pb) was co-opted and charged by eunid to do an extensive review and draft recommendations on infection control in the situations mentioned above. the draft recommendations were amended by the coordination team, and further refi ned jointly by the 27 partners from 15 european member states and the coordination team. the fi nal consensus was reached and validated during two consecutive meetings in rome in may and october, 2007. the draft manuscript was then shared within the network by e-mail and revised to incorporate comments and additional evidence provided by participants until a fi nal version was reached. among the several possible scenarios that may be considered, epidemics outside europe of a yet unknown contagious agent or a known group 3 or 4 agent, such as sars-associated coronavirus or viral haemorrhagic fever, are the most likely to occur. alternatively, a laboratory worker may become sick after being exposed to a known agent in a registered biosafety level 3 or 4 laboratory while doing his or her duty, such as in the last sars outbreak in china and singapore. 23, 27 a third possible situation is intentional release of a bioterrorism agent. if an outbreak of a human-to-human transmissible disease begins in one country, the fi rst case it likely to be missed. thus, other implementations, such as routine respiratory and hand hygiene in health-care settings, healthcare personnel surveillance, and prompt reporting of such patients to national public-health authorities are mandatory. 28 the decision to care for patients in an hliu is based on the capability of the agent to have human-to-human transmission, its transmission rate, and the availability of primary or secondary prophylaxis, such as vaccines or eff ective antimicrobial therapy. although the role of hospital-acquired pneumonia 21 hliu=high-level isolation unit. sars=severe acute respiratory syndrome. *as defi ned by the centers for disease control and prevention, who, and the european community directive. 38 †note that most of the guidelines are based on a very small number of clinical cases and that level of evidence and grading are not necessarily accurate. ‡in vaccinated personnel. §not an offi cial classifi cation, but we recommend this level because laboratory-acquired pneumonia has previously occurred. emergency departments of general hospitals are the primary units where interaction with an unknown hid is most likely, and because many such units were not prepared for this kind of medical situation, they paid a heavy price during the sars outbreak. [46] [47] [48] [49] even now, most of our hospitals are still not prepared to face these kinds of situations. 50 until a suitable network of care for such patients, with communication between health-care workers prior to referral, becomes eff ective in each country, patients suspected of being infected with a highly contagious agent are more likely to be referred to the emergency department of a general hospital by their general practitioner. as a consequence, the emergency department of any hospital should be prepared for such events, and both training and structural features should be implemented. 28 based on studies of sars transmission, measures designed to control respiratory droplets and secretions, along with hand hygiene, would seem to off er signifi cant protection to other patients and health-care workers who have close contact with source patients. 51, 52 given the challenge of recognising early hid cases, and considering the potential for the spread of respiratory infections in health-care settings, the us centers for diseases control and prevention (cdc) recommended a broader strategy to prevent health-care-associated transmission of respiratory illnesses in response to sars. 53 in addition to standard precautions, the cdc suggest that an effi cient measure to reduce the risk of infection transmission needs to be systematically implemented by health-care workers. the cdc described the new standard approach to manage patients with febrile respiratory illness as "respiratory hygiene" or "cough etiquette". 54 patients with cough and fever should be encouraged to report symptoms at admission and should be encouraged or asked to wear a surgical mask and wash their hands after contact with respiratory secretions. these patients should be separated from other patients in the waiting area, and should be examined and assessed as soon as possible by the emergency staff in a single room. 54 signs should be displayed in the waiting areas to promote these measures and to educate patients and health-care workers, and emergency staff should comply with droplet precautions. however, because the modes of infectious agent transmission are often underestimated, as was recently reported for infl uenza and sars, 55 and because tuberculosis cannot be identifi ed without biological testing, eunid recommends that droplet precaution should be upgraded to airborne precaution each time situations in which a patient would need to be admitted to an hliu • patients with an unknown human-to-human transmittable or a potentially transmittable epidemic febrile illness that is native or imported from abroad • patients with a known infectious disease caused by a group 3 or 4 agent* at admission of patients with hid to an emergency department • systematically apply standard precautions and cough and respiratory etiquette • set up at least one single room with a dedicated route and direct access, or an isolation room as recommended by eunid for a referral hospital, † if hliu cannot be used for ruling out hid diagnoses • off er special training to the emergency department team • retain close relationships with the hliu team of the referral hospital • for infection-control reasons, all children suspected to be infected with an hid should be admitted to an hliu • family participation should be minimised • the hliu responsible manager should make all eff orts to be prepared and be able to provide nursing care compatible with children's requirements • if possible, perform intensive-care therapy in the hliu in collaboration with the infectious disease team; the hliu should be pre-equipped for critical care • if a patient with hid is cared for in the intensive-care unit, the unit should be subjected to negative pressure • manual ventilation duration during resuscitation procedures should be reduced to a minimum • use nppv instead of facial mask aerosol therapy when possible and intubation/ mechanical ventilation instead of nppv when safely achievable with maximum precautions • endotracheal intubation should be done with rapid sequence induction by the most skilled person available, who should wear personal protective equipment • meticulous infection-control measures must be followed in case of ventilated patients with hid, particularly during suctioning, tracheotomy, and bronchoscopy with or without bronchoalveolar lavage hliu=high-level isolation unit; nppv=non-invasive positive pressure ventilation. *as summarised in tables 1 and 2. †a hospital to which the patients with hid are usually referred to either by national authority directives or by their own expertise and reference. ‡this remains a matter of debate in our group. that involvement of one of these agents is suspected. consequently, chest radiography should be done separately from other patients, a systematic examination of sputum for acid-fast bacteria should be prescribed when pneumonia is diagnosed (to rule out tuberculosis), and the patient should remain in isolation until the threat of tuberculosis is eliminated. 56 patients with suspected hid should be placed directly in an emergency department isolation room (edir), if available. during admission, such patients should avoid any contact with other patients and unprotected healthcare workers, and therefore direct access to the edir from outside the unit is required. 57 edirs should comply as much as possible with the design and operational management recommendations made by eunid for hliu. 25 whereas general respiratory hygiene rules and cough etiquette should apply to every emergency department of every general hospital, isolation rooms could be available in referral hospitals only. a patient with a possible or confi rmed hid, if not admitted directly to an hliu, should be transferred from the edir to the hliu in a secure manner, by use of a safe isolation transportation system, 58 or at least by staff wearing personal protective equipment and using a clear and secure route. although these general recommendations may be diffi cult to apply in the usual understaff ed, overworked, and busy environments, implementation of these measures is important. these recommendations would also help prevent the transmission of many other important pathogens that are spread by the droplet route, such as infl uenza and mycoplasma pneumoniae. 53 clinicians should remember that the most likely cause of fever in a tropical traveller is malaria, which is far more common than a new or emerging hid. consequently, eunid fi rst advocates that everything possible should be done to rule out the most frequent diff erential diagnoses without delay. to reduce the risk of transmission to health-care workers, samples from patients should be taken in the edir or hliu, depending on availability. severely ill patients may necessitate frequent blood sampling and intravenous line placement for reliable antimicrobial or antiviral drug administration. to reduce exposure due to accidental needle-stick incidents, we recommend the use of a routinely secured arterial line and central venous line access by an hid-trained physician, thus allowing safe serial blood sampling and drug administration without further needle-based procedures. if possible, all routine diagnostic tests should be processed in a biosafety level 3 or 4 laboratory that is located close to the hliu or on the same campus to avoid unnecessary transportation of contaminated samples. 4, 25, 59 once inactivated, the sample can be processed in a routine clinical laboratory with pcr or blood fi lm analysis. under certain handling precautions, the use of a certifi ed autoanalyser in routine tests has been suggested as being safe. however, some experts believe that samples that are likely to be highly contagious, such as blood contaminated by ebola virus, cannot be handled safely in a routine laboratory. the development of a pointof-care test, such as for arterial blood gas, blood electrolyte, or haemoglobin content, and more recently for microbiology (ie, malaria), or for early diagnosis of lower respiratory-tract infections, off ers an alternative to routine tests because they can be done at patients' bedsides. 60 hospital-acquired infections can cause major problems in paediatric wards, and compliance with isolation procedures needs to be ensured. 61, 62 during the sars epidemic, infection-control measures overshadowed family-centred nursing practices in the management of paediatric patients, 63 and created inevitable confl ict. 64 to eff ectively control infection, family participation should be minimised, and all children with suspected hid should be admitted to the hliu. consequently, everything should be organised in the hliu to provide nursing care that respects the privacy of the parents and children. the risk of being infected with sars-associated coronavirus was reported to be about 13-times higher among physicians and nurses who performed or assisted in endotracheal intubations in intensive care units (icus) than in those who did not. 65 nurses who became ill were often exposed to sars-associated coronavirus within 48 h of a patient's admission, during which time the patient usually deteriorated with symptoms, increasing the spread of droplets or aerosols (eg, dyspnoea, cough, etc). 66, 67 patients admitted to the icu are usually severely ill, and are likely to have a high viral load and to be at a point of maximum infectiousness. 68 non-invasive positive pressure ventilation (nppv) is a mode of ventilation assistance used in early acute respiratory failure and acute respiratory distress syndrome. nppv reduces the intubation rate and is eff ective in the treatment of sars-related acute respiratory failure without posing infection risks to health-care workers. [69] [70] [71] [72] in this setting, intubation has been avoided in up to two-thirds of cases in some studies. 73, 74 if aerosolisation and airborne transmission are discounted, respiratory secretions or fl uids, or both, are the main route of sars transmission, ultimately leading to an overwhelming risk of infection via intubation. however, this mode of transmission does not occur with other airborne pathogens, most notably avian infl uenza, which can aerosolise or disperse up to 0·5 m around a patient undergoing nppv. 75 the main problem with dispersion through nppv is not the exhalation portion of the respiratory circuit but the inevitable mask review leaks. consequently, intubation or mechanical ventilation should be preferred to nppv if safely achievable to control droplet aerosolisation and dispersion. consequently, endotracheal intubation should be done by the most skilled person available. in addition, that person should wear personal protective equipment, including eye protection, and use rapid sequence induction. 43 icu rooms should be subjected to negative pressure and a minimum of 15 air changes per hour. 76, 77 some researchers advocate the use of a powered air-purifying respirator, particularly during high-risk manipulations such as endotracheal intubation, because it off ers supplementary protection with a better fi t to the healthcare worker's face. 78, 79 however, its use is a matter of debate because the risk of dysfunction seems important, which may increase the risk of exposure. problems in cleaning, disinfection, and storage of the respirator may also increase the risk of exposure. endotracheal intubation is not the only high-risk procedure in icu patients; cardiopulmonary resuscitation was also reported to be a very high-risk procedure during the sars epidemic. 80 patients with other hids, such as viral haemorrhagic fever, should also be treated in an icu. although the main route of transmission of viral haemorrhagic fever is contact with body fl uids, airborne transmission has been suggested with the reston and zaire strains of ebola virus in monkeys. 81 in an outbreak of ebola in a johannesburg hospital, no subsequent cases of the disease occurred despite the staff being involved in numerous hazardous procedures. 82 despite the fact that this hospital opted for high-level barrier nursing, which entailed the isolation of the patient in a cubicle and the use of protective clothing plus high-effi ciency particulate air-fi ltered respirators to minimise exposure to aerosols, universal blood and body-fl uid precautions may have been suffi cient for protection. 82 the management of invasive diagnostic or therapeutic procedures in a patient with hid is a challenge. however, there were few reports on hospital-acquired hids during invasive procedures before the sars era. in fact, until now, hid outbreaks had only occurred in countries or at times when such techniques were not available, and the recent sars epidemic has revealed the risks in such situations. 83 of note, available evidence on risk factors is weak and somewhat indirect, according to the commonly accepted hierarchy of evidence. much work needs to be done to separate the essential risk factors from the superfl uous ones. high-risk aerosolgenerating procedures were well summarised in the last who interim guidelines. 43 panel 2 shows the eunid recommendations for infection control in the management of patients with hid during these special procedures. although diagnostic bronchoscopy or fl exible lung endoscopy is not necessary in some scenarios (ie, an ongoing outbreak of a known disease), some situations need such invasive procedures to rule out diff erential diagnoses or to collect samples for laboratory • the examination should be kept as short as possible to answer the clinical questions • for hliu-admitted patients, bedside radiography should be provided to avoid transport of patients; radiographic equipment should then be kept in the hliu • radiographs should be interpreted only by a designated radiologist who is aware of infection-control procedures, and by use of a picture-archiving and communication system, if available 84 • for ultrasound scanning, a sonographic scanner should be designated as a portable radiograph to be used only for hid patients • for ct or mri, we strongly recommend that the department appoints a staff member to monitor and ensure that all department staff fully comply with the infection-control measures according to the guidelines • designated sessions or hours, either out of offi ce hours or at the end of a session, should be assigned for such patients • treat hid patients who require dialysis at their bedside with either peritoneal dialysis or haemodialysis • designate dedicated haemodialysis machines • decontaminate dialysate as infectious waste post-mortem examination • risks and benefi ts must be carefully considered • limited autopsy or post-mortem collection of blood and percutaneous biopsy are preferred • the biosafety precautions recommended for clinicians and laboratory staff working with infected patients and specimens must also be followed during post-mortem examination • perform the autopsy only if necessary and in a biosafety level 3/4 isolation room, which can serve as the hliu hliu=high-level isolation unit. investigation. sars transmission has been reported or suggested after the intubation of patients, 84 and use of a nebuliser by health-care workers in patients with sars resulted in a major outbreak of the disease. 85 in a retrospective study among critical-care nurses in toronto, 86 the probability of a sars infection was 6% in nurses who assisted during intubation, suctioning, and manipulating the oxygen mask. in the same study, wearing a mask, especially an n95 mask, was deemed protective. 86 a high-fl ow-rate oxygen mask may also result in health-care worker infection, 83 and we have thus suggested that nppv is preferred to facial mask aerosol therapy if available. bronchoscopy has also been suggested to increase sars-associated coronavirus transmission in health-care workers. 71 the aerosolisation of lung pathogens during fl exible endoscopy and hospital-acquired infections during these procedures are both well documented and have led to standard guidelines for fl exible endoscopy. 87 similar transmissions would probably occur with other respiratory agents, such as avian infl uenza and hantavirus pulmonary syndrome. bronchoscopy, airway suctioning, and other procedure that may induce coughing and expose health-care workers to potentially infected aerosolised respiratory droplets pose an increased risk of transmission of those agents. in most hospitals, rooms dedicated for bronchoscopy are under negative pressure, but these rooms are not necessarily air fi ltered. 88 in addition to airborne transmission, sars-associated coronavirus may also be transmitted by direct contact with infected respiratory secretions and other body fl uids, similar to viral haemorrhagic fever viruses. 49, [89] [90] [91] contact with contaminated environmental surfaces and inanimate objects is suspected to have resulted in the transmission of sars, as suggested by reports that some health-care workers became infected even though they had no direct contact with sars-infected patients. 47, 89, [92] [93] [94] data also suggest that sars-associated coronavirus and orthopoxvirus can survive on hard surfaces such as plastic and stainless steel for several hours, if not days. 40, 47, 94 moreover, many group 3 and 4 viruses, including sars-associated coronavirus, have been identifi ed in human faeces. 47, 90, 93, 94 although there is no published report of transmission of sars to health-care workers and other patients during gastrointestinal endoscopy, the potential for such transmission exists. 95 most of our knowledge on managing infection control in radiology departments comes from experience of tuberculosis and sars. radiology technicians have a relative risk for a positive tuberculin skin test of 1·7 compared with other health-care workers, and those working for less than 1 year have a lower risk of infection, indicating that radiology technicians are more exposed to tuberculosis during their practice. 96 at the prince of wales hospital in hong kong in march, 2003, at least 50 health-care workers, including radiographers, were infected by sars. 97 because imaging plays an important part in the diagnosis and management of hid, the role of the radiology department is to provide an immediate and effi cient radiological service for patients with suspected or confi rmed hids. chest radiography is mandatory in such a situation. to minimise the risk of cross-infection, transportation of patients with hid should be as limited as possible. for ambulatory patients with suspected hids, a satellite radiography centre should be set up with portable radiography equipment in the vicinity of the edir dedicated to hid patients in order to confi rm or to reject the diagnosis. 98 for patients in the hliu, bedside radiography should be provided to avoid transportation of patients. 97, 99 to avoid transmission of fomites, the radiograph fi lm should be handled with care and should only be interpreted by a designated radiologist who is aware of infection control. the fi lm-processing area, where cassettes are brought back to the department after bedside radiography in the hliu, should be considered as high risk, unless the cassettes were processed in the hliu by following a protocol of double bag sealing, and should be disinfected. 98, 99 images should be interpreted through a picture-archiving and communication system, if available. 84, 97, 100 for ultrasound scanning, a sonographic scanner should be designated as a portable radiograph that is only used for hid patients. one machine should be dedicated for a specifi c area such as the hliu. the examination should be kept as short as possible to answer the clinical questions. the transducer should be covered with disposable covers that are discarded between patients. the value of a ct scan in assessing the diagnosis of hids, such as sars, has been established, and a ct scan is sometimes mandatory for a patient's assessment. 101 because this examination can be done only in the radiology department, stringent infection-control measures need to be followed, and the examination should be done only if absolutely necessary for the patient's recovery. we strongly recommend that the radiology department appoint a staff member to monitor and ensure that all staff fully comply with the infection-control measures according to the guidelines. designated sessions or hours, either outside offi ce hours or at the end of a session, should be assigned for such patients. patients should be transported in a special isolation carrier or in a defi ned way to avoid any contact with other patients or unprotected personnel. 58, 100 the department should be divided into low-risk and high-risk areas. after a ct scan, the gantry table and fl oor should be cleaned, and any bed linen should be changed. film cassettes should be decontaminated properly before fi lm processing. in all review cases, radiology technicians, radiologists, and other radiology personnel should comply with universal precautions, including the wearing of a mask, cap, gown, and gloves during direct contact with patients. the main reported dialysis-associated infection is viral hepatitis. as a consequence, guidelines have been edited to prevent nosocomial transmission of this agent to personnel and patients. 102 when the guidelines were followed in a european study, 103 no reported hospitalacquired cases of hantavirus haemorrhagic fever with renal syndrome were reported, despite 30-50% of patients being in need of haemodialysis. most of our knowledge in the management of hid with renal failure has been acquired from sars outbreaks. by comparison with other patients, the care of patients undergoing renal dialysis poses several additional infection-control issues in the disposal of spent dialysate (both haemodialysis and peritoneal dialysis) and in the prevention of cross-contamination within the dialysis unit. 104 during the sars episode, patients receiving dialysis were kept in the sars isolation ward with the other patients with sars. all patients with peritoneal dialysis were treated with intermittent peritoneal dialysis during their hospital stay. the dialysis exchange was done by the ward staff , who wore full protective gear, as recommended by who, including waterproof disposable gowns, caps, gloves, face shields, and n95 face masks. 104 the spent peritoneal dialysis effl uent was decontaminated with a 2% sodium hypochlorite solution. haemodialysis was also done by the ward staff , who wore full protective gear, in a room especially equipped for sars patients in the isolation ward. 104 dedicated haemodialysis machines were used with an ordinary tap water supply that passed through a fi lter without reverse osmosis or any other water treatment. spent dialysate was decontaminated as described above, and all of the blood tubing was discarded as infectious waste. because they were potentially contaminated, unspent dialysate concentrate and the sodium bicarbonate cartridge were also discarded as infectious waste. the dialysis machine was disinfected after each haemodialysis session with a sodium hypochlorite solution according to the manufacturer's instructions. particular attention should be paid to the infection control of dialysate effl uents and decontamination of the machines. although autopsies have been done safely on patients with hid in some circumstances, without prior knowledge of diagnoses such as ebola haemorrhagic fever, hid agents are transmissible at autopsy, which raises concerns about the protection of pathologists and autopsy personnel. 105 tuberculosis was the fi rst autopsytransmitted disease to be reported in the literature, 106 and transmission is also thought possible with multidrugresistant or extremely drug-resistant tuberculosis. aerosol production during autopsy, particularly from ruptured organs, had been recognised early in this situation and has led to some precautions. 107 during the fi rst reported episode of hantavirus pulmonary syndrome, the fi rst fi ve suspected patients were autopsied with standard precautions only, even though the agent was isolated and classifi ed as a group 3 agent. 108 fortunately, no transmission to autopsy personnel occurred. during the sars episode, many autopsies were done, and although there was no case of transmission, several investigators have raised concerns over biosafety in autopsy rooms. 105, 108, 109 recent guidelines have been published to prevent infection during autopsy. 110 before an autopsy is done on a patient suspected to have died from an hid, the possible risks and benefi ts must be carefully considered. 58 limited autopsy or post-mortem collection of blood and percutaneous liver biopsy material may be appropriate. several pathologists suggest that safety measures applied to laboratory workers should also be applied during and after autopsy. furthermore, patients who have died from an unknown hid or from a known group 3 or 4 agent should be autopsied only if necessary and in a biosafety level 3 or 4 isolation room. 58, 108, 109 the literature on hids, particularly sars, indicates that there is a need for hospitals to be prepared for these events and that hlius urgently need to be built in european member state hospitals. research and development of universal, bedside, reproducible, and transferable diagnostic tools are mandatory. prompt reporting to the authorities is needed so that a rapid response can be organised. these measures should be accompanied by harmonised recommendations for the the literature review was done by use of medline and the web of science with the following key words: "sars [all]", "laboratory-acquired infection", "laboratory-associated infection", "imported [each agent's name]", and "[each group 3 and 4 agent's name]". more than 1400 references were obtained. selection criteria within the topics were the impact factor and half-life of the journal, followed by journal availability. the topics selected were as follows: patient's criteria for admission to the hliu, admission to the emergency department, safe sampling for laboratory investigation, isolation of suspected hid patient in hliu, intensive-care management of hid patients, hid in paediatric patients, practice of invasive procedures such as bronchoscopy and gastroscopy, radio imaging, renal dialysis, and post-mortem examination of hid patients. only english-language articles were reviewed. websites such as who, cdc, and other scientifi c and international research society's guidelines were also used. review safe care of these unusual patients. the recommendations reported here by our group will hopefully help establish consensual protocols. networking for the standardisation of procedures and the management of these patients is mandatory. we declare that we have no confl icts of interest. laboratory-acquired infections with hantaan virus, the etiologic agent of korean hemorrhagic fever laboratory acquired tick-borne meningoencephalitis: characterisation of virus strains brief report: treatment of a laboratory-acquired sabia virus infection laboratory-acquired sabia virus infection laboratory-acquired vaccinia infection laboratory infection of a technician by mimivirus biological safety cabinetry laboratory-acquired severe acute respiratory syndrome european network of infectious diseases framework for the design and operation of high-level isolation units: consensus of the european network of infectious diseases designing a biocontainment unit to care for patients with serious communicable diseases: a consensus statement recent singapore sars case a laboratory accident severe acute respiratory syndrome: preparedness, management, and impact using models to identify routes of nosocomial infection: a large hospital outbreak of sars in hong kong risk group classifi cation for infectious agents nosocomial transmission of q fever q fever in pregnancy q fever during pregnancy-a risk for women, fetuses, and obstetricians risks and prevention of nosocomial transmission of rare zoonotic diseases nosocomial infection due to pseudomonas pseudomallei: two cases and an epidemiologic study guidelines for preventing the transmission of mycobacterium tuberculosis in health-care settings extensively drug-resistant tuberculosis: current challenges and threats on the protection of workers from risk related to exposure to biological agents at work. offi cial journal of the european communities smallpox as a biological weapon: medical and public health management. working group on civilian biodefense bichat guidelines for the clinical management of haemorrhagic fever viruses and bioterrorismrelated haemorrhagic fever viruses novel chikungunya virus variant in travelers returning from indian ocean islands human-to-human transmission of hantaviruses infection prevention and control of epidemic-and pandemic-prone acute respiratory diseases in health care: who interim guidelines who issues guidelines to manage any future sars outbreak b virus (herpesvirus simiae) infection in humans: epidemiologic investigation of a cluster sars exposure and emergency department workers sars outbreak in the greater toronto area: the emergency department experience responding to the severe acute respiratory syndrome (sars) outbreak: lessons learned in a toronto emergency department emergency departments (eds) in the united kingdom (uk) are not prepared for emerging biological threats and bioterrorism eff ectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) guideline for isolation precaution: preventing transmission of infectious agents in healthcare settings respiratory hygiene/ cough etiquette in healthcare settings transmission of infl uenza: implications for control in health care settings foundations of the severe acute respiratory syndrome preparedness and response plan for healthcare facilities hospital preparedness for severe acute respiratory syndrome in the united states: views from a national survey of infectious diseases consultants hospital preparedness to bioterrorism and other infectious disease emergencies early diagnosis of lower respiratory tract infections (point-of-care tests) preventing nosocomial infection in paediatric wards pediatric nosocomial infections: children are not little adults balancing infection control practices and family-centred care in a cohort of paediatric suspected severe acute respiratory syndrome patients in hong kong bowlby and robertson revisited: the impact of isolation on hospitalized children during sars transmission of severe acute respiratory syndrome in critical care: do we need a change? transmission of severe acute respiratory syndrome during intubation and mechanical ventilation illness in intensive care staff after brief exposure to severe acute respiratory syndrome 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 clinical analysis of 45 patients with severe acute respiratory syndrome description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china a hospital outbreak of severe acute respiratory syndrome in guangzhou, china a retrospective study of 78 patients with severe acute respiratory syndrome non-invasive versus invasive mechanical ventilation for respiratory failure in severe acute respiratory syndrome noninvasive positive-pressure ventilation: an experimental model to assess air and particle dispersion icu management of severe acute respiratory syndrome practical guidelines for infection control in health care facilities sars: ventilatory and intensive care severe acute respiratory syndrome-singapore possible sars coronavirus transmission during cardiopulmonary resuscitation kass handbook of infectious diseases: exotic viral infections unexpected ebola virus in a tertiary setting: clinical and epidemiologic aspects the epidemiology of the outbreak of severe acute respiratory syndrome (sars) in hong kong-what we do know and what we don't application of ris/ pacs in the prevention and treatment of sars at the hospital radiology sars: experience at prince of wales hospital, hong kong sars among critical care nurses american college of chest physicians and american association for bronchology [corrected] consensus statement: prevention of fl exible bronchoscopy-associated infection the american institute of architects. guidelines for design and construction of hospital and health care facilities sars transmission, risk factors, and prevention in hong kong sars: epidemiology, clinical presentation, management, and infection control measures listening to sars: lessons for infection control an outbreak of severe acute respiratory syndrome among hospital workers in a community hospital in hong kong managing sars amidst uncertainty management of hospital-acquired severe acute respiratory syndrome with diff erent disease spectrum recommendations for the prevention of transmission of sars during gi endoscopy risk of tuberculous infection among healthcare workers in a tertiary-care hospital in ankara eye of the storm: the roles of a radiology department in the outbreak of severe acute respiratory syndrome severe acute respiratory syndrome: avoiding the spread of infection in a radiology department rapid creation of a temporary isolation ward for patients with severe acute respiratory syndrome in taiwan severe acute respiratory syndrome: management and reconfi guration of a radiology department in an infectious disease situation value of ct in assessing probable severe acute respiratory syndrome european best practice guidelines expert group on hemodialysis (era) hantavirus infections in europe severe acute respiratory syndrome in dialysis patients the modern autopsy: what to do if infection is suspected tuberculosis acquired in laboratories and necropsy rooms aerosols in the mortuary hantaviral biosafety issues in the autopsy room and laboratory: concerns and recommendations biosafety level 3 laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects health and safety at necropsy key: cord-321949-s1qu3odd authors: anderson, evan j; weber, stephen g title: rotavirus infection in adults date: 2004-01-28 journal: lancet infect dis doi: 10.1016/s1473-3099(04)00928-4 sha: doc_id: 321949 cord_uid: s1qu3odd rotavirus has been recognised for 30 years as the most common cause of infectious gastroenteritis in infants and young children. by contrast, the role of rotavirus as a pathogen in adults has long been underappreciated. spread by faecal-oral transmission, rotavirus infection in adults typically manifests with nausea, malaise, headache, abdominal cramping, diarrhoea, and fever. infection can also be symptomless. rotavirus infection in immuno-compromised adults can have a variable course from symptomless to severe and sustained infection. common epidemiological settings for rotavirus infection among adults include endemic disease, epidemic outbreak, travel-related infection, and disease resulting from child-to-adult transmission. limited diagnostic and therapeutic alternatives are available for adults with suspected rotavirus infection. because symptoms are generally self-limiting, supportive care is the rule. clinicians caring for adults with gastroenteritis should consider rotavirus in the differential diagnosis. in this review we intend to familiarise clinicians who primarily provide care for adult patients with the salient features of rotavirus pathophysiology, clinical presentation, epidemiology, treatment, and prevention. infective gastroenteritis causes substantial morbidity and mortality worldwide. although various bacterial species have long been associated with gastrointestinal disease, 1 specific viral causes of these infections were not delineated until the early 1970s. however, with the discovery of norwalk virus in 1972 2 and rotavirus in 1973, 3 the causative agents for most non-bacterial gastroenteritis infections were identified. almost immediately, the spectrum of viruses causing gastrointestinal infection in adults was recognised as differing from that in children. among children younger than 2 years, nearly half of all cases of diarrhoea requiring admission to hospital can be attributed to rotavirus infection. 4 by contrast, among adults most non-bacterial outbreaks of gastroenteritis can be linked to the norwalk-like viruses. 5 the important part played by viral pathogens besides the norwalk-like viruses in adults with gastroenteritis is not yet fully appreciated. specifically, the contribution of pathogens that typically affect children is not recognised by most clinicians who take care of adults. such is the case for adult infections caused by the common paediatric pathogen rotavirus. here we review important features of rotavirus microbiology and pathophysiology, along with relevant clinical and epidemiological features of rotavirus infection. in 1973, bishop and colleagues 3 described unique viral particles obtained from the duodenal mucosa of children with gastroenteritis. viruses with similar morphological appearance had been seen in 1963 in the intestinal tissue of mice with diarrhoea. 6 under the electron microscope, the 70 nm diameter viral particles first described in these reports had a wheel-like appearance, prompting the name rotavirus, from the latin rota (figure). 7 rotavirus is a non-enveloped virus now classified within the reoviridae family. 11 segments of doublestranded rna reside within the core. the rna encodes six viral proteins (vp) that make up the viral capsid, and six non-structural proteins (nsp). the core is surrounded by an inner capsid, composed mostly of vp6, the primary group antigen, 1, 8 and includes the epitope detected by most common diagnostic assays. other structural proteins also seem to confer some degree of group specificity. 9 the outer capsid is primarily composed of vp4 and vp7. 10 vp4 contributes the spoke-like projections to the wheel-shaped appearance of rotavirus. this vp is cleaved by trypsin in vitro to yield vp5* and vp8*, which appear to play an important part in cellular attachment. 9 the inner and outer capsids give the viral particle the double-layered icosahedral structure visualised on negative-stain electron microscopy. 10 seven distinct groups of rotavirus (named a to g) have been shown to infect various animal species. of these, only groups a, b, and c have been reported as human pathogens. 9 group a is the primary pathogen worldwide and is the group detected by commercially available assays. additional subgroups and serotypes can be identified by further characterisation of vp4, vp6, and vp7 antigens. 10 group b seems to be limited to causing epidemic infection in asia and the indian subcontinent, whereas group c rotavirus causes endemic infections that frequently go unrecognised. rotavirus spreads from person to person, mainly by faecaloral transmission. although rotavirus has been detected in urine and upper-respiratory samples, 11, 12 these body fluids are not believed to be commonly associated with transmission. after ingestion, rotavirus particles are carried to the small intestine where they enter mature enterocytes 13 through either direct entry or calciumdependent endocytosis. 14 after cytolytic replication in the mature enterocytes of the small intestine, new rotavirus particles can infect distal portions of the small intestine or be excreted in the faeces. more than 10 10 -10 11 viral particles per gram of faeces are excreted by children during infection. 8, 15 the amount of rotavirus excreted by adults might be more variable. in at least one study shedding was 10-100-fold lower in travellers' diarrhoea. 16 symptom-free adults can shed rotavirus in quantities so low as to be undetectable by most routine assays. 17 the mechanism by which rotavirus induces diarrhoea is poorly understood. few investigations have incorporated the study of human mucosal samples. the reports that are available describe various findings: villous shortening, flattening, and atrophy, denudation of microvilli, mitochondrial swelling, distension of the endoplasmic reticulum, depressed disaccharidase concentrations, and infiltration of mononuclear cells. 14, 18, 19 additional hypotheses about the pathophysiology of rotavirus gastroenteritis have been generated from animal studies. in one review the diminished ability of the intestinal epithelium to absorb fluid and nutrients, 14 stimulation of the enteric nervous system, 20 and local villous ischaemia and shortening resulting in impaired nutrient absorption were noted. a murine model of rotavirus infection suggests that rotavirus nsp4 acts as an enterotoxin, potentially by increasing calcium-dependent signalling of chloride secretion. 21, 22 the diarrhoea induced by rotavirus is unlikely to be completely explained by any one process, rather that several mechanisms contribute simultaneously. 14 these mechanisms are summarised in panel 1. 3, 13, 14, [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] resolution of rotavirus gastroenteritis depends greatly on the immunological response of the host. in a normal host, rotaviral antigens are transported to peyer's patches, undergo processing by b cells, macrophages, or dendritic cells and are presented to helper t cells. this cascade culminates in stimulation of rotavirus-specific b cell and cytotoxic t-lymphocyte-precursor expansion. 29 bernstein and colleagues 30 noted that stool rotavirus iga concentrations peaked 14-17 days after infection and persisted for longer than 1 year, but at declining concentrations. the researchers suggested that serum rotavirus iga is a more consistent marker of rotavirus immunity than other antibody measurements. 30 however, rotavirus-specific iga is frequently undetectable in duodenal fluid or faeces in the first week of infection, although symptoms might resolve within that time. 29 this pattern suggests a mechanism independent of humoral immunity. offit 29 notes that infected mature villous epithelial cells are steadily replaced by less-mature enterocytes, which may be less susceptible to rotavirus invasion. increased peristalsis improves clearance of viral particles and the non-specific activity of interferons can prevent vp translation. 29 de bouissieu 31 has reported that interferon ␣ concentrations correlate with a trend towards shorter duration of diarrhoea among patients who have rotavirus infection. although many physicians presume that rotavirus infection will confer lifelong immunity, multiple investigations show that re-infection can occur. bishop and colleagues 32 noted that infection with rotavirus during the neonatal period did not protect against developing rotavirus infection during the first 3 years of life but did lessen the severity of such infections. in a prospective study of 200 mexican infants followed up from birth, valazquez and colleagues 33 noted that by age 2 years 96% of infants had experienced a primary rotavirus infection. during the same period, nearly 70% of the infants experienced a second infection. more than 10% of the children studied had five or more rotavirus infections during the first 2 years of life. yolken and colleagues 34 had previously noted that by age reduced absorptive surface denudation of microvilli; shortening, flattening, and atrophy of villi; invasion of villi by rotavirus causing ischaemia and shortening 3, 18, 19 functionally impaired absorption depressed disaccharidase concentrations; impaired co-transport of glucose and sodium; decreased sodium-potassium atpase activity impairing electrochemical gradient 3, 18, 19, [23] [24] [25] cellular damage impairing absorption mitochondrial swelling; distension of endoplasmic reticulum; mononuclear cell infiltration 3, 13, 18, 19 enterotoxigenic effects of rotavirus protein nsp4 induces increased intracellular calcium concentrations; in murine models, acts like a toxin to induce diarrhoea 21, 22 stimulation of enteric nervous system stimulation of intestinal secretion of fluid and electrolytes; stimulation of intestinal motility resulting in decreased intestinal transit time 14, 20 altered epithelial permeability increased paracellular permeability by weakening tight junctions between cells [26] [27] [28] 2 years, more than 85% of children had antibodies to two different rotavirus serotypes. therefore, although nearly all adults have antibodies to rotavirus, 35 they might still be susceptible to infection. rotavirus can elude host defences and induce repeat infection through several mechanisms. there are multiple groups, subgroups, and serotypes of rotavirus. initial antibody response to infection is serotype specific, with limited production of cross-reactive antibodies. 36 subsequent rotavirus infections increase antibodies that cross-react with multiple serotypes. 36 additionally, certain elements of the rotavirus-specific immune response are short-lived. rotavirus-specific secretory iga is sometimes not detectable in faeces as early as 1 year after infection. 37 elias 38 reported that rotavirus fluorescent antibody titres peaked in children at age 1-3 years but subsequently fell to almost undetectable concentrations in individuals older than 70 years. 38 in a review of multiple studies, jiang and colleagues 36 an appreciation of the typical presentation of rotavirus infection in children is critical to understanding the spectrum of disease among adults. primary infection with rotavirus typically occurs in infants between ages 6 months and 2 years, although infection in neonatal intensive-care units and severe infection in infants younger than 6 months are well documented. 51, 52 in all age-groups, the classic presentation of rotaviral infection is fever and vomiting for 2-3 days, followed by non-bloody diarrhoea. 53 the diarrhoea may be profuse, and 10-20 bowel movements per day are common. when examined, the stool from affected patients is generally devoid of faecal leucocytes. especially when associated with vomiting, the diarrhoea of rotavirus infection can precipitate severe and even life-threatening dehydration. infants with repeat rotavirus infections are generally less-severely affected than those with primary disease. 33 among adults, rotavirus infection has been associated with a wide spectrum of disease severity and manifestations. as such, it is difficult to provide a concise description of a typical clinical presentation. nevertheless, prospective studies of voluntary rotavirus ingestion have provided some insight, although the participants in these studies were primarily young healthy adults. data from several such trials are summarised in the table. 35, [46] [47] [48] 54 for these volunteers, illness most frequently began 2-6 days after ingestion and continued for 1-4 days. about two-thirds of study participants had an antibody response, with more than half of all participants eventually shedding rotavirus. symptoms were less common than evidence of infection, but most frequently included diarrhoea, fever, headache, malaise, nausea, or cramping. one participant passed 11 stools in 1 day. 35 among the volunteers, rotavirus particles were detectable in the stool from the start of symptom onset and persisted for more than 10 days in some. three studies document rotavirus readministration to volunteers, noting that symptoms and antibody response were much less common. 35, 47, 54 in several reports of rotavirus outbreaks among adults similar symptoms have been described. in a prospective study of 98 families with neonates followed up from shortly after birth, only 17 of 43 adults who had serological evidence of rotavirus infection were symptomless. 55 14 had diarrhoea and 11 had abdominal cramping. none had symptoms that necessitated medical care or absence from work. in a separate study, 14 parents of children with rotavirus gastroenteritis developed serological evidence of infection, but only three had diarrhoea. 56 grimwood and colleagues 57 found, in a study of children with rotavirus in 28 families, that 18 of 54 adult family members exposed to rotavirus developed evidence of infection, and all but four were symptomatic. in a study of college students during a rotavirus outbreak, of the 83 individuals who met the criteria for rotavirus infection, 93% had diarrhoea, 90% abdominal pain or discomfort, 83% loss of appetite, 81% nausea, and more than 50% had fatigue, vomiting, headache, chills, subjective or low-grade fever, or myalgia. 58 patients with underlying immunodeficiency are at risk of sustained symptoms and rotavirus dissemination, a phenomenon already recognised among children. this pattern was first described in 1980 when two of four children with underlying primary immunodeficiency who had rotavirus infection developed chronic diarrhoea that at least temporarily responded to administration of human milk containing a high titre of rotavirus antibodies. 59 a geriatric patient with impaired naturalkiller-cell activity and impaired cellular and humoral immunity had rotavirus shedding for at least 35 days. 60 gilger and colleagues 61 noted that in four children who had various immune deficits and chronic diarrhoea from rotavirus, rotavirus was identified in the liver and kidney. whether the involvement of liver or kidney was important is unclear. natural infection quantity of cross-reactive neutralising antibody responses against multiple serotypes, 40-42 of virus-specific secretory iga at intestinal mucosa surface, 37, 43 and of virus-specific iga and igg in serum 41, 44 challenge studies serum serotype specific neutralising antibody; 35, 45 intestinal neutralising antibody; 46,47 serum rotavirus igg, but not necessarily with large doses; [46] [47] [48] and raised prechallenge titres of antibody to specific epitope of rotavirus vp7 49 vaccine-induced serum antibodies might be important in protection from natural infection, but interpretation is complicated because of differences in methods of existing studies; 36 and heterotypic antibody response to rotavirus vaccination might be protective against usual infecting serotypes 36, 50 other investigators have assessed the course of rotavirus infection in patients with malignant disease. a wide spectrum of clinical manifestations and severity of illness have been reported. bolivar 62 screened 90 adults who had various solid tumours and leukaemia, with and without diarrhoea. he noted that two patients had rotavirus infection, both of whom had undergone bone-marrow transplantation and had developed graft-versus-host disease. diarrhoea lasted for 10 days before resolution. in three subsequent studies in bone-marrow-transplant patients results varied. yolken and colleagues 63 prospectively assessed patients undergoing bone-marrow transplantation for infectious gastroenteritis and found that rotavirus occurred less frequently than clostridium difficile or adenovirus, but still occurred in nine of 78 patients. in one patient, adenovirus occurred concomitantly with rotavirus infection. of the eight remaining patients, all developed vomiting, seven had abdominal cramps, six had respiratory illness (infiltrates on chest radiography with appropriate clinical signs and symptoms) and four had diarrhoea. five of the eight rotavirus-infected patients eventually died. troussard and colleagues 64 also prospectively assessed patients undergoing bone-marrow transplantation and noted rotavirus infection in eight of 94 patients. adenovirus occurred concomitantly in two patients. seven of the patients had isolates positive for rotavirus in the winter months, with acute onset, vomiting, and diarrhoea. in a later study of the same topic, rotavirus was noted in four of 60 adult asymptomatic stem-cell-transplant patients followed up prospectively. 65 no patient with gastroenteritis had rotavirus isolated. rotavirus infections in adult patients infected with hiv-1 frequently present as a chronic diarrhoea with sustained viral shedding in stools. albrecht and colleagues, 66 between 1987 and 1991, detailed a retrospective assessment of 106 samples from 66 patients infected with hiv-1 who had otherwise unexplained diarrhoea. 35 samples from patients without diarrhoea served as controls. 13 samples from nine case patients were positive for rotavirus; two of these samples were rotavirus recurrences 6 months after the initial episode. no symptom-free patients had rotavirus infection. rotavirus was associated with diarrhoea of 2-8 weeks' duration in all patients and with abdominal cramping in eight patients. thomas and colleagues 67 looked prospectively at 862 samples from 377 uk hiv-1-positive patients with diarrhoea. rotavirus was third in frequency to adenovirus and coronavirus, occurring in 11 (2·4%) samples. the median cd4 count of patients with rotavirus infection was nine. hrdy 68 described five typical settings for rotavirus infections in adults. we propose modification of these classifications to the following: endemic disease, epidemic outbreaks, travelrelated gastroenteritis, and infections transmitted from children to adults. although substantial overlap exists between the groups, our classifications clarify separate risk factors and clinical features. rotavirus infection in children is seasonal, with peak incidence in winter months in temperate climates. iturriza-gomara and colleagues 69 noted that, in the uk between 1995 and 1998, notable numbers of infections began in december or january, peaking in march or april and falling to almost zero by july. in the usa, kapikian and colleagues 56 found that rotavirus cause 59% of diarrhoea cases necessitating admission to hospital in children between november and april, but could not be linked to cases from may to october. in several studies findings suggest that adult disease is not as season-specific as childhood disease. cox ffu=focus forming unit. nr=data not recorded in original paper and taken as not having occurred in calculation of summary percentages. *data included when >50% infectious dose ingested (>9 ffu). †15 of 38 patients had mild illness (including one patient with no antibody response or shedding). ‡one of four volunteers experienced illness but no specific symptoms were recorded. §summary percentages after rotavirus ingestion calculated only from data from studies in which full clinical syndrome of illness described. all percentages have been rounded to the nearest whole percentage point. adult serum in routine hospital samples are present throughout the year. igm concentrations increased with older age, and antibodies reached 20% in march and fell to 10-11% during the summer months. cox and medley attributed the high rates to igm persistence, igm crossreactivity, or possibly to non-seasonal high infection rates in adults. other researchers have also found that rates of adult disease do not mirror the winter seasonality of infection in paediatric patients. 71 these studies suggest that endemic disease in adults may not arise solely from unrecognised transmission of rotavirus from children to adults. the contribution of rotavirus as a cause of endemic gastrointestinal disease varies according to geographic distribution and characteristics of patients. in a small prospective study in the uk, rotavirus caused 4·1% of acute diarrhoea in adults admitted to hospital. 72 similarly, 3% of acute diarrhoea in switzerland, 73 3% of infectious diarrhoea pathogens in a swedish clinic for infectious diseases, 74 5% of adults with gastroenteritis requiring admission in thailand, 75 2-4% of adults older than 15 years with gastroenteritis presenting to their family physician in the netherlands, 76 and nearly 4% of individuals older than 45 years in michigan 77 were due to rotavirus. in studies in other geographic areas even higher rates of infection have been seen. in japan, nakajima and colleagues 71 reported that group a rotavirus had a role in 14% of patients with diarrhoea. pryor and colleagues 78 noted that rotavirus was second only to campylobacter spp as a cause of diarrhoea among australian adults, accounting for 17% of all cases. in indonesia, 42% of patients presenting with diarrhoea had rotavirus-positive stools compared with 11% of control samples. 79 in a study of mexican adults, 63% of patients presenting with acute gastroenteritis during winter months were positive for rotavirus. 80 even these results might underestimate the true prevalence of endemic rotavirus infection. group c rotavirus is not routinely detected by commercial assays but it does contribute to endemic rotavirus infection worldwide. in a study in the uk, 43% of patients were seropositive for group c rotavirus. 81 among adults, clusters of rotavirus infections most frequently occur in communities that are otherwise sheltered from more routine exposure to rotavirus-infected children. 82 one of the largest outbreaks involved nearly 3500 people in 1964, in an isolated area of micronesia. 83 since then, other outbreaks have occurred among closed communities, including a finnish military base, 84 an israeli kibbutz, 85 and an isolated south american indian community. 86 outbreaks of rotavirus infection have also occurred in long-term health-care facilities, particularly those with close living quarters; compromised host immunity and multiple comorbid disorders might help facilitate the spread of infection. 87-91 cubitt and colleagues 88 described an epidemic of rotavirus among staff and patients in an extended-stay geriatric hospital, in which 15 of 39 residents developed symptoms and seven had confirmed rotavirus infection. halvorsrud and orstavik 92 described an outbreak of 92 cases of acute gastroenteritis among nursing-home patients with identification of rotavirus by comparing acute and convalescent antibody titres. rotavirus has been suggested as the causal pathogen in 5% of diarrhoea outbreaks in a study of institutions caring for elderly residents. 93 among adults, rotavirus outbreaks are not confined to geriatric populations. group a rotavirus was associated with an outbreak of gastroenteritis among college students in the district of colombia. 58 rotavirus also caused a waterborne outbreak of gastroenteritis in 1981 in eagle-vail and avon, co, usa in which severity of symptoms correlated with the amount of tap water consumed. 94 finally, griffin and colleagues 95 screened 263 outbreaks of gastroenteritis in the usa between 1998 and 2000 and found that rotavirus was implicated in three outbreaks. uniquely affecting asia, group b rotavirus has been associated with outbreaks affecting large numbers of adults in broad geographic distributions of china and india. 96, 97 rotavirus has been implicated as an important contributor to travellers' diarrhoea among adults, especially among those visiting central america and the caribbean. in a study of travellers returning from jamaica, rotavirus was identified in 9% of individuals with diarrhoea, making the virus second only to enterotoxigenic escherichia coli as a cause. 98 in two studies of us students travelling in mexico, electron microscopy identified rotavirus in about 25% of patients who had diarrhoea, compared with 3% and 15%, respectively, of symptom-free patients. 99, 100 in a third study, a substantial rise of antibodies to rotavirus was seen in 17% of two student groups travelling to mexico. 101 by contrast, only 5-6% seroconverted to norwalk virus. 101 ryder and colleagues 102 found rotavirus in 26% of panamanian travellers to mexico who had diarrhoea. sheridan and colleagues 103 similarly found that 36% of us peace corps volunteers and 30% of panamanian travellers visiting mexico had at least a four-fold increase in rotavirus antibody titres. adult travellers with rotavirus shed 10-100 times less rotavirus than do paediatric patients. 99, 100 although rotavirus can be linked to adult gastroenteritis in each of the other settings, adults who are in contact with children are at particularly high risk of infection. transmission of rotavirus within families from children to parents seems to be a common event. wenman and colleagues 55 showed prospectively that rotavirus infection occurred in 36 of 102 adults caring for children with rotavirus infection. by contrast, only four of 86 adults whose children had no documented rotavirus infection became infected. 55 grimwood and colleagues 57 confirmed this finding in a report that a third of adult family members in new zealand developed evidence of rotavirus infection. the same phenomenon has been seen among parents of more severely ill children. kim and colleagues 104 found evidence of rotavirus infection in 55% of adult contacts of children who were admitted to hospital with rotavirus, compared with 17% of adult contacts whose children were not infected. more casual contact might also be sufficient to facilitate rotavirus transmission from children to adults. rodriguez and colleagues 105 reported that nine of 12 adults experienced illness after exposure to children infected with rotavirus in a playgroup. although substantial evidence is lacking, child-to-adult transmission of rotavirus is accepted to occur with some frequency on paediatric wards. many paediatric nurses, medical students, and house officers experience symptoms of gastroenteritis during the winter months when most paediatric rotavirus infections are encountered. von bonsdorff and colleagues 106 described paediatric nurses at several different locations with acute gastroenteritis caused by rotavirus. among seven hospital staff that developed diarrhoea after direct contact with children with diarrhoea staying in hospital, a rise in antibody titres was detected in three. 104 interestingly, in the same study, six of 45 medical students reported gastroenteritis. three of the students had rotavirus particles present on electron microscopy and were noted to be more ill than the parents of the children who were infected. all had diarrhoea for 3-6 days and two of the three had low-grade fever and vomiting. another case report supports transmission of rotavirus from children to hospital caretakers. 107 electron microscopy, which permits visualisation of the pathognomonic wheel-like appearance, was initially used for diagnostic purposes, but elisa or eia have become more commonly used. commercial assays are reliable, convenient, and inexpensive, but require at least 10 4 -10 7 virions to generate a positive result. 108, 109 the false-positive rate of commercial assays is 3-5%. 69 one of the biggest limitations of most commercial assays is that they do not detect non-group-a rotavirus. 68, 110 other more sensitive and newer methods are being used in research. one such method is pcr, which is up to 1000 times more sensitive than immunoassays. 109 in one study using pcr, 30% of otherwise healthy children shed virus for 25-57 days after symptoms developed. 111 although stool cultures are routinely tested for bacterial pathogens, the low frequency of detecting a positive result calls the usefulness of this practice into question. rotavirus infection can occur in a similar number of patients to some bacterial pathogens. sending a sample of rotavirus antigen for testing by elisa or eia could potentially cut costs if by doing so either hospital stay or procedures could be avoided. such a cost-benefit analysis in adult patients has not been published. one limitation is that adults might shed less rotavirus in faeces than do children, further hampering diagnosis. 100 we suggest that obtaining rotavirus antigen testing for patients admitted to hospital with risk factors for rotavirus infection will be cost effective if additional inpatient studies can be avoided. determination of rotavirus infection may also be beneficial if infectious patients can be isolated to prevent nosocomial spread. a positive rotavirus antigen test might also allow physicians to avoid prescribing antibiotics for travel-related rotavirus infections. treatment of rotavirus infections is primarily directed at symptom relief and restoration of normal physiological function. oral rehydration should be attempted initially. in most developing countries, oral rehydration salt solutions are used extensively in children. most adults can be managed by encouraging them to drink fluids. an additional intervention that has been used is administration of lactobacillus spp bacteria to shorten the duration of diarrhoea. 112, 113 although seldom used in children, codeine, loperamide, and diphenoxylate can help with symptom relief and control of the volume of diarrhoea. 114 bismuth salicylate, in a placebo-controlled double-blinded trial, was efficacious in treating the symptoms of rotavirus diarrhoea. 115 trial use of bismuth salicylate can be considered in adults when other coexistent infectious causes have been ruled out. if symptoms cannot be controlled and the patient becomes dehydrated, administration of intravenous fluids and hospital admission might be necessary. rarely, extraordinary measures have been attempted to help resolve rotavirus infections. for example, human breastmilk has been provided to immunodeficient infected children to help resolve chronic diarrhoea. 59 this option, however, is not practical in adults. several groups report oral administration of human serum immunoglobulins possessing antirotavirus activity to bind free rotavirus antigen. guarino and colleagues 116 noted a mean duration of diarrhoea of 76 h in children who received one oral dose of 300 mg/kg human serum immunoglobulin, compared with 131 h in children who did not. in a study involving three immunocompromised children with chronic rotavirus diarrhoea, oral administration of human serum immunoglobulins (igg 150 mg/kg) cleared rotavirus antigen in all three, but rotavirus antigen recurred in two. 117 among adults, oral immunoglobulin administration of 5-6 g daily for 5 days to bone-marrow-transplant recipients has been successful. 118 prevention of rotavirus infection can be facilitated by avoiding exposures and faecal-oral spread. contact with sick children and potentially contaminated food and water should be avoided. since 43% of rotavirus virions placed on human fingers survive for 60 min, thorough hand washing is critical in prevention. 119 contact isolation for patients diagnosed with rotavirus infection is necessary, generally for the duration of hospital stay, because of sustained faecal shedding of low concentrations of virus. 120 gloves, gowns, isolation, and rigorous hand washing should be used in the care of individuals infected with rotavirus. 121 sattar and colleagues 122 reported that rotavirus survives best in low humidity on non-porous surfaces at room temperature or cooler. phenolic disinfectants do not inactivate rotavirus; instead hypochlorite or sodium dichloroisocyanurate tablets with a free chlorine concentration of at least 20 000 parts per million are recommended. 110 a 70% ethanol solution is also effective in inactivation of rotavirus and can help to prevent environmental spread. 120 rotavirus infection in adults has been successfully prevented by use of a commercially available review disinfectant spray on rotavirus contaminated fomites under experimental conditions. 123 given the substantial disease-related morbidity and mortality associated with rotavirus, the development of an effective vaccine is a priority. although multiple vaccines were under development, the tetravalent rhesus-human reassortant rotavirus vaccine (rrv-tv) seemed to produce the best results. the rrv-tv vaccine prevented about half of rotavirus infections, but was much more effective in preventing severe disease. 124, 125 shortly after the vaccine was approved, the vaccine adverse-event monitoring system noted by mid-1999 an excess of cases of intussusception among recently vaccinated infants, eventually prompting the vaccine to be withdrawn. two sharply differing perspectives on the risk and benefits of the rrv-tv vaccine and the advisory committee on immunization practices' confirmation of its decision to withdraw its recommendation for the vaccine have been put forward. 126, 127 other vaccines are under development. vaccines have been primarily developed to attempt to decrease the severity of rotavirus infections in children. although vaccines seem to be fairly safe in adults from the vaccine trials, we are unaware of any plan to consider vaccination in adult patients. vaccination could theoretically be used in adult patients considering travel to central america or the caribbean or among immunocompromised patients to prevent or lessen the severity of rotavirus diarrhoea. despite recognition as an important cause of gastroenteritis in children, rotavirus's role in adult gastroenteritis is underappreciated. immunity to rotavirus is incomplete and most people have multiple infections over their lifetime. adults with rotavirus can be asymptomatic, but the most common symptoms are nausea, malaise, headache, abdominal cramping, diarrhoea, and fever. adults at particular risk of rotavirus infection are travellers, adults exposed to infected children, and immunocompromised people. group a rotavirus, the most common human pathogen, can be diagnosed with many different commercial assays, but all have limited sensitivity. rotavirus testing might be beneficial in certain clinical settings if detecting rotavirus would change management of patients or prevent nosocomial spread. treatment is primarily symptomatic. rotavirus should be considered in the differential diagnosis of adult infectious gastroenteritis. we have no conflicts of interest. we identified sources for this review by searches of medline with use of the key words "rotavirus infection" and "adult." the search strategy and selection criteria included all english and human studies from the year 1970 until the present. we further reviewed the abstracts for relevance before inclusion. review of the references of the papers retrieved by the initial search allowed for additional studies to be identified and considered for inclusion. visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis viral gastroenteritis molecular epidemiology of "norwalk-like viruses" in outbreaks of gastroenteritis in the united states epizootic diarrhea of infant mice: identification of the etiologic agent relationship between viruses from acute gastroenteritis of children and newborn calves rotavirus infections: guidelines for treatment and prevention rotaviruses and their replication fields' virology nonenteric sources of rotavirus in acute diarrhea rotavirus infection of the oropharynx and respiratory tract in young children infantile enteritis viruses: morphogenesis and morphology pathogenesis of rotavirus diarrhea rotaviruses of man and animals human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption excretion of serotype g1 rotavirus strains by asymptomatic staff: a possible source of nosocomial infection duodenal mucosal damage in 31 infants with gastroenteritis structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein diarrhea induction by rotavirus nsp4 in the homologous mouse model system rotavirus infection impairs intestinal brush-border membrane na(+)-solute cotransport activities in young rabbits d-glucose transport in piglet jejunal brush-border membranes: insights from a disease model absence of a campmediated antiabsorptive effect in an undifferentiated jejunal epithelium rotavirus alters paracellular permeability and energy metabolism in caco-2 cells intestinal permeability assessed with polyethylene glycols in children with diarrhea due to rotavirus and common bacterial pathogens in a developing community rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal caco-2 cell monolayers host factors associated with protection against rotavirus disease: the skies are clearing induction and persistence of local rotavirus antibodies in relation to serum antibodies rotavirus induces alpha-interferon release in children with gastroenteritis clinical immunity after neonatal rotavirus infection: a prospective longitudinal study in young children rotavirus infections in infants as protection against subsequent infections epidemiology of human rotavirus types 1 and 2 as studied by enzyme-linked immunosorbent assay oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance the role of serum antibodies in the protection against rotavirus disease: an overview role of coproantibody in clinical protection of children during reinfection with rotavirus distribution and titres of rotavirus antibodies in different age groups mechanisms of protection against rotavirus in humans and mice protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies anti-rotavirus g type-specific and isotype-specific antibodies in children with natural rotavirus infections protection against rotavirus disease after natural rotavirus infection: us rotavirus vaccine efficacy group fecal antibody responses to symptomatic and asymptomatic rotavirus infections serum antibody as a marker of protection against natural rotavirus infection and disease studies in volunteers with human rotaviruses effects of antibody to rotavirus on protection of adults challenged with a human rotavirus protection of adults rechallenged with a human rotavirus human rotavirus studies in volunteers: determination of infectious dose and serological response to infection identification of vp7 epitopes associated with protection against human rotavirus illness or shedding in volunteers homotypic and heterotypic epitope-specific antibody responses in adult and infant rotavirus vaccinees: implications for vaccine development clinical manifestations of rotavirus infection in the neonatal intensive care unit rotavirus gastroenteritis in infants aged 0-6 months in melbourne, australia: implications for vaccination comparison of serum and mucosal antibody responses following severe acute rotavirus gastroenteritis in young children orbivirus acute gastroenteritis of infancy rotavirus infection in adults. results of a prospective family study human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children spread of rotavirus within families: a community based study foodborne outbreak of group a rotavirus gastroenteritis among college students: district of columbia chronic rotavirus infection in immunodeficiency prolonged shedding of rotavirus in a geriatric inpatient extraintestinal rotavirus infections in children with immunodeficiency rotavirus screening in adult cancer patients infectious gastroenteritis in bone-marrow-transplant recipients virus recovery from stools of patients undergoing bone marrow transplantation infectious gastroenteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients rotavirus antigen detection in patients with hiv infection and diarrhea enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome epidemiology of rotaviral infection in adults molecular epidemiology of human group a rotavirus infections in the united kingdom between 1995 and 1998 serological survey of antigroup a rotavirus igm in uk adults winter seasonality and rotavirus diarrhoea in adults aetiology of acute diarrhoea in adults etiology of acute infectious diarrhea in a highly industrialized area of switzerland enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a swedish clinic for infectious diseases rotavirus as a cause of severe gastroenteritis in adults etiology of gastroenteritis in sentinel general practices in the netherlands the tecumseh study. xv: rotavirus infection and pathogenicity acute diarrhoea in adults: a prospective study enteropathogens associated with acute diarrhea in community and hospital patients in jakarta, indonesia acute gastroenteritis associated with rotavirus in adults seroepidemiology of human group c rotavirus in the uk rotavirus infections in adults in association with acute gastroenteritis gastroenteritis due to rotavirus in an isolated pacific island group: an epidemic of 3,439 cases rotavirus epidemic in adults two sequential outbreaks of rotavirus gastroenteritis: evidence for symptomatic and asymptomatic reinfections an outbreak of rotavirus diarrhea among a nonimmune, isolated south american indian community rotavirus infection in a geriatric population an outbreak of rotavirus infection in a long-stay ward of a geriatric hospital epidemic of viral gastroenteritis in an elderly community an outbreak of rotavirus infection in a geriatric hospital outbreaks of astrovirus type 1 and rotavirus gastroenteritis in a geriatric inpatient population an epidemic of rotavirusassociated gastroenteritis in a nursing home for the elderly outbreaks of infectious intestinal disease in residential institutions in england and wales 1992-1994 a community waterborne gastroenteritis outbreak: evidence for rotavirus as the agent outbreaks of adult gastroenteritis traced to a single genotype of rotavirus investigation of an outbreak of adult diarrhea rotavirus in china emergence of adult diarrhoea rotavirus in calcutta, india epidemiology, etiology, and impact of traveler's diarrhea in jamaica rotavirus in travelers' diarrhea: study of an adult student population in mexico human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption norwalk virus and rotavirus in travellers' diarrhoea in mexico travelers' diarrhea in panamanian tourists in mexico traveler's diarrhea associated with rotavirus infection: analysis of virus-specific immunoglobulin classes human reovirus-like agent infection: occurrence in adult contacts of pediatric patients with gastroenteritis common exposure outbreak of gastroenteritis due to type 2 rotavirus with high secondary attack rate within families rotavirus associated with acute gastroenteritis in adults rotavirus-associated gastroenteritis in two adults probably caused by virus reinfection viral infections of the gastrointestinal tract improved detection of rotavirus shedding by polymerase chain reaction control of outbreaks of viral diarrhoea in hospitals: a practical approach extended excretion of rotavirus after severe diarrhoea in young children oral bacterial therapy reduces the duration of symptoms and of viral excretion in children with mild diarrhea bacteriotherapy with lactobacillus reuteri in rotavirus gastroenteritis rotavirus infections: guidelines for treatment and prevention bismuth subsalicylate in the treatment of acute diarrhea in children: a clinical study oral immunoglobulins for treatment of acute rotaviral gastroenteritis oral administration of human serum immunoglobulin in immunodeficient patients with viral gastroenteritis: a pharmacokinetic and functional analysis severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces red book: 2003 report of the committee on infectious diseases infection control for hospitalized children institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray randomised placebo-controlled trial of rhesushuman reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis efficacy of the rhesus rotavirus-based quadrivalent vaccine in infants and young children in venezuela reappraisal of the association of intussusception with the licensed live rotavirus vaccine challenges initial conclusions the first rotavirus vaccine and intussusception: epidemiological studies and policy decisions for personal use. only reproduce with permission from the lancet. a 73-year-old woman presented with a 2-year history of persistent ulcerous nasal injury that had progressed slowly and without fever, respiratory, or any general symptoms, until the upper lip was affected. a painless ulcero-vegetating injury was seen but the patient was otherwise normal; chest radiographs did not show abnormalities. further examination showed soft tissue attenuation and an ulcered mass lesion in the nasal cavity (figure). the definitive diagnosis was made by isolating mycobacterium tuberculosis from tissue removed during biopsy and antituberculous therapy was started. the lesion had resolved at completion of treatment.tuberculosis of head and neck occurs infrequently and involvement of the nose is rare. granulomatous lesions within the nasal cavity may represent either local disease or a manifestation of a systemic disorder and the differential diagnosis must include tuberculosis. although almost forgotten in industrialised countries, this unusual form of tuberculosis can appear mainly in females and the elderly. it is not thought to be contagious, or to produce noticeable symptoms or physical signs. key: cord-331244-zaguyxm5 authors: stephenson, iain; nicholson, karl g; wood, john m; zambon, maria c; katz, jacqueline m title: confronting the avian influenza threat: vaccine development for a potential pandemic date: 2004-07-30 journal: lancet infect dis doi: 10.1016/s1473-3099(04)01105-3 sha: doc_id: 331244 cord_uid: zaguyxm5 sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. vaccination is the principal means to combat the impact of influenza. during an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. an emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. the manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirusexpressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. in clinical trials, conventional surfaceantigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen. sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. vaccination is the principal means to combat the impact of influenza. during an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. an emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. the manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirusexpressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. in clinical trials, conventional surfaceantigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen. few infectious diseases cause such a huge annual toll of morbidity, mortality, and economic loss as influenza. in addition, influenza can unpredictably emerge to cause pandemics. the 1918 spanish pandemic spread around the world within 9 months causing up to 40 million deaths. 1 the transmission of avian influenza h5n1 to at least 32 people during the 2004 asian h5n1 epizoonotic period 2,3 prompted concerns that the next pandemic is imminent. as highlighted by the recent severe acute respiratory syndrome (sars)coronavirus outbreak, international air travel increases global vulnerability to infectious respiratory pathogens. our ability to combat influenza and its complications depends primarily on vaccination. annual influenza vaccine production is a wellplanned process that takes up to 6 months. current facilities may not be suitable for rapid bulk manufacture of avian influenza virus vaccines in response to a world threat. influenza viruses are enveloped negative-sense rna viruses with a segmented genome belonging to the orthomyxoviridae family. they are classified on the basis of their core proteins into three distinct types: a, b, and c. 4 influenza a viruses infect a range of mammalian and avian species, whereas type b and c are essentially restricted to human beings. influenza a viruses are responsible for annual epidemics and occasional pandemics, whereas influenza b viruses cause outbreaks every 2-4 years, but are not associated with pandemics. the main antigenic determinants of influenza a and b viruses are two surface glycoproteins: the neuraminidase and the haemagglutinin, both capable of eliciting immune responses in human beings. the haemagglutinin is involved with receptor binding and membrane fusion. the neuraminidase facilitates cleavage of virus progeny from infected cells, prevents viral aggregation, and aids movement through the mucosal respiratory-tract epithelium. virus strains are classified according to host species of origin, geographic site and year of isolation, serial number, and, for influenza a, by serological properties of subtypes of haemagglutinin and neuraminidase. influenza a h1 and h3 subtypes circulating in human beings evolve and undergo antigenic variability continuously. a lack of effective proofreading by the viral rna polymerase leads to a high rate of transcription errors that can result in aminoacid substitutions in surface glycoproteins. virus variants with substitutions in the antibody-binding sites can evade humoral immunity and reinfect individuals. this is termed "antigenic drift". the segmented viral genome allows for a second type of antigenic variation. if two influenza viruses simultaneously infect a host cell, genetic reassortment may generate a novel virus with new surface or internal proteins. pandemic influenza viruses arise by this process of "antigenic shift", when a virus with a new haemagglutinin subtype emerges and spreads efficiently in a naive human population. comparisons of pandemic and interpandemic influenza are shown in table 1. bird populations. 5, 6 avian influenza a viruses generally do not cause disease in these natural hosts. the principal site of influenza virus replication in aquatic birds is the gastrointestinal tract resulting in high faecal viral titres and viral transmission in migratory feeding areas. despite the range of virus subtypes, only a few haemagglutinin (h1, h2, h3) and neuraminidase subtypes (n1, n2) have established in human beings and have caused widespread respiratory disease. the haemagglutinin of human influenza viruses preferentially binds to sialic acid receptors containing ␣2,6-galactose linkages, whereas avian influenza viruses preferentially bind to those containing ␣2,3-galactose linkages. these binding preferences correlate with the predominance of sialic acid ␣2,6-galactose linkages on human epithelial cells, and ␣2,3-galactose linkages on avian intestinal epithelial cells. [7] [8] [9] although the molecular mechanisms responsible for receptor-binding specificity are poorly defined, it is believed that haemagglutinin of avian origin must acquire human receptor-binding specificity to generate influenza strains capable of sustained human-to-human transmission. site-directed mutagenesis studies have shown that only one or two aminoacid mutations are required for this change. 10 limited passage in human beings of a virus possessing an avian haemagglutinin, such as occurring in asia currently, 2,3 may be sufficient to generate such a change. during the 20th century, an h1n1 virus in 1918, an h2n2 virus in 1957, and an h3n2 virus in 1968 caused influenza pandemics. human-avian reassortant viruses seem to have caused the pandemics of 1957 and 1968. the 1957 h2n2 virus differed by three genes (haemagglutinin, neuraminidase, and the rna polymerase pb1) from the h1n1 virus that infected people between 1918 and 1957. the 1968 h3n2 virus differed by two genes (haemagglutinin and pb1) from the h2n2 virus that infected people between 1957 and 1968. in both cases, the h2 and h3 haemagglutinin genes were contributed by avian viruses. 11 sequence analyses of early h2 and h3 isolates indicate receptor-binding specificity was altered by a single aminoacid substitution soon after human introduction. 10 since pig trachea contains receptors for both avian and human influenza viruses, and can support replication of viruses of both human and avian origin, it has been suggested that genetic reassortment between avian, swine, and human influenza viruses may occur in pigs, and that they represent a "mixing bowl" for the evolution of human pandemic strains. 12 haemagglutinin and neuraminidase of h5n1 viruses isolated from human beings, poultry, and wild ducks have distinguishable properties. 13 chickens seem to support a separate natural reservoir of influenza viruses, indicating a possible role as intermediate hosts in zoonotic transmission. some avian h9 viruses established in poultry are capable of two-way transmission between domestic ducks, where they are able to generate multiple reassortants with other cocirculating viruses. 14 these reassortant viruses have haemagglutinin receptor-binding sequences potentially capable of human infection, suggesting that new viruses may emerge directly from the avian pool. the close proximity of people to high concentrations of waterfowl, poultry, and swine in southeast asia, and avian influenza activity, has identified this region as a hypothetical influenza epicentre (figure 1). 15 avian influenza viruses generally do not replicate efficiently in human beings, even after experimental infection. 16 before 1997, direct transmission of avian influenza viruses to the human respiratory system was not considered possible. however, it is now recognised that at least some subtypes of avian influenza viruses can replicate within the human respiratory tract. although it is unclear whether the recent reported increase in transmission of avian influenza to people (table 2) is the result of heightened surveillance, the geographical expansion of h5n1 poultry outbreaks across asia is an unprecedented and new event. more than 30 confirmed cases of transmission of avian h5n1 virus to human beings has increased the possibility that an avianhuman reassortant virus may emerge to effectively transmit among people. caused by immunity in population result pandemic influenza a antigenic shift: emergence of novel or little or no background immunity high attack rates, excess mortality and re-emerging subtype of influenza a (maybe partial immunity in older morbidity in all age groups people if re-emerging virus) interpandemic influenza antigenic drift: evolution of existing little immunity in infants. partial variable outbreaks or epidemics influenza (a or b) strains immunity in adults by cross-reacting with variable morbidity and antibody to previously seen mortality, usually in elderly and young and related strains (h3 greatest severity) the first association of avian influenza viruses with respiratory illness in human beings was during 1997 when six deaths from 18 human cases of highly pathogenic influenza h5n1 occurred during an outbreak among live-bird markets (table 2) . [18] [19] [20] all viral genes were of avian origin, indicating that h5n1 had crossed the species barrier without adaptation or reassortment with human viruses. despite the elimination of ducks, geese, and quail (sources of h5n1 and its donor genes) and cleaning days in the markets, h5n1 viruses have subsequently reemerged in hong kong poultry markets, 32 although hong kong remains free from infection in the 2004 h5n1 outbreak across asia. 29, 33 in late 2002, highly pathogenic h5n1 viruses were isolated from dead waterfowl in hong kong (personal communication, m peiris, university of hong kong), which was notable since h5n1 viruses do not typically cause disease in waterfowl. in february 2003, h5n1 reemerged in hong kong in two family members returning from a trip to china. 24 in 2004, pathogenic h5n1 viruses are causing extensive poultry outbreaks in asia with 23 deaths (68%) of 34 human cases reported in vietnam and thailand. 2, 3, 30 in hong kong in 1999, and again in 2003, influenza h9n2 viruses were isolated from children with mild respiratory illnesses. 22, 28 as with h5n1, no human-to-human transmission was evident. 23 additional human h9 infections in china, 34 and detection of antibody to h9 in human serum samples in china and hong kong 35 suggest that further human h9 infection has occurred. eurasian h9 viruses circulating since the late 1990s have been classified into three phylogenetic sub-lineages: g1, y280 (g9-like), and y439. 36, 37 those established in live-bird 38 and have also transmitted to swine in hong kong and china. [38] [39] [40] some avian h9 viruses have acquired receptor-binding characteristics typical of human strains, increasing the potential for reassortment within both human and pig respiratory tracts. 41 in early 2003, outbreaks of highly pathogenic avian influenza h7 among poultry occurred in the netherlands and extended to belgium and germany. 26, 27 more than 80 workers involved in control of the outbreak developed viral h7n7 conjunctivitis and a few developed respiratory illness. evidence of limited human-to-human spread, and a fatal respiratory infection, highlighted a significant threat to human health. more recently, cases of avian influenza (h7n3) infections have been reported in two cullers during control of an h7n3 poultry outbreak in canada. 30 avian influenza (h10n7) seems to have crossed the species barrier from poultry to people for the first time. in egypt in april 2004 two infants presenting with mild febrile respiratory symptoms had h10n7 influenza viruses isolated from respiratory samples. 31 as well as h5, h7, and h9 viruses, the h6 subtype has acquired the ability to infect chickens and is rapidly becoming endemic in poultry populations. phylogenetic analyses of h6n1 viruses isolated from wildfowl, showing high nucleotide homology of the internal genes to human h5n1 and h9n2 viruses, suggests these subtypes can transfer genetic material between each other and are potential sources of new strains. 42 h2n2 viruses were responsible for the 1957 influenza pandemic and circulated as the only human subtype until 1968 when it was replaced by h3n2 subtype. the h2 haemagglutinin gene, along with the neuraminidase and pb1 genes, were derived from avian sources. 11 since people born after 1968 lack immunity to the h2 subtype, h2 viruses pose a pandemic threat to this susceptible population. h2 viruses continue to circulate in wild ducks, 43 although the conditions required for the re-emergence of human h2 influenza viruses are unclear. although the virulence of avian influenza viruses has been well studied in avian species, their virulence in mammals is not well understood. infection with avian influenza a viruses in birds causes a wide spectrum of disease ranging from subclinical to overwhelming systemic illness (figure 2). both h5 and h7 subtypes have the ability to evolve into highly pathogenic forms. although virulence is a polygenic trait, a major contributing factor in birds is the haemagglutinin. cleavage of the haemagglutinin into two subunits is essential for viral infectivity. 44 haemagglutinin from strains of low pathogenicity is cleaved by proteases limited to the respiratory tract of mammalian species and the intestinal tract of avian species. by contrast, haemagglutinin from highly pathogenic viruses can be cleaved by proteases present in a range of tissues, resulting in multi-system infection. structural features at the cleavage site determine the cleavability of the haemagglutinin. the acquisition of multiple basic aminoacids in highly pathogenic h5 or h7 haemagglutinin enables cleavage of the protein by these ubiquitous proteases and confers virulence. carbohydrate side chains in the vicinity of the cleavage site may also affect the access of the proteases and hence virulence. 45 other factors in addition to the haemagglutinin cleavage site are likely to contribute to virus pathogenicity in mammals. virulence of mouse-adapted influenza a viruses have been associated with the interferon antagonist properties of the ns1 protein 46 or the ability of the neuraminidase glycoprotein to sequester circulating plasminogen and promote haemagglutinin cleavage. 47 balb/c mice [47] [48] [49] [50] and ferrets 51 are useful mammalian hosts for the evaluation of human h5n1 pathogenesis. in these mammalian hosts, the multibasic aminoacid motif in the haemagglutinin is necessary but not sufficient for virulence. 47, 52, 53 a single aminoacid substitution in pb2 is associated with high pathogenicity of human h5n1 viruses in mice 52 although substitutions in other gene products are likely to play a part. 49 in human h5n1 infection, disease progression to respiratory failure is unusually severe, with features of haemophagocytosis, leucopenia, and multiple organ failure. 3, 19, 30 in-vitro infection of human macrophages with 1997 h5n1 human viruses induces high levels of cytokines compared with some human virus strains. 54 in pig respiratory epithelial cells, the 1997 h5n1 human viruses were also shown to be relatively resistant to the inhibitory effects of host antiviral cytokines such as interferons. 55 thus, the severity of h5n1 infection in people is likely to be related to the induction of excessive proinflammatory responses that exacerbate tissue injury. it has been suggested that the nonstructural gene has a role. 54 gene sequence analyses of the 1918 pandemic virus, which displayed enhanced virulence in human beings has not yet uncovered molecular determinants previously associated with influenza virus virulence. 56, 57 the molecular determinants and gene constellations that confer virulence of avian, swine, and human viruses, and the circumstances under which virulent phenotypes emerge remains unclear. understanding the basis of virulence is important for vaccine design, particularly of live vaccines, so that viruses can be attenuated. current inactivated influenza vaccines are produced from virus grown in embryonated hens' eggs, and are of three types: whole-virus, "split-product", or subunit "surface-antigen" formulations. whole-virus vaccines are associated with increased adverse reactions, especially in children, and are little used. most influenza vaccines are split-product vaccines, produced from detergent-treated, highly purified influenza virus, or surface-antigen vaccines containing purified haemagglutinin and neuraminidase. 58 vaccines are usually trivalent, containing 15 g each of two influenza a subtypes (h1n1 and h3n2) and one influenza b strain. vaccines elicit a relatively strain-specific humoral response, have reduced efficacy against antigenically drifted viruses, and are ineffective against unrelated strains. the who reviews vaccine composition biannually and updates antigenic content depending on prevalent circulating subtypes to provide antigenically well-matched vaccines. protective efficacy of 70-95% in healthy young adults is obtained when there is a good antigenic match between the vaccine and circulating strains. 59 vaccination of the elderly is associated with 19-63% reductions in hospitalisation for pneumonia and influenza, 17-39% reductions for all respiratory conditions, and 27-75% reductions in all-cause mortality. 60 new influenza vaccines must elicit protective immunity. the haemagglutinin-inhibition test is most commonly used for the detection of antibody to influenza, although single radial haemolysis (srh) may also be used, since both are correlated with immune protection. 61, 62 in the european union, interpandemic influenza vaccines should fulfil certain criteria, prepared by the committee for proprietary medicinal products (cpmp), 63 which are usually assessed by haemagglutinin-inhibition tests in limited annual clinical studies (panel 1). in the event of pandemic influenza, vaccine demand would soar. savings made using monovalent rather than trivalent vaccine (15 g haemagglutinin per dose instead of 45 g) would possibly be offset by a two-dose schedule, increased demand, and difficulties with production of egg-grown viruses. new developments include the use of mammalian cell lines to culture influenza virus for vaccines to provide increased flexibility of production at times of heightened demand. 64 immunopotentiating effects of adjuvants and whole-virus vaccine may increase antigenicity, allowing dose content reduction enabling maximum efficient use of limited supplies. the population immune status in a pandemic situation differs from that seen during the interpandemic period. at the onset of the previous pandemics, younger adults were immunologically naive to the new strains, whereas older populations may have been primed by previous infections of related strains that circulated in earlier times. global immune susceptibility to avian influenza subtypes would be expected. the quantity of antigen required to elicit satisfactory immune responses in naive individuals is unclear since few studies have been done after the emergence of a novel virus. current events suggest the urgent need to develop a clearly defined strategy for clinical assessment of safety and immunogenicity of pandemic vaccines. in 1976 and 1977, the emergence of influenza a/new jersey/76 and a/ussr/92/77 (h1n1) triggered pandemic alerts, and afforded the opportunity for vaccine trials in immunologically naive and primed populations. a series of whole-virus vaccine studies [65] [66] [67] [68] [69] reported differences between naive populations (those aged 24 years and not exposed to previous h1n1 strains) and primed populations (older than 24). in naive patients, if one dose of vaccine was administered, large doses (in excess of 60 g haemagglutinin) were required to fulfil cpmp criteria. however, if two doses of vaccine were given, lower antigen doses (5 g) were needed. whole-virus vaccine was significantly more immunogenic than subunit or split-product vaccines. in primed patients, as is the case during interpandemic periods, no difference in immunogenicity between whole-virus vaccine and subunit or split-product vaccines was reported. however, a consistent finding was that whole-virus vaccine was associated with increased reactogenicity, particularly in children, who developed febrile complications even with low doses. 67 although licensed for use in human influenza vaccines, aluminium salts are rarely used since studies have indicated little clinical benefit. 70 however, encouraging findings were more recently reported in a study of whole-virus a/singapore/1/57 (h2n2) vaccine in immunologically naive people. 71 monovalent alumadjuvanted vaccine containing either 7·5, 3·8, or 1·9 g h2 haemagglutinin per dose was compared with unadjuvanted whole-virus 15 g vaccine. although a single dose of any vaccine was unable to elicit responses associated with protection, a second dose of vaccine boosted responses to mean geometric increase in antibody >2·5 (>2 in the у60 years group) number of seroconversions or significant rises in anti-haemagglutinin antibody (ie, four-fold increase in post-vaccination hi titre or 50% increase in srh zone) should be >40% (>30% in the у60 years group) proportion of patients achieving a seroprotective hi titre of у1/40, or srh titre of >25 mm 2 post vaccination should be >70% (>60% in the >60 years group) rates that fulfilled cpmp licensing criteria across all doses, suggesting that up to an eight-fold reduction in antigen content could be achieved with the addition of alum (figure 3). since influenza a viruses possess a segmented genome, simultaneous infection of eggs with two different viruses may result in reassortment of segments to produce a desired vaccine seed strain. the influenza a virus components of annual influenza vaccines are typically derived from egggrown reassortment viruses that have the relevant haemagglutinin and neuraminidase genes of the antigenically relevant strain, and the six remaining gene segments from a/puerto rico/8/34 (h1n1). these pr/8/34 segments confer high growth properties in eggs favoured for inactivated vaccine production. 58 this process requires large numbers of eggs, and in many companies lacks the flexibility to respond rapidly to a pandemic event. highly pathogenic h5 and h7 viruses cannot be grown in large quantities because they are lethal to chicken embryos. 72 such pathogenic strains also impose regulatory and safety issues. as the multibasic sequence cleavage site is believed to contribute to the pathogenesis of human h5n1 infection, vaccine preparation from wild-type h5n1 virus would require heightened biocontainment to protect workers and eliminate the possibility of environmental contamination and infection of susceptible animals. thus, several approaches have been attempted for avian influenza vaccine development. these include: (1) the production of inactivated vaccine from wildtype virus; (2) the selection of an antigenically related nonpathogenic vaccine strain; (3) the use of baculoviruses to express recombinant haemagglutinin; (4) dna-based vaccines; (5) the use of plasmid-based reverse genetics systems to construct vaccine seed strains possessing attenuated haemagglutinin; and (6) plasmid-based reverse genetic systems to construct attenuated donor strain recombinants. reverse genetics is likely to produce the most rapid response in an emerging pandemic. much of the preclinical and clinical development of highly pathogenic avian influenza vaccines has used h5 virus as a model. after the 1997 h5n1 outbreak, inactivated vaccines from wild-type a/hong kong/156/97 (h5n1) virus were prepared in the uk and the netherlands. 73, 74 whole-virus vaccine was effective in protecting mice against lethal challenge with h5n1 virus. 73 conventional surface-antigen vaccine was poorly immunogenic in chickens and did not protect against lethal dose challenge, although an iscom formulation (antigen as immune-complex stimulators) boosted immune responses and protected against lethal h5n1 challenge. 74 because the use of highly pathogenic strains has safety restrictions, the selection of a surrogate non-pathogenic virus capable of evoking crossreactive immunity to the 1997 hong kong h5n1 viruses was investigated. both a/duck/hokkaido/ 67/96 (h5n4) and a/duck/singapore/97 (h5n3) have haemagglutinin proteins antigenically similar to a/hong kong/ 156/97 (h5n1) and were used in experimental vaccines. 72, 73 although antibody titres to h5n1 induced by a/duck/ singapore (h5n3) vaccine were four-fold lower than the homologous strain, 73 inactivated whole-virus vaccine 72,73 and alum-adjuvanted subunit vaccine 48 were capable of protecting mice against lethal h5n1 challenge. although attempts to reassort a/duck/singapore (h5n3) with a/pr/8/34 to produce a high-growth virus suitable for vaccine production failed, 73 conventional and mf59-adjuvanted a/duck/ singapore/97 (h5n3) surface-antigen vaccines were clinically assessed in a randomised phase i trial. 75 two doses of 7·5, 15, or 30 g h5 haemagglutinin were given 3 weeks apart. antibody responses were measured by haemagglutinininhibition, virus microneutralisation, and srh. although both vaccines were well tolerated, non-adjuvanted vaccine was poorly immunogenic, with only one of 11 (9%) recipients seroconverting by haemagglutinin-inhibition and srh h5n1, and four (36%) by microneutralisation and srh h5n3 after two 30 g doses. the addition of mf59 gave significantly higher antibody responses (figure 4), and two doses achieved seroconversion rates of 13/31 (42%), 29(94%), 31(100%), and 26(84%) by haemagglutinin-inhibition, microneutralisation, srh h5n3, and srh h5n1, respectively. antibody titres by srh to h5n1 were about half those to h5n3, showing the need for close antigenic matching between vaccine and pandemic strains to ensure maximum vaccine efficacy. antibody responses after h5n3 revaccination 16 months later were boosted significantly above those achieved after two doses. 76 it is desirable for vaccines to boost responses after initial priming, since second waves occur 3-9 months after the first pandemic wave of infection. 77 one problem in assessing vaccine responses was the insensitivity of the haemagglutinininhibition test, routinely used in assessment of influenza vaccines, for the detection of antibody to h5 when compared with neutralisation tests. 78, 79 haemagglutinin-inhibition relies on the ability of antibody to disrupt the haemagglutinin-sialic-acid-receptor binding interaction between virus and erythrocytes. if the erythrocytes used in the test are not optimised for expression of ␣2,3galactose linkages that are necessary for avian influenza virus binding, the test is insensitive. 80 however, there are no recognised clinical correlates of immune protection for neutralisation antibody. it was only possible to assess vaccine responses with respect to cpmp licensing criteria after development of a specific srh assay. 81 a modified haemagglutinin-inhibition test, using enzymatically altered turkey erythrocytes or horse erythrocytes, was developed to increase the sensitivity for detecting antibody to avian influenza virus antigens. 82 an alternative to egg-derived vaccines involves the use of haemagglutinin protein expressed in insect cells by recombinant baculovirus. potential difficulties include the use of uncleaved rather than cleaved haemagglutinin and differences in glycosylation in insect cells that may effect immunogenicity. nonetheless, antibody responses to 15-45 g doses of recombinant h1 and h3 antigens are similar to those induced by licensed vaccines. 83, 84 baculovirus-derived h5 and h7 haemagglutinin vaccines protect against lethal virus challenge in chickens, even when the haemagglutinin sequence homology differs by up to 16%. 85 however, when clinically assessed in people, a recombinant baculovirus-expressed h5 vaccine was suboptimal. even after two doses of 90 g, only 52% subjects seroconverted by microneutralisation, suggesting improvements in immunogenicity are needed. 86 reverse genetics systems can be used to generate attenuated avian influenza viruses, and are likely to prove pivotal in pandemic vaccine development. the appropriate haemagglutinin and neuraminidase genes from a virus can be cloned and, if necessary, the haemagglutinin may be attenuated by removal of the multi-basic cleavage site sequence, and inserted into plasmids. the plasmids are transfected into a cell line together with plasmids encoding the internal genes from the a/pr8/34 virus to generate an appropriate non-pathogenic vaccine seed strain. vaccine candidates expressing the target haemagglutinin from highly pathogenic viruses could potentially be produced within weeks of an emerging event. recombinant h5 viruses showing desirable properties for h5 vaccine formulation-including loss of egg lethality, virulence, and infectivity in animal models-have been produced, 87, 88 although one attenuated recombinant virus showed limited neurovirulence in mice. 87 reverse genetics is thus capable of generating attenuated viruses from pathogenic strains suitable for vaccine production with only limited enhancement of biosecurity measures and using pre-existing equipment and facilities. it should also be possible to prepare panels of reassortant viruses and vaccine seed candidates containing target genes of potential pandemic viruses in advance of any specific threat. however, there are regulatory, safety, and legal problems to overcome before the technology can be used for vaccine development. mammalian cell lines (eg, vero cells) used for transfection must be of certified quality for human vaccine production. viruses generated by reverse genetics may be considered to be "genetically modified organisms", imposing local and national safety regulations regarding research and development. in addition, intellectual property rights on reverse genetics technology are held, and licences may need to be granted for commercial use of vaccines. inactivated influenza vaccines are poor inducers of cytotoxic t-cell (ctl) responses, which aid in recovery from influenza infection. it has been suggested that crossreactive immunity to several influenza virus subtypes could be induced by ctl responses to conserved epitopes in internal proteins. 89 dna vaccines integrate gene sequences for the antigenic protein of interest into bacterial plasmids that are inoculated into the host. the expression of plasmid dna produces the antigen in its natural configuration, which is more likely to stimulate neutralising antibody and undergo hla class i expression inducing ctl responses. 90 h5 and h7 haemagglutinin-expressing dna vaccine protects mice and chickens against lethal dose virus challenge. 91,92 dna vaccine expressing conserved internal proteins including nucleoprotein give partial protection to mice against h5n1 infection. 93 intradermal dna influenza vaccines are beginning clinical evaluation. iscom-formulated h1n1 vaccine can induce ctl responses and greater longer-lasting antibody responses than conventional vaccine. 94 it offers broad protection against virus challenge with h2, h3, h9, and virulent h5 viruses in mice. iscom vaccines are tolerated in human beings and induce broad ctl and rapid humoral responses. 95 mucosal delivery of inactivated influenza h3n2 vaccine adjuvanted with modified heat-labile enterotoxin from escherichia coli induces b-cell-dependent heterosubtypic immunity against lethal h5n1 virus challenge in mice. 96 in the absence of an antigenically matched vaccine, alternative vaccine strategies that induce crossreactive immunity by iscom, dna, or mucosal vaccines may provide useful firstline defence against an emerging pandemic strain. since h9 viruses do not have a multibasic haemagglutinin cleavage site, they show low pathogenicity for avian species and may be grown to high titres in eggs. both g1-like (a/hong kong/1073/99) and y280 (g9-like; a/hong kong/2018/03) h9n2 viruses are capable of human infection. 22, 28 for an effective h9 vaccine strategy, an understanding of the relative immunogenicity and crossprotection induced by these lineages is required. although infection of mice with g1 or g9 group h9 viruses did not evoke detectable crossreacting neutralising antibody, they were protected from subsequent rechallenge with the homologous or heterologous virus lineage. 97 whole-virus g1 h9 vaccine produced crossreactive antibody responses to both g1 and g9 viruses, and protected mice against rechallenge with either virus. by contrast, whole-virus g9 h9 vaccine induced homologous antibody titres only and was able to protect against g9 challenge, but showed reduced protective efficacy against the heterologous g1 lineage. here, a single dose of h9 vaccine induced adequate immune responses in mice, by contrast with findings with h5n1 vaccine that required a two-dose schedule to elicit adequate immune responses. 74, 97 since some y280 (g9-like) h9 viruses do not grow well in eggs, an a/pr/8/34 reassortant has been produced. 98 one dose of inactivated g9/pr8 vaccine protected mice against g9 challenge. two doses of vaccine increased antibody responses capable of protecting mice against both g1 and g9 h9 challenge. whole-virus and subunit a/hong kong/1073/99 (h9n2) vaccines were clinically evaluated in the uk. 99 60 adults were randomly assigned two doses, administered 3 weeks apart, of 7·5, 15, or 30 g h9 haemagglutinin content. although well tolerated, whole-virus vaccine was more reactive, in keeping with h1n1 vaccines. there was little detectable crossreactive immune response to an antigenically distinct g9 h9 virus (unpublished findings). more than 40% of the prevaccination serum samples showed reactivity to h9n2 by neutralisation and haemagglutinin-inhibition, suggesting preexisting crossreacting antibody from exposure to earlier haemagglutinins. it is unlikely that h9 influenza has circulated widely in the uk. further serological testing correlated h9 reactivity with antibody responses to h2, but not h1 or h3 haemagglutinin. people with baseline reactivity to h9 were born before 1969, and thus had been potentially exposed to h2 during its period of circulation in human beings. subjects were immunologically divided into naive and primed recipients. in truly naive subjects, one dose of either vaccine was poorly immunogenic. although whole-virus vaccine was more immunogenic than subunit vaccine, two doses still left a significant number of vaccinees with serological responses below the protective threshold (table 3) . among primed individuals, one dose of either vaccine boosted anti-h9 responses, fulfilling cpmp criteria. since the second dose was of questionable value, to preserve limited vaccine supplies during the first wave of an emerging pandemic, different schedules in different populations could be considered. a german study among 18-60 year-olds reported one dose of 15 g whole-virus vaccine a/hong kong/1073/99 (h9n2) capable of fulfilling at least one cpmp criterion and that a second dose of vaccine significantly improved responses. 71 however, age-related responses or the effect of pre-existing reactivity to h9n2 were not analysed. in keeping with the h2n2 experience, alum-adjuvant allowed a reduction in h9 content to 1·3 g per dose while maintaining immunogenicity. intranasally delivered live, attenuated cold-adapted influenza vaccines elicit systemic and local mucosal immune responses and display protective efficacy. 100 attenuated cold-adapted strains are generated by reassortment between a wild-type virus expressing target haemagglutinin and neuraminidase, and a cold-adapted donor such as influenza a/ann arbor/6/60 (h2n2). donor strains are cold adapted, temperature-sensitive, and attenuated. these properties are associated with polygenic mutations. these live attenuated viruses display high levels of phenotypic and genotypic stability and are not transmissible to close seronegative contacts. 101 both attenuated h5n1 and h9n2/ann arbor cold-adapted recombinant viruses have been generated and are seen to be non-pathogenic in mammalian and chicken models. 98, 102 concerns over the generation of a reassortant between a live virus vaccine containing an avian influenza virus and a co-infecting human strain, and the possibility of spontaneous genetic change may limit the use of such vaccines in the interpandemic period. while current intramuscular influenza vaccines are effective at inducing relatively strainspecific serum haemagglutination-inhibition igg, they are poor at stimulating secretory iga in nasal wash fluid. 103, 104 as secretory iga exhibits potential heterotypic crossreactivity to influenza virus strains at the point of entry, 104,105 live attenuated virus vaccines may offer wider protection against vaccinedrifted variants that could be advantageous once a pandemic is underway. specific influenza antiviral agents are available for early treatment and prophylaxis of influenza. 106 the adamantanes, amantadine and rimantadine, have been available for more than 30 years and inhibit strains of influenza a including nonhuman subtypes. however, rapid emergence and transmission of drug-resistant virus after treatment may render prophylaxis ineffective. the genetic basis for resistance seems to be single aminoacid substitutions in the viral m2 ion channel. the h5n1 strains isolated from poultry and human cases in 2004 had genotypic changes in the m2 gene associated with resistance, 29 suggesting these agents would be of little clinical value should these strains become capable of humanto-human transmission. neuraminidase inhibitors, such as zanamivir and oseltamivir, are effective in prevention studies 106 and are highly active against a broad range of influenza a viruses of both human and avian origin, including amantadine-resistant strains. although strains with reduced susceptibility to neuraminidase inhibitors have been isolated after sequential passage of virus in presence of drugs, clinically significant resistant strains have not, as yet, been identified. antiviral drugs may be of benefit in protecting individuals in essential services whilst waiting for an effective vaccine to be prepared; however, supply and cost issues would limit their effect on the course of a pandemic. a sufficiently large supply of antivirals to curb pandemic influenza would require international or need for robust surveillance programmes in human and animal populations and sharing of information between animal and human surveillance systems surveillance information to be open and shared in a timely fashion to assess potential threats potential pandemic strains come from animal reservoir. improved understanding of the antigenic and molecular associations between potential pandemic strains of same subtype improved understanding of immunogenicity against drifted avian influenza strains is required as the ability to generate broad crossprotective immunity is desirable in vaccine candidate. intellectual property rights of attenuated viruses produced by reverse genetics must be addressed in advance because licences for commercial use may be required vaccine virus candidate needs to be able to grow well in eggs (or approved cell culture) to improve ability to respond rapidly to emerging threat improved understanding of virulence determinants in mammalian models to be able to attenuate viruses used for vaccine manufacture safety of virus handling for workers involved in preparation of vaccine to assess and approve mammalian cell lines of human vaccine quality ensure that reagents from animal sources are transmissible spongiform encephalopathies compliant "reverse genetics" generated viruses are labelled as genetically modified organisms-implications for national and local regulatory authorities clinical assessment of vaccines derived by reverse genetics ability to organise antigenicity studies rapidly in response to emerging threat may require prepared approved protocols that can be readily adapted. improvement in assessment of antibody responses to avian influenza vaccines to establish licensing criteria standardisation of assays for detection of neutralising antibody to avian influenza establish correlates of immune protection of neutralising antibody whole-virus vaccine more immunogenic than subunit or split-product vaccine in immunological naive populations (h1n1, h9n2) two doses of vaccine required in immunologically naive populations, the first to prime and the second to boost responses (h1n1, h2n2, h5n3, h9n2) in primed populations, a single dose of vaccine can potentially induce responses associated with protection (h1n1, h5n3, h9n2) addition of adjuvants such as mf59 and aluminium salts have the potential to significantly enhance immunogenicity and spare antigen use (h5n3, h9n2 and h2n2) avian haemagglutinin (h5 and to a lesser extent h9) seems to be less immunogenic in people than h1 and h2 assessment of antibody responses to avian influenza may require additional serological methods other than the standard haemagglutinininhibition test (h5n3) potential crossreactivity with pre-existing antibodies complicates interpretation of immune responses in people (h9n2) need to develop understanding of improving vaccine candidates to enhance heterosubtypic crossreactivity and protection need to assess vaccine candidates in advance of pandemic to identify difficulties and establish dosing schedules in different populations national stockpiling before the onset of such an event; this would require considerable expense, and vaccination is likely to remain the principal means of combating pandemic influenza. although pandemic planning and understanding is greater since the first h5 outbreak in 1997, our ability to respond rapidly remains less than optimal. the 2004 asian h5n1 epizoonotic outbreak indicates the urgent need for vaccines against avian influenza viruses. however, regulatory and safety considerations confront their development (panel 2). it is necessary to improve our understanding of the virulence determinants in mammalian systems to be able to attenuate viruses to select appropriate and safe vaccine strains that can generate broad crossreactivity. national and international authorities must urgently confront regulatory issues to allow production and clinical assessment of newly generated virus vaccine candidates. there are important observations from our clinical experience with vaccines for pandemic influenza (panel 3). despite increased reactogenicity, the greater immunogenicity of whole-virus vaccines could be beneficial in a pandemic. vaccines containing avian h5 and h9 haemagglutinin seem to be less immunogenic in human beings than vaccines based on h1 and h2 haemagglutinin. whether this is a general event associated with avian subtypes is at present unclear. enhancement with mf59 or alum salts may provide best antigen use and enhance immunogenicity. overall, so far, clinical trials of avian influenza vaccine candidates have given disappointing results. it remains to be seen how plasmid-derived reverse-genetics influenza vaccines will perform once regulatory hurdles have been overcome. we have no conflicts of interest. chronicle of influenza pandemics avian influenza (h5n1) in 10 patients in vietnam the textbook of influenza evolution and ecology of influenza a viruses characterisation of a novel influenza haemagglutinin h15: criteria for determination of influenza a subtypes single amino acid substitutions in influenza haemagglutinin change receptor binding specificity receptor specificity in human, avian and equine h2 and h3 influenza virus isolates receptor specificity of influenza a viruses correlate with agglutination of erythrocytes from different animal species early alterations of the receptor binding properties of the h1, h2 and h3 avian influenza virus haeamgglutinins after their introduction into mammals avian to human transmission of the pb1 gene of influenza a virus in the 1957 and 1968 pandemic influenza in pigs and their role as the intermediate host the surface glycoproteins of h5 influenza viruses isolated from humans, chickens and wild aquatic birds have distinguishable properties characterisation of h9 subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? an influenza epicenter? replication of avian influenza viruses in humans avian influenza isolated from a woman with conjunctivitis human influenza a h5n1 virus related to a highly pathogenic avian influenza virus clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus antibody responses in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts characterization of the surface proteins of influenza a h5n1 viruses isolated from humans in 1997-1998 human infection with influenza h9n2 lack of evidence for human-to-human transmission of avian influenza a (h9n2) viruses in hong kong re-emergence of fatal human influenza a subtype h5n1 disease antigenic characterization of recent avian influenza a h5n1 viruses isolated from humans avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory respiratory distress syndrome transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands influenza a (h9n2) in hong kong special administrative region of china avian influenza h5n1 infection in humans: urgent need to eliminate the animal reservoir-update 5 cases of influenza a (h5n1)-thailand avian influenza virus a (h10n7) circulating among humans in egypt an update on avian influenza in hong kong in 2002 h5n1 influenza viruses isolated from geese in southeastern china: evidence for genetic reassortment and interspecies transmission to ducks discovery of man infected by avian influenza virus seroprevalence of antibody to h9n2 viruses in poultry workers of hong kong molecular characterization of h9n2 influenza viruses: were they the donors of the internal genes of h5n1 viruses in hong kong influenza a viruses: relationship between h9n2 relationship between h9n2 and h5n1 human isolates emergence and control of zoonotic ortho-and paramyxovirus diseases cocirculation of avian h9n2 and human h3n2 viruses in pigs in southern china seropidemiological evidence of avian h4, h5 and h9 influenza viruses transmission to pigs in south eastern china characterisation of a human h9n2 influenza virus isolated in hong kong molecular evolution of h6 influenza viruses from poultry in south eastern china: prevalence of h6n1 influenza viruses possessing seven a/hongkong/156/97 h5n1-like genes in poultry transmission of eurasian avian h2 influenza virus to shorebirds in north america enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the haemagglutinin polypeptide proteolytic cleavage of influenza virus haemagglutinins: primary structure of the connecting peptide between ha1 and ha2 determines proteolytic cleavability and pathogenicity of avian influenza viruses inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses a novel mechanism for the acquisition of virulence by a human influenza a virus a mouse model for the evaluation of pathogenesis and immunity to influenza a (h5n1) viruses isolated from humans molecular correlates of influenza a h5n1 virus pathogenesis biological heterogeneity, including systemic replication in mice, of h5n1 influenza a virus isolates from humans in hong kong pathogenesis of avian influenza a h5n1 viruses in ferrets molecular basis for high virulence of hong kong h5n1 influenza a viruses pathogenicity and antigenicity of a new influenza a (h5n1) virus isolated from duck meat induction of proinflammatory cytokines in human macrophages by influenza a h5n1 viruses: a mechanism for the unusual severity of human disease? lethal h5n1 influenza viruses escape host anti-viral cytokine responses origin and evolution of the 1918 spanish influenza virus heamagglutinin gene characterisation of the 1918 spanish influenza virus neuraminidase gene vaccine production antigenic drift and efficacy of influenza virus vaccines efficacy/clinical effectiveness of inactivated influenza virus vaccines in adults determinants of immunity to influenza infection in man correlation between rate of infection and pre-existing titer of serum antibody as determined by single radial haemolysis during an epidemic of influenza a/victoria/3/75 note for guidance on harmonisation of requirements for influenza vaccines, cpmp/bwp/214/96. circulaire no 96-0661: 1-22. london: committee for proprietary medicinal products development of a vero cell-derived influenza whole virus vaccine antibody responses and reactogenicity of graded doses of inactivated influenza a/new jersey.76 whole virus vaccine in humans summary of influenza virus vaccine trials in adults trials of influenza a/new jersey/76 virus vaccine in normal children: an overview of age related antigenicity and reactogenicity clinical studies of monovalent inactivated whole virus and subunit a/ussr/77 (h1n1) vaccine: serological responses and clinical reactions responses of volunteers to inactivated influenza virus vaccines lack of adjuvant effect of alpo4 on purified influenza virus haemagglutinin in man pandemic preparedness: lessons learnt from h2n2 and h9n2 candidate vaccines avirulent avian influenza virus as a vaccine strain against a potential human pandemic vaccine against h5n1 influenza iscom vaccine induced protection against a lethal challenge with a human h5n1 influenza virus safety and antigenicity of nonadjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a randomised trial of two potential vaccines against h5n1 influenza boosting immunity to influenza h5n1 with mf59-adjuvanted h5n3 a/duck/singapore/97 vaccine in a primed human population pandemic planning serological evidence of human infections with avian influenza viruses detection of antibody to avian influenza (h5n1) virus in human sera by using a combination of serological assays sialic acid receptor specificity of erythrocytes affects haemagglutinin-inhibition test detection of antibody to avian haemagglutinin a single radial haemolysis assay for antibody to h5 haemagglutinin detection of anti-h5 responses in human sera by hi using horse erythrocytes following mf59-adjuvanted influenza a/duck/singapore/97 vaccine recombinant baculovirus influenza a haemagglutinin vaccines are well tolerated and immunogenic in healthy adults evaluation of recombinant haemagglutinin expressed in insect cells as an influenza vaccine in young and elderly adults baculovirus-derived haemagglutinin vaccines protect against lethal infection by avian h7 and h5 subtypes safety and immunogenicity of a recombinant hemagglutinin vaccine for h5 influenza in humans preparation of a standardized, efficacious agricultural h5n3 vaccine by reverse genetics evaluation of a genetically modified reassortant h5n1 influenza a virus vaccine candidate generated by plasmid-based reverse genetics cross-subtype protection in humans during sequential overlapping and/or concurrent epidemics caused by h3n2 and h1n1 influenza viruses influenza dna vaccines strategies for inducing protection against avian influenza a virus subtypes with dna vaccines impact of glycosylation on the immunogenicity of a dna based influenza h5 vaccine dna vaccine expressing conserved influenza virus proteins protective against h5n1 challenge infection in mice heterotypic immunity against human influenza a viruses including recently emerged avian h5 and h9 viruses induced by iscom vaccine in mice requires both cytotoxic t-lymphocyte and macrophage function a randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza iscom vaccines and conventional vaccines mucosal delivery of inactivated influenza vaccine induces b cell dependent heterosubtypic crossprotection against lethal influenza a h5n1 virus infection immunity to influenza a h9n2 viruses induced by infection and vaccination generation and evaluation of a high-growth reassortant h9n2 influenza a virus as a pandemic vaccine candidate safety and antigenicity of whole virus and subunit influenza a/hong kong/1073/99 (h9n2) vaccine in healthy adults: phase i randomised trial cold-adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody responses and vaccine efficacy: a meta-analysis genotypic stability of coldadapted influenza vaccine in an efficacy clinical trial recombinant influenza a virus vaccines for the pathogenic human a/hong kong/97 (h5n1) viruses comparison of the immune response to inactivated and live, attenuated influenza vaccines the efficacy of live attenuated cold adapted trivalent intranasal influenza virus vaccine in children intranasal immunization with formalin-inactivated virus vaccine induces a broad spectrum of heterosubtypic immunity against influenza a virus infection in mice influenza: vaccination and treatment searches of medline, pubmed, current contents, and references from relevant articles, as well as the extensive files of the authors identified data for this review. search terms were "avian influenza", "influenza vaccine'', "pandemic influenza", "h5 influenza", "h9 influenza", "h7 influenza", "influenza vaccines", "pathogenesis" and "virulence". english language articles were reviewed. key: cord-296573-4c9gch5b authors: to, kelvin kw; chan, jasper fw; chen, honglin; li, lanjuan; yuen, kwok-yung title: the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities date: 2013-08-19 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70167-1 sha: doc_id: 296573 cord_uid: 4c9gch5b infection with either influenza a h5n1 virus in 1997 or avian influenza a h7n9 virus in 2013 caused severe pneumonia that did not respond to typical or atypical antimicrobial treatment, and resulted in high mortality. both viruses are reassortants with internal genes derived from avian influenza a h9n2 viruses that circulate in asian poultry. both viruses have genetic markers of mammalian adaptation in their haemagglutinin and polymerase pb2 subunits, which enhanced binding to human-type receptors and improved replication in mammals, respectively. hong kong (affected by h5n1 in 1997) and shanghai (affected by h7n9 in 2013) are two rapidly flourishing cosmopolitan megacities that were increasing in human population and poultry consumption before the outbreaks. both cities are located along the avian migratory route at the pearl river delta and yangtze river delta. whether the widespread use of the h5n1 vaccine in east asia—with suboptimum biosecurity measures in live poultry markets and farms—predisposed to the emergence of h7n9 or other virus subtypes needs further investigation. why h7n9 seems to be more readily transmitted from poultry to people than h5n1 is still unclear. the infl uenza viruses belong to the rna virus family orthomyxoviridae. on the basis of antigenic and genetic diff erences between the internal proteins, nucleoprotein and matrix protein, infl uenza viruses are classifi ed into a, b, and c. the infl uenza a virus has a negative-sense, single-stranded, and eight-segmented genome. on the basis of two surface proteins, 17 haemagglutinin subtypes and ten neuraminidase subtypes can be found (fi gure 2). 19 except h17 and n10, which have been found in bats only, all haemagglutinin and neuraminidase subtypes have been found in wild waterfowl. 20 unlike other neuraminidase subtypes, n10 does not have neuraminidase activity. 21 the 17 haemagglutinin subtypes are further classifi ed into group 1 and group 2 on the basis of the antigenic and phylogenetic characteristics of haem agglutinin. the ha1 subunit of the haemagglutinin attaches onto host cell sialic acid receptor. the haemagglutinin precursor protein ha0 is cleaved by proteases into ha1 and ha2 before ha2 can mediate virion-cell fusion. the internal proteins are encoded by six other gene segments encoding nucleoprotein np, matrix protein m1, ion channel protein m2, the polymerase complex proteins pb1, pb2, pa, including pb1-f2, pa-x, pa-n155, pa-n182, and non-structural proteins including pb1-f2, pa-x, pa-n155, pa-n182, ns1, and ns2. 19, 22 the eight gene segments can reassort when two diff erent viruses infect an animal host cell. phylogenetic analysis shows that the 1997 h5n1 virus infecting human beings was a reassortant with goose h5n1 haemagglutinin, teal h6n1 neuraminidase, and internal genes from quail h9n2 and teal h6n1. 23, 24 the 2003 h7n7 virus that infected human beings in the netherlands was a reassortant between avian infl uenza a h7n3, infl uenza a h10n7, and other eurasian avian infl uenza viruses (fi gure 3). 9 on the basis of available sequences in the public domain, the 2013 h7n9 virus is also a reassortant of avian infl uenza viruses consisting of haemagglutinin and neuraminidase most closely related to h7n3 viruses isolated from ducks in zhejiang and h7n9 viruses from wild birds in korea, respectively. the m, np, and ns genes are most closely related to h9n2 viruses from chickens in eastern china, whereas personal view the pa, pb1, and pb2 genes are most closely related to those of h9n2 viruses from brambling in beijing. 11, 12, 13, 25, 26 because the origin of all gene segments of the 2013 h7n9 virus are avian, gene reassortment probably took place in an avian host. although reassortment events of avian infl uenza viruses have been recorded in pigs in korea, 27 the 2013 h7n9 virus and the immediate precursors have not been found in pigs. 28 the haemagglutinin sequence of one of the 2013 h7n9 virus human isolates (a/shanghai/1/2013) was phylogenetically distinct from other human and avian isolates, 12, 29 suggesting that this new reassortant might have been circulating for some time to achieve this degree of diversity. because of the roughly 5% nucleotide diff erences in the h7 and n9 genes between the 2013 h7n9 virus and the closest avian virus genes, more extensive virological surveillance of wild or domestic avian and non-avian animal species, as has been done for the severe acute respiratory syndrome coronavirus, 30 is needed to understand the evolutionary pathway of this h7n9 virus, which is the fi rst n9 subtype virus infecting human beings. up to now, only one wild pigeon from nanjing has tested positive for the 2013 h7n9 virus, 31 but the epidemiological and genomic details of this virus isolate are not yet available. one unique feature of the 2013 h7n9 human epidemic is the absence of preceding die-off s in poultry or wild birds, which makes epidemiological control diffi cult. the start of the h5n1 outbreak in 1997 was marked by three highly pathogenic avian infl uenza virus outbreaks on farms 1 month before the fi rst human case and about 32 avian infl uenza virus can be divided into highly pathogenic avian infl uenza virus and low pathogenic avian infl uenza virus on the basis of pathogenicity in chickens. the world organization for animal health has defi ned highly pathogenic avian infl uenza virus as any infl uenza virus that is lethal for six or more of eight 4-8-week-old susceptible chickens by 10 days after intravenous inoculation with 0·2 ml of a 1/10 dilution of a bacteriafree infective allantoic fl uid, or that has an intravenous pathogenicity index of greater than 1·2, which is calculated by scores on the severity of illness. 33 one key feature of highly pathogenic avian infl uenza virus is the presence of multibasic aminoacids at the haemagglutinin cleavage site, which render haemagglutinin susceptible to cleavage by many diff erent proteases. 34 hence, h5 or h7 viruses that have low pathogenicity in chickens are still regarded as highly pathogenic avian infl uenza virus if their ha0 cleavage sequence is similar to that of other highly pathogenic avian infl uenza viruses. the haemagglutinin cleavage site from all reported 2013 h7n9 viruses do not possess multibasic aminoacids. 11, 12, 35 data from preliminary pathogenicity testing suggest that chickens and quails infected with h7n9 do not show signs of illness. 36 a low pathogenic avian infl uenza virus can mutate to become highly pathogenic during an outbreak in domestic poultry. for the h7n3 virus outbreaks in 2002 and 2004, the diff erence in the cleavage site between low pathogenic avian infl uenza virus and highly pathogenic avian infl uenza virus was speculated to be a sequence insertion from the neuraminidase or m gene at the haemagglutinin cleavage site through intersegmental recombination. 37 close monitoring of the evolution of the 2013 h7n9 is needed. another possible explanation for the apparent absence of a preceding infection with h7n9 in poultry in the 2013 outbreak is that previous infection by a closely related low pathogenic avian infl uenza h7 virus elicited crossprotection for this h7n9 virus because h7n3 virus was recently reported in ducks in zhejiang, china. 38 furthermore, prevalent h9n2 infections in poultry or the extensive use of the h5n1 vaccine in poultry in china might induce cell-mediated immunity against highly conserved internal viral proteins without inducing any neutralising antibody. this process might be suffi cient to militate against poultry death, but not asymptomatic viral shedding. 39 the eff ect of previous infection by other avian infl uenza virus subtypes and previous h5n1 vaccine on the susceptibility to infection by h7n9 deserves further investigation. interspecies jumping of an avian infl uenza virus into human beings is favoured by several factors: viral mutations that allow the virus to bind, replicate, and spread in human beings; a high inoculum of virus during the transmission between the poultry and human being; host susceptibility such as the presence of underlying diseases; extremes of age; and genetic predispositions. one key viral factor governing host adaptation and transmission is the relative binding affi nity between haemagglutinin and the sialic acid receptor on the host cell surface. 40 avian infl uenza viruses generally prefer α-2,3 sialic acid receptors abundantly found in avian alimentary tract and human infl uenza viruses generally prefer α-2,6 sialic acid receptors abundantly found in the human respiratory tract. h5n1 viruses can invade the lower respiratory tract of human beings because the viruses retain strong affi nity for the α-2,3-linked receptors present in the lower respiratory tract of human beings. 41 an increased affi nity for the α-2,6-linked receptors, which are abundant in the upper respiratory tract of human beings, is believed to be responsible for transmission between birds and human beings. in egypt, which has had the highest number of h5n1 infections since 2009, 99% of virus isolates had a thr160ala (h3 numbering) mutation, which is associated with loss of glycosylation at position 158-160, and increased transmissibility in ferrets and guinea pigs. 42, 43 thr160ala was also present in all reported strains of 2013 h7n9 (table 2). the haemagglutinin mutations gly186val and gln226leu, which increase the binding of h5n1 and h7 viruses to α-2,6-linked receptor and h5n1 virus transmission between ferrets, are present in most strains of the human and avian 2013 h7n9, but none of the 1997 h5n1 strains (table 2). 11,12,35,45-47 however, a recent structural modelling and binding study with human tracheal epithelium and alveolar tissue sections suggested that 2013 h7n9 has weak binding to both α-2,3-linked and α-2,6-linked receptors. 48 further studies should confi rm whether various strains of h7n9 diff er in binding affi nities. neuraminidase enables release of virus particles from the host cell surface. deletion in the stalk region of neuraminidase, which was found in the human h5n1 viruses and previous h7 viruses, is associated with increased virulence and replication in the respiratory tracts of chickens. 49 2013 h7n9 also has a fi ve aminoacid deletion in the stalk region, which might improve its adaptation to poultry. 11, 12, 29, 35 comparative studies in wild waterfowl and domestic chickens will ascertain the degree of adaptation by this virus in these avian species. mutations in the polymerase complex aff ect viral replication. pb2 glu627lys mutation is associated with increased viral replication at 33°c, the nasal body temperature in human beings, which is markedly lower than that of avian species. 50 furthermore, this mutation is important in aerosol transmission of avian h1n1 virus between ferrets. 51 glu627lys was found only in human strains, but not the avian strains, of 1997 h5n1 or 2013 h7n9 (table 2) . however, glu627lys was specifi cally found in the qinghai outbreak of h5n1 in 2005. 52 pb2 asp701asn is another important mutation associated with mammalian adaptation and transmission. 42, 53 notably, most virus isolates from individuals infected with 2013 h7n9 had either glu627lys or asp701asn, but not both. asp701asn can compensate for the lack of glu627lys for transmission between guinea pigs. 53 other mutations aff ect viral replication, transmission, and virulence of infl uenza viruses. ns1 protein is the key virulence factor counteracting innate host immunity. ns1 protein contains a four-aminoacid motif at the c terminal that disrupts cellular signalling by binding to the host pdz domain-containing proteins. recombinant infl uenza virus with pdz-binding motif has increased pathogenicity in mice. 54 ns1 protein from human seasonal infl uenza viruses cannot bind to human proteins containing pdz domain. 55 the ns1 of all 2013 h7n9 strains has a deletion of the pdz motif. the structural m1 protein is needed for viral assembly and budding. two mutations in m1, asn30asp and thr215ala, associated with increased virulence in a mice model, are found in all strains of the 2013 h7n9 virus. 56 before the 2013 outbreak, the h7n9 virus had never been reported to cause human infection, although other h7 viruses had been associated with sporadic infections since 1979 (table 3) . 5, 7, 9, [57] [58] [59] [60] [61] [62] [63] serological evidence of h7 infection was present in 38% of a rural population in the jiangsu province near shanghai. 64 however, a study including 1544 serum samples from poultry workers in eastern china in 2012 did not identify any seropositive samples by microneutralisation assay, suggesting that subclinical h7n9 infection was unlikely before 2013. 65 infl uenza a subtypes h7n1, h7n2, h7n3, h7n7, and h7n8 were previously isolated from poultry and wild birds from china. 38, 66 although studies of seroprevalence did not fi nd any evidence of h7 infection among sparrows or pigs in china, 67,68 h7n9 viruses were isolated from birds in nearby countries, including mongolia and south korea in 2008. 69, 70 moreover, in the usa, h7n9 viruses have caused outbreaks in chickens. 71 other h7n9 viruses were reported in wild birds or poultry from four continents. 10 most of the laboratory-confi rmed cases of 2013 h7n9 were older adults living in the city, with a median age of 61 years. by contrast, in the 1997 h5n1 outbreak in hong kong, 61% of patients were younger than 18 years of age 1,74 and the mean age of patients with h5n1 in china between 2003 and 2008 was 28·1 years (table 4) . 78 the severe disease in older adults and the small number of cases in children in the 2013 h7n9 outbreak can be accounted for by several factors. first, h7n9 infection in children was mainly mild or asymptomatic (appendix); 14,79,80 therefore, infection in this age group might have been underdiagnosed because confi rmatory laboratory tests were usually reserved for severe cases. two children were found to be infected with h7n9 during an enhanced surveillance that included 6333 children younger than 4 years of age with infl uenza-like illness. 79 second, some adult patients were poultry workers; generally only adults go to live poultry markets and therefore adults might have had a greater dose or prolonged duration of exposure to h7n9 than did children. additionally, adults might have an increased chance of exposure to pet birds. finally, older adults might have impaired development of specifi c immune response to the h7n9 virus because of the immunological phenomenon of original antigenic sin. 81 previous exposure to other infl uenza viruses might also lead to rapid increase in non-neutralising antibodies against these viruses, which can be associated with more severe h7n9 disease. 83, 84 notably, most fatal or severe cases of 2013 h7n9 have comorbidities, and up to a quarter of adult patients were smokers. 15 the eff ect of genetic predisposition (eg, the ifitm3 and cd55 polymorphisms) on h7n9 infection warrants further investigation. 85, 86 a higher percentage of elderly patients aged 65 years or older and a higher proportion of males were infected during the 2013 h7n9 than were in the 1997 h5n1 outbreak. one explanation for this fi nding could be that most grandmothers in shanghai take care of their grandchildren at home while the retired grandfathers go to live poultry markets for grocery shopping every day. 87 another unique feature is that no poultry workers were infected with h5n1 in the hong kong epidemic. seroepidemiology might ascertain the cause of this diff erence. human beings probably acquired the h7n9 virus via direct contact with infected poultry or their surroundings, although aerosol transmission between ferrets was reported. 77 ser variant associated with adaptation to pigs 9/10 all all all increased binding to α-2,6-linked sialic acid receptor all all 3 of 5 6 of 10 gly186val increased binding to α-2,6-linked sialic acid receptor 9 of 10 all nd nd increased binding to α-2,6-linked sialic acid receptor 8 of 10 * 5 of 6 nd nd cleavage by ubiquitous proteases nd nd all all deletions in stalk region increased virulence all all all all neuraminidase resistance 2 of 11 nd nd nd enhanced polymerase activity all all all all improved viral replication at 33°c 7 of 9 nd 2 of 9 nd mammalian adaptation 1 of 9 nd nd nd enables droplet transmission in ferrets nd nd nd nd enables droplet transmission in ferrets 5 of 7 all nd nd full-length full-length pb1-f2 needed for virulence in mice all 3 of 4 all all increased virulence in a mice model nd nd nd nd asn30asp, thr215ala increased virulence in a mice model all all all all amantadine resistance all all nd nd increased virulence in mice all all all all pdz-binding motif signalling of host proteins deleted deleted avian type avian type personal view that 18% of transmission between farms was related to wind. 89 up to may 22, 2013, 899 758 animal and environmental samples had been tested. 53 samples were positive for the 2013 h7n9 virus; 51 of these were collected from poultry or environmental samples in poultry markets, one sample was collected from a wild pigeon in nanjing, and one sample was collected from a domestic racing pigeon at a household farm in nantong. 28 2013 h7n9 viruses with very similar gene sequences have been found in pigeons, chickens, ducks, and environmental samples from poultry markets in aff ected areas. 11, 35, 36, 44 as in the h5n1 outbreak in hong kong, most confi rmed cases of h7n9 (55·9%) had history of contact with poultry. 15 although some patients had contact with pigs, 2013 h7n9 has not been isolated from pigs. 14, 36 in the 2013 outbreak, the h7n9 virus might have originated from poultry farms and was amplifi ed in overcrowded live poultry markets before its transmission to human beings. slaughtering and preparation of infected poultry constitute the highest risks of exposure to infected poultry secretions and excreta. although transmission to human beings through contact with wild birds or consumption of inadequately cooked poultry is possible, documentation of such occurrences is scarce even though h7 viruses are able to survive and remain infective for a prolonged period. 90 no defi nitive evidence of continuing person-to-person transmission exists, although some fi rst-generation transmission might have occurred in four case clusters of which three were familial clusters. 14,91 however, exposure to a common avian or environmental source could not be excluded in these clusters. few reports of person-to-person trans mission of h5n1 have been recorded. 92 the predilection of the 2013 h7n9 virus for the lower respiratory tract-shown by the higher virus burden in patients sputum than throat swab-might explain the low number of person-to-person transmissions as in the case of h5n1. 132 reported h7n9 infections in patients from march to may 2013 has far exceeded the 45 reported h5n1 infections in china within the past 10 years. 61 the crude case fatality of 2013 h7n9 infection is 30%, which is lower than that for h5n1 infection (60%), but much higher than the 0·1% of the 2009 pandemic or seasonal infl uenza. 3, 19 the true case fatality rate is uncertain because many of the patients are still receiving care in the intensive care units. enhanced surveillance between march 7 and april 28, 2013, in which 20 739 patients with infl uenza-like illness were tested, identifi ed only two patients with h7n9 who were not hospitalised. 79 the fi rst human cases of h7n9 were detected in shanghai, which is served by the farming and industrial establishments along the yangtze river delta. shanghai, a cosmopolitan megacity hardest hit by this novel virus, is similar to hong kong, a region that is served by the pearl river delta and where the fi rst human cases of h5n1 were detected in 1997. 1,75,76 both cities have well established health-care infrastructure with the necessary diagnostic methods for the detection and charac terisation of new viruses. furthermore, both cities are located along the asian-australasian fl yway, where migratory birds stop at their wetlands and have surrounding poultry farms serving the densely populated areas that have many live poultry markets (table 4) . between 1985 and 2011, consumption of poultry per person has increased 3·3 times from 3·2 kg to 10·6 kg in urban areas, whereas overall consumption of poultry increased 8·8 times from 1·53 to 13·54 million tonnes in china between 1987 and 2012. [93] [94] [95] additionally, of all municipal areas or provinces in china, shanghai has the highest densities of both people and poultry. 94 this is similar to the situation in hong kong, where poultry consumption increased greatly before the 1997 h5n1 outbreak (fi gure 4). 75, 93, 96 the emergence of the h7n9 virus in 2013 might also be related to the selective pressure induced by the widespread use of the h5n1 vaccine because h5 and h7 are the common subtypes that generally cause poultry outbreaks. china uses more than 90% of the h5n1 vaccine worldwide. 97 9 netherlands 89 human infections with one fatality; an outbreak in chicken arose before the human cases in the h7n9 and h5n1 outbreaks, infected individuals presented with acute severe community-acquired pneumonia that did not respond to typical and atypical antimicrobial coverage. in an analysis of 111 of 132 reported cases of human h7n9 infections in 2013, more than 90% of patients had fever or cough, and 24% of the 111 patients had haemoptysis. 15 71% of the patients had acute respiratory distress syndrome. other complications associated with h7n9 infection are rhabdomyolysis, acute kidney injury, encephalopathy, and multiorgan dysfunction. 11, 15 unlike the 1997 h5n1 outbreak, in which nearly 40% of patients had only fever and upper respiratory tract symptoms, none of the 111 patients with 2013 h7n9 infection had sore throat, rhinorrhoea, or conjunctivitis, although mild rhinorrhoea was reported in a 3·5-year-old boy. 80 gastrointestinal symptoms (13·5%) were less common in people infected with h7n9 than in those with the 1997 h5n1 infection (50%). 1,15 reye's syndrome was reported in a child with 1997 h5n1 infection. 1 gastro intestinal haemorrhage, reactive haemo phagocytosis, and haemorrhagic pleural eff usion, which were present in some cases of 1997 h5n1, have not been reported for h7n9 infection. coinfection at presentation or during admission to hospital can occur, as reported for h5n1. 2, 15, 98 laboratory abnormalities in patients with either infections were prominent lymphopenia, thrombocytopenia, coagulo pathy, and raised serum transaminase, creatine kinase, and c-reactive protein concentrations. radio logical features were similar in both infections: pulmonary consolidation (which progressed to involve bilateral lung fi elds), diff use ground glass opacities, pleural eff usions, and mediastinal emphysema. none of these clinical, laboratory, and radiological manifestations are pathognomonic for h7n9 infections. the defi nitive diagnosis depends on viral culture or real-time-pcr (rt-pcr) for the h7 and n9 gene targets from respiratory tract secretions. because of the predilection of this virus for the lower respiratory tract, if available, sputum, endotracheal aspirate or bronchoalveolar lavage might give a better sensitivity than throat or nasopharyngeal specimens. however, point-of-care rapid antigen tests and multiplex rt-pcr assays have low sensitivity. 99 acute and convalescent serum antibody testing by haemagglutination inhibition or neutralisation can be useful for epidemiology or retrospective diagnosis. any febrile patients with history of travel to aff ected areas, or contact with poultry or infected patients can be tested for h7n9 as a screening strategy for the early identifi cation of imported cases. 2013 h7n9 is probably resistant to adamantanes because of the presence of an m2 ser31asn mutation (table 2). 12 both genetic analysis and in-vitro testing showed that most strains of 2013 h7n9 are susceptible to neuraminidase inhibitors. 100 however, two reported strains carry the neuraminidase arg292lys (n2 numbering) mutation, which is associated with resistance to oseltamivir and zanamivir (increase in half maximal inhibitory concentration [ic 50 ] more than 9000-fold for oseltamivir and between four-fold and 25-fold for zanamivir). 101 personal view therapy of more than 3 days after symptom onset was not an independent risk factor for acute respiratory distress syndrome in 2013 h7n9 infections, 15 previous experience from h5n1, 2009 pandemic, and seasonal infl uenza infections suggest that early oseltamivir treatment started less than 2 days after symptom onset can reduce morbidity and mortality. 19, 102 inhalational neuraminidase inhibitors such as zanamivir and laninamivir are unlikely to be helpful in patients with respiratory failure. 103, 104 because oseltamivir might not aff ect outcomes in patients with late and severe infl uenza pneumonia, intravenous zanamivir and peramivir should also be investigated in future treatment trials. 105 other investigational antivirals include those acting on the haemagglutinin receptor (das181), nucleoprotein (nucleozin), poly merase (viramidine and t705), and protease (aprotinin). 106 antivirals targeting the host machinery including nitazoxanide were also reported. [106] [107] [108] although 2013 h7n9 is susceptible to neuraminidase inhibitors, the virus might develop resistance during treatment. 19 mice infected with the infl uenza virus resistant to amantadine had improved survival when treated with the triple combination of amantadine, oseltamivir, and ribavirin. 109 empirical antibiotics are initially needed to cover other common pathogens, such as streptococcus pneumoniae and staphylococcus aureus, but should be stopped when bacterial tests are negative. the role of immunomodulation remains controversial. glucocorticoids have been used in many patients with 2013 h7n9, but the benefi t is unclear. 12, 15 non-steroidal anti-infl ammatory drugs such as celecoxib improved survival of mice with h5n1 infection. 110 confl icting outcomes of patients treated with statins and macrolides have been reported. 111 the plasma of convalescent patients, which contains specifi c neutralising antibodies, has been used in patients with the h5n1 virus, 1918 pandemic h1n1, and the 2009 pandemic h1n1. hyperimmune globulin was also used in the 2009 pandemic. both treatments have reduced morbidity and mortality in small studies. [112] [113] [114] [115] supportive measures in intensive care are especially important for patients with respiratory or multiorgan failure. extracorporeal membrane oxygenation could be benefi cial in patients with respiratory failure despite maximum mechanical ventilatory support. 116 in view of the many similarities between the hong kong and shanghai outbreaks, many of the measures implemented in hong kong can be, and have already been, applied to the current outbreak in eastern china. in the 1997 hong kong outbreak, temporary closure of live poultry markets and cessation of poultry trading were instituted. subsequently, diff erent poultry species were segregated to reduce the risk of further genetic shanghai (registered population) hong kong 1 9 7 3 1 9 7 7 1 9 8 1 1 9 8 3 1 9 8 9 1 9 9 3 1 9 9 5 2 0 0 1 2 0 0 5 2 0 0 7 2 0 1 1 year 1 9 7 5 1 9 7 9 1 9 8 5 1 9 8 7 1 9 9 1 1 9 9 7 1 9 9 9 2 0 0 3 2 0 0 9 reassortment. the regular cleansing of designated transport cages in live poultry markets was introduced to stop the virus traffi cking between farms and markets. to interrupt the amplifi cation of infl uenza virus, a monthly rest day with no live poultry allowed in the wet market was implemented in hong kong in july 2001. the isolation rate of h9n2 from poultry in live markets before the rest day could be as high as 10%, but reduced to less than 1% after the rest day. 117 to further reduce the spread of infl uenza virus in wet markets, overnight poultry storage was banned in july 2008. 118 a further 84% reduction in the isolation rate of h9n2 was reported. as in the h5n1 outbreak, poultry vaccination specifi cally against the h7n9 virus could probably reduce its transmission. a mathematical model showed that poultry vaccination with a matched h5n1 vaccine with coverage of only 30% of the poultry reduces the basic reproductive number to less than one. 117 the 1997 h5n1 human outbreak was stopped after a cull of all poultry in hong kong. 117 subsequently, only registered farms with stringent biosecurity measures in china can import chickens to hong kong. if a highly pathogenic avian infl uenza virus outbreak arose, all live poultry and poultry products from the aff ected province would be suspended for up to 21 days. for unaff ected farms within 3 km of the index farm aff ected by the highly pathogenic avian infl uenza virus, there would be a 90 day suspension for live poultry and poultry products. for farms where 2013 h7n9 is detected by virological testing, similar depopulation and perimetric moratorium strategies of poultry control are necessary to control the source. the general public was educated to avoid contact with birds or poultry, to cook poultry thoroughly, and to comply with hand hygiene because consumption of raw poultry might be a route of transmission. public education and administrative measures have also led to a slow change in eating habits from live chicken to chilled or frozen chicken. to reduce the contact between human beings and poultry in hong kong, the hong kong government implemented a voluntary surrender scheme in 2004, and subsequently a buyout scheme in 2008 for poultry retailers, wholesalers, transporters, and farmers. in 2006, domestic households were prohibited to keep any live poultry. with these measures, between 1997 and 2013, the number of local poultry farms in hong kong decreased from about 800 to 30 and the number of retail poultry stalls decreased from about 800 to 131. since 1997, the number of imported live chickens was reduced from about 100 000 per day to 7000 per day. enhanced surveillance of h5n1 and h7n9 in both live and dead wild birds, farm, and market poultries might be important for geographical areas along the migratory route of wild birds. with these measures, no local cases of h5n1 in hong kong have been identifi ed since 2007, despite the failure to implement central slaughtering because of cultural resistance. the 2013 h7n9 outbreak in human beings resembles the 1997 h5n1 outbreak in many ways, including substantial mortality associated with severe pneumonia, multiorgan dysfunction, and cytokine dysregulation, appearance in cities along the route of migratory birds with high density of poultry, predominant poultry-to-human transmission, and the predilection for the lower respiratory tract. 11, 119 the high proportion of elderly men infected with h7n9 could be attributable to diff erent social circumstances leading to this age and sex bias. the rapid increase in human beings infected with severe h7n9 to more than 130 cases within 2 months from march to may, 2013, is unprecedented for avian infl uenza viruses and could be related to enhanced transmissibility from poultry to human or improved virological testing. however, h7n9 is diffi cult to control because fl ock die-off did not precede human cases. stringent measures to control the poultry outbreak will stop the human epidemic and perhaps decrease the risk of h7n9 evolving to become a pandemic agent. even though this dangerous scenario has not happened with h5n1, the virus has spread from hong kong to the whole of china, southeast asia, middle east, and africa. 2 although the number of human beings infected with h7n9 was greatly reduced in may 2013 (possibly related to the rising ambient temperature or the epidemiological control measures in live poultry markets), high vigilance is still needed in view of the interval of 6 months between the fi rst and second 1997 h5n1 human case. biosecurity measures for live poultry markets, farms, and traffi cking should be commensurate with increasing demand for poultry. transmission cycles within markets or between markets and farms could be interrupted by a no overnight poultry policy with daily poultry-stall cleansing and ultimately central slaughtering in cities. precautionary research and development of poultry and human h7n9 vaccines are important. control in rural areas with many backyard farms relies on poultry vaccination. virological surveillance of wild birds and domestic poultry in farms and markets is important in understanding the emergence of the 2013 h7n9 and other avian infl uenza viruses. over 30 years from 1981 to 2011, the consumption of poultry in hong kong has tripled to 60 kg per person per year. in 2012, the consumption of poultry in china reached 10 kg per person per year, and the mainland cities such as shanghai are likely to catch up with the amount of consumption in hong kong in coming years (fi gure 4). 95 increasing problems with avian infl uenza in poultry and human beings are anticipated. we declare that we have no confl icts of interest. a/mallard/ohio/11os2010/2011(h7n8) a/common goldeneye/wisconsin/10os4202/2010(h7n6) a/northern pintail/illinois/10os3959/2010(h7n3) a/chicken/new york/sg-00299/1997(h7n2) a/american black duck/maryland/424/2001(h7n3) a/ruddy turnstone/delaware bay/124/2007(h7n3) a/northern shoveler/california/44287-164/2007(h7n7) a/american green-winged teal/california/28228/2007(h7n6) a/starling/victoria/1/1985(h7n7) a/turkey/england/647/1977(h7n7) a/duck/hong kong/301/1978(h7n2) a/turkey/ireland/pv74/1995 (h7n7) a/duck/hokkaido/w62/2011(h7n7) a/duck/taiwan/4201/99(h7n7) a/duck/hokkaido/143/2003(h7n1) a/duck/korea/bc10/2007(h7n3) a/mallard/geumgang/1/2007(h7n7) a/duck/mongolia/119/2008(h7n9) a/magpie/korea/yjd174/2007(h7n7) a/duck/tsukuba/700/2007(h7n7) a/duck/zhejiang/12/2011(h7n3) a/hangzhou/1/2013(h7n9) a/duck/korea/a349/2009(h7n2) a/chicken/new jersey/sg-00341/2000(h7n2) a/chicken/pennsylvania/143586/2002(h7n2) a/duck/zhejiang/10/2011(h7n3) a/chicken/california/755/1999(h10n7) a/ruddy turnstone/new jersey/1148676/2004(h5n7) a/starling/victoria/1985(h7n7) a/wild goose/dongting/pc0360/2012(h7n7) a/duck/mongolia/583/02(h4n7) a/duck/hong kong/562/1979(h10n9) a/turkey/france/03295/2003(h9n9) a/shearwater/australia/2576/1979(h15n9) a/swan/shimane/190/2001(h6n9) a/pintail/shimane/324/1998(h1n9) a/duck/mongolia/119/2008(h7n9) a/duck/hunan/1590/2007(h6n9) a/duck/vietnam/g30/2008(h11n9) a14/2011(h7n9) a/hangzhou/1/2013(h7n9) clinical features and rapid viral diagnosis of human disease associated with avian infl uenza a h5n1 virus avian infl uenza a h5n1 virus: a continuous threat to humans world health organization. cumulative number of confi rmed human cases for avian infl uenza a(h5n1) reported to who laboratory-confi rmed case of human infection with avian infl uenza a(h6n1) virus in taiwan recovered; taiwan cdc urges public to take precautions to stay healthy low pathogenic avian infl uenza a (h7n2) virus infection in immunocompromised adult infl uenza virus a (h10n7) in chickens and poultry abattoir workers human illness from avian infl uenza h7n3, british columbia infection of immunocompromised patients by avian h9n2 infl uenza a virus avian infl uenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome chinese center for disease control and prevention. questions and answers about human infection witha(h7n9) avian infl uenza virus human infections with the emerging avian infl uenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome human infection with a novel avian-origin infl uenza a (h7n9) virus the fi rst case of h7n9 infl uenza in taiwan preliminary report: epidemiology of the avian infl uenza a (h7n9) outbreak in china clinical fi ndings in 111 cases of infl uenza a (h7n9) virus infection shanghai reports 1 h7n9 death world health organization. global alert and response disease outbreak news. human infection with avian infl uenza a(h7n9) virus-update mapping the h7n9 avian fl u outbreaks two years after pandemic infl uenza a/2009/h1n1: what have we learned? a distinct lineage of infl uenza a virus from bats structural and functional characterization of neuraminidase-like molecule n10 derived from bat infl uenza a virus identifi cation of novel infl uenza a virus proteins translated from pa mrna emergence of multiple genotypes of h5n1 avian infl uenza viruses in hong kong sar characterization of the infl uenza a virus gene pool in avian species in southern china: was h6n1 a derivative or a precursor of h5n1? origin and diversity of novel avian infl uenza a h7n9 viruses causing human infection: phylogenetic, structural, and coalescent analyses evolutionary characteristics of a/hangzhou/1/2013 and source of avian infl uenza virus h7n9 subtype in china genetic characterization of h7n2 infl uenza virus isolated from pigs ministry of agriculture of the people's republic of china environmental connections of novel avianorigin h7n9 infl uenza virus infection and virus adaptation to the human severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats no h7n9 virus found in poultry farms: moa characterization of avian h5n1 infl uenza viruses from poultry in hong kong avian infl uenza. manual of diagnostic tests and vaccines for terrestrial animals 2012 avian infl uenza virus hemagglutinins h2, h4, h8, and h14 support a highly pathogenic phenotype genetic analysis of novel avian a(h7n9) infl uenza viruses isolated from patients in china emergence of avian infl uenza a (h7n9) virus causing severe human illness-china intersegmental recombination between the haemagglutinin and matrix genes was responsible for the emergence of a highly pathogenic h7n3 avian infl uenza virus in british columbia sequence and phylogenetic analysis of h7n3 avian infl uenza viruses isolated from poultry in china in 2011 strategies and challenges for eliciting immunity against avian infl uenza virus in birds interspecies transmission and emergence of novel viruses: lessons from bats and birds avian fl u: infl uenza virus receptors in the human airway identifi cation of amino acids in ha and pb2 critical for the transmission of h5n1 avian infl uenza viruses in a mammalian host egyptian h5n1 infl uenza viruses-cause for concern live-animal markets and infl uenza a (h7n9) virus infection quantitative description of glycan-receptor binding of infl uenza a virus h7 hemagglutinin experimental adaptation of an infl uenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets airborne transmission of infl uenza a/h5n1 virus between ferrets glycan receptor binding of the infl uenza a virus h7n9 hemagglutinin length variations in the na stalk of an h7n1 infl uenza virus have opposite eff ects on viral excretion in chickens and ducks biochemical impact of the host adaptation-associated pb2 e627k mutation on the temperature-dependent rna synthesis kinetics of infl uenza a virus polymerase complex human ha and polymerase subunit pb2 proteins confer transmission of an avian infl uenza virus through the air properties and dissemination of h5n1 viruses isolated during an infl uenza outbreak in migratory waterfowl in western china transmission of infl uenza virus in a mammalian host is increased by pb2 amino acids 627k or 627e/701n a new infl uenza virus virulence determinant: the ns1 protein four c-terminal residues modulate pathogenicity large-scale sequence analysis of avian infl uenza isolates two amino acid residues in the matrix protein m1 contribute to the virulence diff erence of h5n1 avian infl uenza viruses in mice update: infl uenza activity-united states, 2003-04 season assessing the role of contact tracing in a suspected h7n2 infl uenza a outbreak in humans in wales serological analysis of serum samples from humans exposed to avian h7 infl uenza viruses in italy between 1999 and outbreak of low pathogenicity h7n3 avian infl uenza in uk, including associated case of human conjunctivitis update on human cases of infl uenza at the human-animal interface conjunctivitis in human beings caused by infl uenza a virus of seals avian infl uenza virus isolated from a woman with conjunctivitis pandemic infl uenza: a zoonosis? serologic study for infl uenza a (h7n9) among high-risk groups in china characterization of an avian infl uenza virus of subtype h7n2 isolated from chickens in northern china seroprevalence and genetic characteristics of fi ve subtypes of infl uenza a viruses in the chinese pig population: a pooled data analysis a survey of avian infl uenza in tree sparrows in china in 2011 low pathogenic h7 subtype avian infl uenza viruses isolated from domestic ducks in south korea and the close association with isolates of wild birds and their relationships with korean isolates from domestic poultry and wild birds avian infl uenza in north america infl uenza a viruses from wild birds in guatemala belong to the north american lineage hofl e u. detection of low pathogenic avian infl uenza viruses in wild birds in castilla-la mancha (south central spain) outbreak of avian infl uenza a(h5n1) virus infection in hong kong in 1997 shanghai municipal health and family planning commission demographic trends in hong kong case-control study of risk factors for avian infl uenza a (h5n1) disease, hong kong update: who-confi rmed human cases of avian infl uenza a (h5n1) infection monitoring avian infl uenza a(h7n9) virus through national infl uenza-like illness surveillance avian infl uenza a(h7n9) virus infections original antigenic sin responses to infl uenza viruses virus-host interactions and the unusual age and sex distribution of human cases of infl uenza a(h7n9) in china high titer and avidity of nonneutralizing antibodies against infl uenza vaccine antigen are associated with severe infl uenza severe pandemic 2009 h1n1 infl uenza disease due to pathogenic immune complexes interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe infl uenza in chinese individuals a functional variation in cd55 increases the severity of 2009 pandemic h1n1 infl uenza a virus infection h7n9 mystery: why does age profi le tilt older? infectivity, transmission, and pathology of human h7n9 infl uenza in ferrets and pigs genetic data provide evidence for wind-mediated transmission of highly pathogenic avian infl uenza infectivity of h7 lp and hp infl uenza viruses at diff erent temperatures and ph and persistence of h7 hp virus in poultry meat at refrigeration temperature transcript of media briefi ng by dr michael o'leary, who representative in china probable limited person-to-person transmission of highly pathogenic avian infl uenza a (h5n1) virus in china united states department of agriculture foreign agricultural service. production, supply, and distribution online database poultry sector in china: structural changes during the past decade and future trends impact of vaccines and vaccination on global control of avian infl uenza human co-infection with novel avian infl uenza a h7n9 and infl uenza a h3n2 viruses in jiangsu province evaluation of analytical sensitivity of seven point-of-care infl uenza detection kits and two molecular tests for detection of avian-origin h7n9 and swine-origin h3n2 variant infl uenza a viruses association between adverse clinical outcome in human disease caused by novel infl uenza a h7n9 virus and sustained viral shedding and emergence of antiviral resistance neuraminidase sequence analysis and susceptibilities of infl uenza virus clinical isolates to zanamivir and oseltamivir impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 infl uenza a (h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients clinical effi cacy of long-acting neuraminidase inhibitor laninamivir octanoate hydrate in postmarketing surveillance long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of infl uenza: a double-blind, randomized, noninferiority clinical trial newer infl uenza antivirals, biotherapeutics and combinations thiazolides, a new class of anti-infl uenza molecules targeting viral hemagglutinin at the post-translational level the lipid mediator protectin d1 inhibits infl uenza virus replication and improves severe infl uenza obatoclax, saliphenylhalamide, and gemcitabine inhibit infl uenza a virus infection effi cacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant infl uenza a viruses delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of infl uenza a/h5n1 virus eff ect of immunomodulatory therapies in patients with pandemic infl uenza a (h1n1) 2009 complicated by pneumonia hyperimmune intravenous immunoglobulin treatment: a multicentre double-blind randomized controlled trial for patients with severe a(h1n1)pdm09 infection convalescent plasma treatment reduced mortality in patients with severe pandemic infl uenza a (h1n1) 2009 virus infection hoff man sl. meta-analysis: convalescent blood products for spanish infl uenza pneumonia: a future h5n1 treatment? treatment with convalescent plasma for infl uenza a (h5n1) infection referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 infl uenza a(h1n1) a model to control the epidemic of h5n1 infl uenza at the source avian infl uenza and ban on overnight poultry storage in live poultry markets, hong kong h7n9 is a virus worth worrying about we thank wing-man ko of the food and health bureau of the hong kong special administrative region for helping with some data collection in this manuscript. hc is partially supported by the areas of excellence of the university grants committee 367 (grant 